U.S. patent application number 10/008140 was filed with the patent office on 2003-07-03 for simultaneous quantification of nucleic acids in diseased cells.
Invention is credited to Stuyver, Lieven.
Application Number | 20030124512 10/008140 |
Document ID | / |
Family ID | 27399486 |
Filed Date | 2003-07-03 |
United States Patent
Application |
20030124512 |
Kind Code |
A1 |
Stuyver, Lieven |
July 3, 2003 |
Simultaneous quantification of nucleic acids in diseased cells
Abstract
This invention pertains to a novel method to screen the efficacy
of various anti-viral, and especially anti-HIV and HCV agents by
using a novel real-time polymerase chain reaction technique. This
method can also be applied to toxicity screening, especially of
mitochondrial toxicity of these compounds as well.
Inventors: |
Stuyver, Lieven;
(Snellville, GA) |
Correspondence
Address: |
Sherry M. Knowles, Esq.
KING & SPALDING
45th Floor
191 Peachtree Street, N.E.
Atlanta
GA
30303
US
|
Family ID: |
27399486 |
Appl. No.: |
10/008140 |
Filed: |
October 18, 2001 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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60241488 |
Oct 18, 2000 |
|
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60256067 |
Dec 15, 2000 |
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60282156 |
Apr 6, 2001 |
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Current U.S.
Class: |
435/5 ; 435/6.16;
435/91.2; 536/24.3 |
Current CPC
Class: |
C07H 19/048 20130101;
C12Q 1/701 20130101; C07H 21/04 20130101; C12Q 2600/142 20130101;
C12Q 2545/101 20130101; C12Q 2561/101 20130101; C12Q 2537/143
20130101; C12Q 1/6895 20130101; C07H 19/06 20130101; C12Q 1/6876
20130101; C12Q 1/689 20130101; C12Q 2600/136 20130101; C07H 19/20
20130101; Y10S 435/81 20130101; C07H 19/10 20130101; C12Q 1/6809
20130101; C07H 19/16 20130101; C12Q 1/701 20130101; C12Q 2600/158
20130101 |
Class at
Publication: |
435/5 ; 435/6;
435/91.2; 536/24.3 |
International
Class: |
C12Q 001/70; C12Q
001/68; C07H 021/04; C12P 019/34 |
Claims
We claim:
1. A process for identifying a compound which inhibits viral
replication that includes contacting nucleic acids from a virus
infected host with an amplification reaction mixture that contains
at least two primers and/or probes that provide detectable signals
during a polymerase chain reaction, wherein the first primer and/or
probe provides a detectable signal on the occurrence of the
transcription of viral nucleic acids; and the second primer and/or
probe provides a second detectable signal on the occurrence of the
transcription of host nucleic acids.
2. The process of claim 1, wherein the host nucleic acid is nuclear
nucleic acid.
3. The process of claim 1, wherein the host nucleic acid is
mitochondrial nucleic acid.
4. The process of claim 3, wherein the mitochondrial nucleic acid
is mitochondrial DNA.
5. The process of claim 3, wherein the mitochondrial nucleic acid
is mitochondrial RNA.
6. The process of claim 1, wherein the viral nucleic acid is a
non-coding sequence.
7. The process of claim 6, wherein the non-coding sequence is a
5'-non-coding sequence.
8. The process of claim 6, wherein the non-coding sequence is a
3'-non-coding sequence.
9. The process of claim 6, wherein the non-coding sequence is an
intron.
10. The process of claim 6, wherein the non-coding sequence is from
.beta.-actin.
11. The process of claim 6, wherein the non-coding sequence is from
GAPDH.
12. The process of claim 1, wherein the viral nucleic acid is a
coding sequence.
13. The process of claim 12, wherein the coding sequence is from
HIV.
14. The process of claim 12, wherein the coding sequence is from
HBV.
15. The process of claim 12, wherein the coding sequence is from
HCV.
16. The process of claim 12, wherein the coding sequence is from
BVDV.
17. The process of claim 12, wherein the coding sequence is from
West Nile Virus.
18. The process of claim 12, wherein the coding sequence is from
herpes.
19. The process of claim 12, wherein the coding sequence is from
influenza.
20. The process of claim 12, wherein the coding sequence is from
RSV.
21. The process of claim 12, wherein the coding sequence is from
EBV.
22. The process of claim 12, wherein the coding sequence is from
CMV.
23. A process for assessing the toxicity of a compound that
includes contacting nucleic acids from a host with an amplification
reaction mixture that contains at least two primers and/or probes
that provide detectable signals during a polymerase chain reaction,
wherein the first primer and/or probe provides a detectable signal
on the occurrence on the transcription of host mitochondrial
nucleic acids; and the second primer and/or probe provides a second
detectable signal on the occurrence on the transcription of host
nuclear nucleic acid.
24. The process of claim 23, wherein the host mitochondrial nucleic
acid is mitochondrial DNA.
25. The process of claim 23, wherein the host mitochondrial nucleic
acid is mitochondrial RNA.
26. The process of claim 23, wherein the host mitochondrial nucleic
acid is a non-coding sequence.
27. The process of claim 26, wherein the non-coding sequence is a
5'-non-coding sequence.
28. The process of claim 26, wherein the non-coding sequence is a
3'-non-coding sequence.
29. The process of claim 26, wherein the non-coding sequence is an
intron.
30. The process of claim 26, wherein the non-coding sequence is
from .beta.-actin.
31. The process of claim 26, wherein the non-coding sequence is
from GAPDH.
32. The process of claim 23, wherein the host mitochondrial nucleic
acid is a coding sequence.
Description
FIELD OF THE INVENTION
[0001] This application is in the area of processes for the
detection and analysis of viral infections and mitochondrial
toxicity, and for processes for the identification of active
compounds for the treatment of viral infections and processes to
measure mitochondrial toxicity resulting from drug therapies.
[0002] This application claims priority to U.S. Provisinal
Application No. 60/241,488, filed on Oct. 18, 2000, U.S. Provisinal
Application No. 60/256,067 filed on Dec. 15, 2000 and U.S.
Provisinal Application No. 60/282,156, filed on Apr. 6, 2001.
BACKGROUND OF THE INVENTION
[0003] The detection and quantification of nucleic acid sequences
is of importance for a wide range of applications. The most widely
used method to detect nucleic acids are based on the polymerase
chain reaction (PCR). PCR is used to amplify a segment of DNA
flanked by stretches of known sequences. Two oligonucleotides
binding to these known flanking sequences are used as primers for a
series of in vitro reactions that are catalyzed by a DNA
polymerase. These oligonucleotides typically have different
sequences and are complementary to sequences that lie on opposite
strands of the template DNA and flank the segment of DNA that is to
be amplified. The template DNA is first denatured by heat in the
presence of a large molar excess of each of the two
oligonucleotides and the four 2'-deoxynucleotide triphosphates. The
reaction mixture is then cooled to a temperature that allows the
oligonucleotide primers to anneal to their target sequences.
Afterwards, the annealed primers are extended by the DNA
polymerase. The cycle of denaturation, annealing, and DNA-synthesis
is then repeated about 10 to 50 times. Since the products of one
cycle are used as a template for the next cycle the amount of the
amplified DNA fragment is theoretically doubled with each cycle
resulting in a PCR-efficiency of 100%.
[0004] "Real-time PCR" refers to a polymerase chain reaction that
is monitored, usually by fluorescence, over time during the
amplification process, to measure a parameter related to the extent
of amplification of a particular sequence, such as the extent of
hybridization of a probe to amplified target sequences. The DNA
generated within a PCR is detected on a cycle by cycle basis during
the PCR reaction. The amount of DNA increases faster the more
template sequences are present in the original sample. When enough
amplification products are made a threshhold is reached at which
the PCR products are detected. Thus amplification and detection are
performed simultaneously in the same tube.
[0005] In biological research, PCR has accelerated the study of
testing for communicable diseases. Medical applications of PCR
include identifying viruses, bacteria and cancerous cells in human
tissues. PCR can even be used within single cells, in a procedure
called in situ (in-site) PCR, to identify specific cell types. PCR
can also be applied to the amplification of RNA, a process referred
to as reverse transcriptase PCR (RT-PCR). RT-PCR is similar to
regular PCR, with the addition of an initial step in which DNA is
synthesized from the RNA target using an enzyme called a reverse
transcriptase. A wide variety of RNA molecules have been used in
RT-PCR, including ribosomal RNA, messenger RNA and genomic viral
RNA.
[0006] PCR itself is quite simple, but sample preparation can be
laborious. The goals of sample preparation include the release of
nucleic acid (DNA or RNA), concentration of the nucleic acid to a
small volume for PCR, and removal of inhibitors of PCR. Inhibitors
of PCR are naturally occurring substances which reduce the
efficiency of PCR, and which are often present in clinical samples.
When the specimen contains a large amount of target nucleic acid,
sample preparation is trivial. But sample preparation is more
difficult in most clinical specimens, particularly when a large
volume specimen must be processed and only a few pathogens are
present. Complex protocols are often required.
[0007] Since PCR detects the presence or absence of a particular
nucleic acid target, it will only detect a pathogen if its nucleic
acid is present in the particular specimen. PCR detects nucleic
acids from living or dead microbes. This must be recognized if PCR
is used to monitor response to therapy. PCR provides at most
nucleic acid sequence information. PCR can be used to screen for
drug resistance mutations, but it does not provide direct
antibiotic susceptibility data.
[0008] Appropriate controls are necessary when PCR is used
diagnostically. These include negative controls, positive controls
and specificity controls. Negative controls (no target DNA) are
needed to detect contamination. Contamination can occur during
sample preparation or reagent mixing, so negative controls need to
be processed in parallel with clinical samples. Negative controls
should be interspersed among the samples to detect
cross-contamination from sample to sample. Contamination is
frequently intermittent; a sufficient number of negative controls
must be included to detect low rates of contamination. Most
published studies have not included a sufficient number of negative
controls.
[0009] Positive controls include a small number of target DNA
copies. Positive controls are needed to ensure efficient release of
target DNA from pathogens, to guard against loss of DNA during
sample processing, and to identify the presence of inhibitors
(natural substances sometimes present in clinical samples that
reduce PCR efficiency). Positive controls should be processed in
parallel with clinical specimens. Clinical specimens vary in the
presence of inhibitors of PCR, and it may be necessary to add an
internal positive control for each sample. The internal positive
controls have the same recognition sites as the target DNA, but are
designed with some difference in the internal sequence.
Amplification of the internal positive controls can be
distinguished from that of the real target DNA.
[0010] Specificity controls are needed to determine the range of
target DNAs that will be amplified by the PCR assay. For assays
designed to detect pathogens in clinical samples, human DNA samples
must be tested to ensure that the PCR primers do not recognize a
human DNA target by chance. Related pathogens must be tested to
determine the range of species/strains that will be amplified.
Specificity controls are needed only once, when a new PCR assay is
designed. Negative and positive controls must be included every
time samples are processed, and should be processed simultaneously
with the clinical samples.
[0011] PCR has been used in three broad categories of diagnostic
procedures, namely detection, characterization and
quantification.
[0012] Detection is the most difficult PCR procedure, especially
when the number of pathogens in the specimen is low. The PCR must
be conducted under conditions of high sensitivity. Many temperature
cycles are used, or a nested protocol is used in which the products
from the first reaction are re-amplified with a second set of
primers. This makes PCR for detection especially prone to carryover
contamination. Sample preparation may be laborious, as there is an
attempt to process as large a specimen volume as possible.
Inhibitors of PCR occur naturally in many clinical samples, and are
a major limitation. Numerous positive and negative controls must be
included as described above.
[0013] In a characterization procedure, nucleic acid variants are
identified based on the nucleic acid sequence between the two PCR
primers. Many techniques can be used to detect variable sequences,
including length polymorphism, changes in restriction sites, and
direct DNA sequencing. This is often the easiest type of PCR to
carry out clinically. Ample quantities of nucleic acid target can
be present in the specimen, either an already grown bacterial or
viral culture or a clinical sample with large numbers of microbes.
Goals can include rapid detection of drug resistance mutations,
assignment of strains to clinically meaningful phylogenetic groups,
or epidemiological tracing.
[0014] Quantitation (indicationg how many copies of the target
nucleic acid are present) has primarily been applied to chronic
viral infections, especially hepatitis C virus (HCV) and human
immunodeficiency virus (HIV) infections. The level of viremia has
prognostic implications, and has been used to demonstrate response
to antiviral drugs. PCR is quite sensitive, but it is not
inherently quantitative. The amount of the final PCR product is
usually similar from an initial sample containing 10 or 10,000
copies. This limitation can be overcome by serial dilution of the
clinical sample until no target DNA is detected, or by the addition
of synthetic competitor DNA molecules. The competitor molecules
have regions complementary to the two primers, but differ in some
way from the natural target (e.g., a different length). By
comparing the amount of the natural and competitor PCR products, a
rough estimation of the number of target molecules in the sample is
possible.
[0015] PCR has been applied in the research setting to hundreds of
pathogens, and has yielded important insights into pathogenesis and
epidemiology of many infectious diseases. For clinical purposes,
PCR-based diagnostic tests are best applied when the following
conditions are fulfilled: (1) The results of the test will make a
clear clinical difference and a therapy will be given or withheld
based on the results of PCR; (2) routine culture methods are
limited because the microbe cannot be grown (e.g., Mycobacterium
leprae, HCV), grows slowly (e.g., M. tuberculosis), or is difficult
to culture (e.g., Brucella species, HIV); and (3) there is an
accessible clinical specimen which contains large numbers of
microbes (e.g., blood for HCV or HIV).
[0016] PCR has been useful in a variety of chronic virus infections
(HIV, HCV, hepatitis B virus, human papillomavirus and
cytomegalovirus). PCR has been crucial for the detection of HIV
infection in neonates, since maternal antibodies complicate
serologic diagnosis. Quantitation of HIV and HCV viremia by PCR has
important prognostic implications, and has been used to monitor
response to drug therapy. PCR is useful for the rapid diagnosis of
pulmonary infections in immuno-compromised hosts, particularly for
cytomegalovirus and Pneumocystis carinii.
[0017] HIV
[0018] The human immunodeficiency virus type-1 (HIV-1) is a
retrovirus belonging to the family of the Lentiviridae. One of the
characteristic features of this virus group is that the members
replicate over a DNA intermediate through the viral encoded reverse
transcriptase (RT) enzyme activity. The high replication rate
combined with the low fidelity of that reverse transcriptase enzyme
provides the virus with an extremely high genomic flexibility. As a
consequence, different levels of genetic variability are observed
for HIV-1. The epidemic is characterized by the presence of clades
within the M-group virus, but there is also an O-group and an
N-group virus described, each of them again harboring a variety of
clades. Quasispecies populations within the infected individual are
also seen. Clinically, there are some important consequences to
this quasispecies concept, for example, in vaccine development and
immune escape. This concept contributes to the emergence of drug
resistant variants that surface under antiviral treatments.
[0019] In order to control the course of the disease in infected
individuals, potent highly active anti-retroviral therapies (HAART)
have been designed. Due to the ongoing replication of the virus,
anti-retroviral drug resistance eventually develops, leading to
therapy failure. Therefore, there is an ongoing need for more and
more potent anti-HIV-1 drugs.
[0020] To assess the efficacy of drugs in the treatment of patients
in vivo, clinical markers of virus replication needed to be
defined. In the past, some surrogate markers, like CD-cell count,
have been used. More recently, some commercial assays like
Quantiplex (Chiron), NucliSense (Organon-Teknika) and Amplicor
HIV-1 Monitor (Roche) were developed to directly measure viral
load. These viral load determinations proved to be an excellent
tool in monitoring therapeutic efficiency for HAART and for
clinical trials with new experimental drugs.
[0021] The design of an HIV-1 viral load test is a real challenge.
Ideally, a viral load test should fulfill to the following
criteria:
[0022] i) be able to detect the huge variability of clades within
one group with the same efficiency;
[0023] ii) have a dynamic range of at least five logs or higher;
and
[0024] iii) the lower limit of detection should be as low as a few
viral copies/mL.
[0025] Although variability at the PCR-primer binding sites is a
real concern in assay development, RT-PCR based assays are
considered as the most sensitive technologies.
[0026] Mitochondrial Toxicity
[0027] Mitochondrial toxicity is clearly recognized as an adverse
effect of long-term use of antiviral agents, in particular reverse
transcriptase inhibitors. Clinical features of this mitochondrial
toxicity vary depending on the tissues that are affected. It is
largely dependent on the aerobic metabolism needed for energy
supply required for that particular tissue. Most toxic events are
reversible at an early stage, however lactic acidosis is often
irreversible and can result in death.
[0028] The common pathway of antiviral agent induced toxicity is
mitochondrial dysfunction. The antiviral agent (most likely the
triphosphate form of a nucleoside analogue) inhibits the
mitochondrial DNA polymerase .gamma. leading to the loss of
mitochondria. This enzyme is essential for the replication of the
mitochondrial genome. Tissues with high ATP demand are most
susceptible to mitochondrial toxicity.
[0029] The mechanism underlying this mitochondrial dysfunction
includes failure of energy dependent ionic balance. Subsequently,
there is an increase in intracellular calcium, initiating lipolysis
and proteolysis, and leading to the accumulation of lactic acid and
partial reduction of the respiratory activities.
[0030] Since the mitochondrial dysfunction develops over months and
symptoms are initially mild, it is important to develop sensitive
diagnostic tests that allow determination of the enzyme activity
and inhibition by the selected antiviral agent. Evenly important,
new candidate antiviral agents need to be evaluated for their
unfavorable DNA polymerase .gamma. inhibiting capacities.
[0031] Hepatitis C
[0032] Hepatitis C virus (HCV) infection is a pandemic infection,
and is a major cause of liver disease. Reports of successful
treatment of HCV infection with interferon have increased interest
in applications of RT-PCR.
[0033] Available tests for HCV infection are limited. Initial
serologic tests for HCV had poor sensitivity. Second and
third-generation serologic tests have improved sensitivity, but are
still not completely dependable. HCV RNA is readily detected in
serum using RT-PCR. Viremic patients typically have very high viral
titers.
[0034] PCR has been applied to the diagnosis of HCV infection in a
variety of clinical settings. HCV can be detected as early as one
week after infection, and PCR can be used to detect HCV infection
during the "window" period between infection and seroconversion.
HCV PCR is useful for detecting HCV in seronegative individuals
with liver disease. It can be used to confirm maternal to fetal
spread of HCV. HCV PCR may be useful in the evaluation of
seropositive individuals as candidates for interferon or other
therapies. Portions of HCV-seropositive patients are negative by
HCV PCR, and may have resolved their infections. PCR-negative
individuals have lower serum transaminase concentrations and less
histologic activity on liver biopsies. Long-term follow-up studies
are needed, but it may be reasonable to withhold therapy from
patients with negative HCV PCR results.
[0035] The amount of HCV viremia can be determined by either
quantitative PCR. PCR is sensitive and is quantitative over a wide
range of viral titers. High-titer viremia is correlated with an
advanced disease stage. The prognostic value of HCV quantitation
awaits prospective studies, but the level of viremia may be useful
in selecting candidates for therapy. Quantitative HCV PCR also
appears to be useful in monitoring the response to therapy.
[0036] WO 00/44936 filed by Bavarian Nordic Research Institute A/S
describes a real-time PCR method for the detection and
quantification of variants of nucleic acid sequences which differ
in the probe-binding site. The method is based in the complete or
partial amplification of the same region of the variants and the
addition of two or more oligonucleoitde probes to the same PCR
mixture, each probe being specific for the probe-binding site of at
least one variant.
[0037] WO 01/66799 filed by E.I. DuPont Nemours and Company
discloses a PCR-based dsDNA quantification method that monitors the
fluorescence of a target, whose melting characteristics is
predetermined, during each amplification cycle at selected time
points. By selecting targets with distinguishing melting curve
characteristics, multiple targets can be simultaneously
detected.
[0038] WO 00/68436 filed by Nationales Zentrum fur Retroviren
discloses sequences allowing the detection and quantification of
human immunodeficiency virus.
[0039] U.S. Pat. No. 6,235,504 assigned to the Rockefeller
University describes methods for identifying genetic sequences
useful as genomic equivalent markers for organisms.
[0040] U.S. Pat. No. 6,210,875 discloses a process for determining
the efficacy of antiviral therapy in an HIV-infected host that
includes detecting the level of transcriptionally active HIV in the
monocytes of the subject at a plurality of times by simultaneously
exposing the monocytes to an oligonucleotide probe that
specifically binds to at least a portion of HIV mRNA and exposing
the monocytes to an antibody, wherein the oligonucleotide probe is
labeled with a fluorescent label, comparing the detected HIV
levels, and correlating the HIV levels over time with the therapy
regimen.
[0041] U.S. Pat. No. 5,843,640 discloses an in situ process of
simultaneously detecting a specific predetermined nucleic acid
sequence and a specific predetermined cellular antigen in the same
cell.
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Zhang, D. D. Ho, and M. Markowitz 1999. Use of real-time PCR and
molecular beacons to detect virus replication in human
immunodeficiency virus type 1-infected individuals on prolonged
effective antiretroviral therapy. J Virol. 73:6099-6103. Locatelli,
G., F. Santoro, F. Veglia, A. Gobbi, P. Lusso, and M. S. Malnati
2000. Real-time quantitative PCR for human herpesvirus 6 DNA. J
Clin Microbiol. 37:4042-4048; Machida, U., M. Kami, T. Fukui, Y.
Kazuyama, M. Kinoshita, Y. Tanaka, Y. Kanda, S. Ogawa, H. Honda, S.
Chiba, K. Mitani, Y. Muto, K. Osumi, S. Kimura, and H. Hirai 2000.
Real-time automated PCR for early diagnosis and monitoring of
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Microbiol. 38:2536-2542; Martell, M., J. Gomez, J. I. Esteban, S.
Sauleda, J. Quer, B. Cabot, R. Esteban, and J. Guardia 1999.
High-throughput real-time reverse transcription-PCR quantitation of
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F., D. Thouvenot, and B. Lina 2001. Development of a real-time PCR
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A. Cubie 2001. Detection of herpes viruses in clinical samples
using real-time PCR. J Virol Methods. 96:25-31; Niesters, H. G., J.
van Esser, E. Fries, K. C. Wolthers, J. Cornelissen, and A. D.
Osterhaus 2000. Development of a real-time quantitative assay for
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Nitsche, A., N. Steuer, C. A. Schmidt, O. Landt, H. Ellerbrok, G.
Pauli, and W. Siegert 2000. Detection of human cytomegalovirus DNA
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Ohyashiki, J. H., A. Suzuki, K. Aritaki, A. Nagate, N. Shoji, K.
Ohyashiki, T. Ojima, K. Abe, and K. Yamamoto 2000. Use of real-time
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A. Gruters, A. D. Osterhaus, and H. G. Niesters 2000. Development
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Tanaka, and M. Kohara 1999. Real-time detection system for
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Tyagi, S. Dube, B. J. Poiesz, and F. R. Kramer 1999. Multiplex
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Smets, M. Wessel, L. Fischer, G. Offner, H. Kirchner, and P. Bucsky
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[0043] Although assays exist for the diagnosis and evaluation of
viral infections, additional assays and kits are needed that
provide a more sensitive or precise analysis of the condition of a
diseased cell. More sensitive and precise methods are also needed
to assess the activity of a compound or substance against a target
virus and to assess host toxicity induced by the compound or
substance.
[0044] It is therefore an object of the present invention to
provide a process for the identification of active compounds for
the treatment of viral infections.
[0045] It is another object of the present invention to provide a
process to measure mitochondrial toxicity.
[0046] It is another object of the present invention to provide a
process for the detection and analysis of viral infections.
[0047] It is a further object of the invention to provide a process
for the detection and analysis of mitochondrial toxicity.
SUMMARY OF THE INVENTION
[0048] Processes and methods for the simultaneous quantification of
nucleic acids in diseased cells that are based on real-time PCR are
provided. The real-time-PCR protocol is an excellent tool for
reliable quantification of in vitro drug screening and evaluation
protocols to determine the efficacy of potential anti-viral agents.
Quantification using these simulateous PCR cycle threshold (Ct)
detection techniques during one-step real-time RT-PCR (Applied
Biosystems, CA) eliminates the variability resulting from
quantification of end-point RT-PCR products. In addition, the
mitochondrial toxicity assay is an added tool to assess potential
side-effects for these chemotherapeutic agents.
[0049] This real time multiplex PCR system includes the
simultaneous measurements of cellular DNA (for example rDNA) or
cellular RNA (for example rRNA or .beta.-actin m-RNA), and viral
RNA or DNA. In one embodiment, the simultaneous real time analysis
of host and viral nucleic acid allows the calculation of a
sensitivity assay that indicates the comparative condition of the
host cell and the virus. In a separate aspect of the invention,
multiplex PCR is used to simultaneously measure the nuclear and the
mitochondrial nucleic acid of a cell to provide information on drug
toxicity, or to evaluate a cell (in vivo or in vitro) that may
exhibit a disease that involves mitochondrial toxicity, such as
peripheral neuropathy, peripheral lipodystrophy, or a genetic
disease that causes a disruption in mitochondrial DNA or RNA
synthesis.
[0050] The methods and processes are economic, non-radioactive,
rapid, accurate, reproducible, and amenable to large through-put.
It can provide a dynamic range of quantification with linearity of
over 5-7 logs. One way to express the antiviral effectiveness of a
compound is to subtract the threshold RT-PCR cycle of the test
compound with the average threshold RT-PCR cycle of the negative
control. This value is called DeltaCt (.DELTA.Ct). A .DELTA.Ct of
3.3 equals a 1-log reduction (equals EC.sub.90) in viral nucleic
acid production. Compounds that result in a reduction of viral
nucleic acid greater than 1.5, or more preferred, 2 Ct values (75%
reduction of viral nucleic acid) are typically useful compounds for
the inhibition of viral growth.
[0051] With the availability of both the viral .DELTA.Ct data and
the host .DELTA.Ct, a specificity parameter can be introduced. This
parameter is obtained by subtracting the host .DELTA.Ct value from
the viral .DELTA.Ct value. This results in .DELTA..DELTA.Ct values;
a value above 0 means that there is more inhibitory effect on the
viral nucleic acid, a .DELTA..DELTA.Ct value below 0 means that the
host nucleic acid is more affected. As a general rule,
.DELTA..DELTA.Ct values above 2 are considered as significantly
different from the no-drug treatment control, and hence, exhibits
useful antiviral activity. However, compounds with a
.DELTA..DELTA.Ct value of less than 2, but showing limited
molecular cytotoxicty data (rRNA .DELTA.CT between 0 and 2) may
also be desired for certain applications requiring compounds with
low toxicity.
[0052] As an example, a compound might reduce the host RNA
polymerase activity, but not the host DNA polymerase activity.
Therefore, quantification of rDNA or .beta.-actin DNA (or any other
host DNA fragment) and comparison with DNA levels of the no-drug
control is a relative measurement of the inhibitory effect of the
test compound on cellular DNA polymerases. With the availability of
both the HCV .DELTA.Ct data and the rDNA .DELTA.Ct, a specificity
parameter can be introduced. This parameter is obtained by
subtracting both .DELTA.Ct values from each other. This results in
.DELTA..DELTA.Ct values; a value above 0 means that there is more
inhibitory effect on the viral encoded polymerase, a
.DELTA..DELTA.Ct value below 0 means that the host rDNA levels are
more affected than the viral nucleic acid levels. As a general
rule, .DELTA..DELTA.Ct values above 2 are considered as
significantly different from the no-drug treatment control, and
hence, is an interested compound for further evaluation. However,
compounds with a .DELTA..DELTA.Ct value of less than 2, but with
limited molecular cytotoxicty (rDNA .DELTA.CT between 0 and 2) are
also possible active candidate compounds for further evaluation
[0053] In a first embodiment, a process for assessing a viral
disease is provided that includes contacting nucleic acid from a
viral infected host cell with an amplification reaction mixture
that contains at least two primers and/or probes that provide
detectable signals during a polymerase chain reaction, wherein
[0054] the first primer and/or probe provides a detectable signal
on the occurrence on the transcription of host nucleic acid;
and
[0055] the second primer and/or probe provides a second detectable
signal on the occurrence on the transcription of viral nucleic
acid.
[0056] In a particular embodiment, the level of transcription of
the viral and host nucleic acid is compared to that of a standard,
including but not limited to, a known viral infected host cell, or
alternatively, an internal standard can be established by comparing
the extent of transcription of the host and viral nucleic acid over
a number of samples from the host to monitor and measure the change
in infection. In another embodiment, the data can be assessed as
described above through the use of .DELTA.CT and .DELTA..DELTA.Ct
values.
[0057] In a preferred embodiment, the nucleic acid is a consensus
or non-coding sequence, which can be either 5' or 3' to the target
expressed sequence. In one embodiment, the non-coding sequence is
an intron or a part thereof. Non-limiting examples are non-coding
sequences from .beta.-actin or GAPDH.
[0058] The host nucleic acid can be nuclear or cytoplasmic, and in
particular, mitochondrial nucleic acid, and the viral nucleic acid
can be either DNA or RNA.
[0059] This process can be used to evaluate the ability of the
compound or substance to inhibit the replication of any virus,
including but not limited to a virus from the Retroviridae,
Flaviviridae, Orthomyxoviridae, Paramyxoviridae, Herpesviridae,
Hepadnaviridae, Picornaviridae, Reoviridae, Poxviridae,
Adenoviridae, Papoviridae, Parvoviridae, Bunyaviridae, Filoviridae,
Arenaviridae or Togaviridae family. In particular, the virus is
HIV, hepatitis (including but not limited to A, B, C, D and G),
BVDV (bovine diarrhea virus), herpes simplex, Adenovirus type 1,
influenza, including influenza A (HINI), influenza A (H3N2),
influenza B, influenza C and influenza D, measles, mumps,
parainfluenza type 3, RSV (respiratory syncytial virus), HSV
(herpes simplex virus), EBV (Epstein Barr virus), CMV
(cytomegalovirus) or West Nile Virus.
[0060] In a second embodiment, a process for assessing a disease
state that includes a disruption in mitochondrial DNA or RNA
synthesis is provided that includes contacting nucleic acid from a
host with an amplification reaction mixture that contains at least
two primers and/or probes that provide detectable signals during a
polymerase chain reaction, wherein
[0061] the first primer and/or probe provides a detectable signal
on the occurrence on the transcription of host mitochondrial
nucleic acid; and
[0062] the second primer and/or probe provides a second detectable
signal on the occurrence on the transcription of host nuclear
nucleic acid.
[0063] In a third embodiment, a process for identifying a compound
or substance that inhibits viral replication is provided that
includes (i) contacting nucleic acid from a virus infected host
that has been treated with the compound with (ii) an amplification
reaction mixture that contains at least two primers and/or probes
that provide detectable signals during a polymerase chain reaction,
wherein
[0064] the first primer and/or probe provides a detectable signal
on the occurrence of the transcription of viral nucleic acid;
and
[0065] the second primer and/or probe provides a second detectable
signal on the occurrence of the transcription of host nucleic
acid.
[0066] In a fourth embodiment, a process for assessing the
mitochondrial toxicity of a compound is provided that includes
contacting nucleic acid from a host that has been treated with the
compound with an amplification reaction mixture that contains at
least two primers and/or probes that provide detectable signals
during a polymerase chain reaction, wherein
[0067] the first primer and/or probe provides a detectable signal
on the occurrence on the transcription of host mitochondrial
nucleic acid; and
[0068] the second primer and/or probe provides a second detectable
signal on the occurrence on the transcription of host nuclear
nucleic acid.
[0069] In a fifth embodiment, a process for assessing the tendency
of a compound to induce peripheral neuropathy or peripheral
lipodystrophy is provided that includes contacting nucleic acid
from a host cell that has been treated with the compound with an
amplification reaction mixture that contains at least two primers
and/or probes that provide detectable signals during a polymerase
chain reaction, wherein
[0070] the first primer and/or probe provides a detectable signal
on the occurrence on the transcription of host mitochondrial
nucleic acid; and
[0071] the second primer and/or probe provides a second detectable
signal on the occurrence on the transcription of host nuclear
nucleic acid.
[0072] These processes and methods optimally utilize the conserved
regions in the genome of the virus and host to design unique
combinations of a PCR primer/probe-sets. In one embodiment, this
probe contains a detectable signal, so that upon exonucleic
degradation, the signal, indicating target nucleic acid, can be
detected in real-time. This technique has been found to be
sensitive and accurate; in addition, quantification using PCR cycle
threshold (Ct) detection during one-step real-time RT-PCR (Applied
Biosystems, CA) has eliminated the variability resulting from
quantification of end-point RT-PCR products.
[0073] In a particular embodiment of the present invention, process
of simulatneous real-time PCR includes the following steps:
[0074] a) contacting at least a portion of a target nucleic acid
sequence in a sample with
[0075] i) a suitable amplification reaction mixture; and
[0076] ii) two or more independently labeled oligonucleotides or
probes that hybridizes to the target nucleic acid sequence, such
that the when the target nucleic acid sequence is amplified, each
independently labeled probe releases an unique detectable
signal;
[0077] iii) wherein at least one independently labeled
oligonucleotide or probe that hydrbiridizes to a target viral
nucleic acid sequence; and
[0078] iv) at least one independently labeled oligonucleotide or
probe that hydrbiridizes to a target host nucleic acid
sequence;
[0079] b) carrying out an amplification procedure on the
amplification mixture; and
[0080] c) detecting in real time the release of the unique
signals.
[0081] The presence of the amplicon, of course, indicates that the
target nucleic acid is present in the sample; the target RNA or DNA
in the sample can be quantitated based on signal intensity.
[0082] The current invention can also be applied to a new method
for sensitive and accurate determination of mitochondrial toxicity
of candidate chemotherapeutic compounds using real-time-PCR by
determining the ratio of nuclear (or endogenous control) DNA or RNA
to mitochondrial DNA or RNA. In a preferred embodiment, this
toxicity screening assay is used to determine toxicity of potential
anti-viral agents, and in particular anti-HIV, especially
anti-HIV-1, and anti-hepatitis viruses, especially HBV and HCV.
[0083] In order to quantify the total amount of mitochondrial DNA
or RNA, amplification of an endogenous control needs to be
performed to standardize the amount of such mitochondrial DNA or
RNA. This endogenous control is an RNA or DNA that is present in
each experimental sample and is representative of the total amount
of nuclear DNA or RNA. By using this endogenous control as an
active reference, quantities of mitochondrial DNA or RNA can be
normalized for differences in the amount of total DNA or RNA added
to each reaction. Some non-limiting examples of endogenous controls
are any human gene, but especially .beta.-actin,
glyceraldehyde-3-phosphate dehydrogenase or ribosomal RNA.
[0084] This method includes the following steps:
[0085] a) contacting at least a portion of a nuclear nucleic acid
sequence in a sample with
[0086] i) an amplification reaction mixture; and
[0087] i) two or more independently labeled oligonucleotides or
probes that hybridizes to the target nucleic acid sequence, such
that the when the target nucleic acid sequence is amplified, each
independently labeled probe releases an unique detectable
signal;
[0088] ii) wherein at least one independently labeled
oligonucleotide or probe hybridizes to a target nuclear nucleic
acid sequence; and
[0089] iii) at least one independently labeled oligonucleotide or
probe that hybridizes to a target mitochondrial nucleic acid
sequence;
[0090] b) carrying out an amplification procedure on the
amplification mixture;
[0091] c) detecting in real time the release of the signal.
[0092] The quantity of the nuclear amplicon can be compared to the
quantity of mitochondrial amplicon based on differences in signal
intensity, thereby indicating the level of mitochondrial
toxicity.
BRIEF DESCRIPTION OF THE FIGURES
[0093] FIG. 1 is an illustration of a calibration of standard curve
for HIV-1 (1a), HCV (1b), BVDV (1c), mitochondrial DNA (1d) and
molecular toxicology (1e) RT-PCR. The attenuated clinical samples
were diluted in DMEM-F12/10% FBS. The Ct value indicates the
threshold cycle where the one-step RT-PCR detection of the target
becomes positive. The Log cp/mL value is the logarithm of the
amount of target copies per mL sample. The .diamond-solid. line
indicates the Roche Amplicor HIV-1 Monitor, while the .box-solid.
line indicates real-time HIV-1 RT-PCR.
[0094] FIG. 2 is a graph that depicts the correlation of real-time
RT-PCR for HIV-1 with NASBA HIV-1 technology. HIV-1 infected
samples were taken from SCID-mice experiments. The 99% confidence
intervals are indicated with dashed lines.
[0095] FIG. 3 are illustrations of the effect of antiviral
compounds on viral load and RT activity in culture supernatant. The
.diamond-solid. line indicates data from a traditional RT assay,
while the .box-solid. line represents data obtained from using
HIV-1 RT-PCR.
[0096] FIG. 4 is a non-limiting illustration of RT-PCR standard
curves and relative efficiency plot. In this particular example,
quantities of .beta.-actin DNA and mitochondrial DNA were measured
in real-time to generate the following plots: 1) the
.diamond-solid. line is the .beta.-actin standard curve; 2) the
.box-solid. line is the mitochondrial DNA standard curve; and 3)
the .tangle-solidup. line is a .DELTA.Ct plot (Ct .beta.-actin-Ct
mitochondrial).
[0097] FIG. 5 are illustration of the effect of antiviral compounds
on mitochondrial DNA polymerase .gamma.. 2.sup.-.DELTA..DELTA.CT is
the arithmetic formula used to express the differences in
mitochondrial DNA after calibration (no drug) and normalization
(.beta.-actin). Concentrations are in .mu.M.
[0098] FIG. 6 is an illustration of the quantitative detection of
viral nucleic acids by real-time PCR. A fluorogenic probe is shown
during the extension phase of PCR. If the target sequence is
present, the probe anneals downstream from one of the primer sites
and is cleaved by the 5' nuclease activity of Taq DNA polymerase as
this primer is extended. This cleavage of the probe separates the
reporter dye from quencher dye, increasing the reporter dye signal.
Cleavage removes the probe from the target strand, allowing primer
extension to continue to the end of the template strand. Thus,
inclusion of the probe does not inhibit the overall PCR process.
Additional reporter dye molecules are cleaved from their respective
probes with each cycle, effecting an increase in fluorescence
intensity proportional to the amount of amplicon produced.
[0099] FIG. 7 is an illustration of the organization of the HCV
genome as compared to the Hepatitis C Virus replicon, indicating
the location of cleavage sites within the polyprotein and the
nontranslated regions (NTRs). The open reading frame (ORF) is
flanked on the 5' end by an NTR that functions as an internal
ribosome entry site (IRES) and at the 3' end by a highly conserved
sequence essential for genome replication.
[0100] FIG. 8 is a graph of the changes in the amounts of cellular
and viral nucleic acids over a seven day incubation period in
Huh7/HCV Replicon cells.
[0101] FIG. 9 is a bar graph of the effect of test compounds on HCV
RNA levels in the Huh7 HCV replicon system.
[0102] FIG. 10 contains two bar graphs showing the changes in
nucleic acid levels in Huh7 cells in terms of the amount of
mitochondrial RNA and, in the other, the changes in mitochondrial
DNA, after a seven day incubation period with various drugs.
DETAILED DESCRIPTION OF THE INVENTION
[0103] Processes and methods for the simultaneous quantification of
nucleic acids in diseased cells that are based on real-time PCR are
provided. The real-time-PCR protocol is an excellent tool for
reliable quantification of in vitro drug screening and evaluation
protocols to determine the efficacy of potential anti-viral agents.
Quantification using these simulateous PCR cycle threshold (Ct)
detection techniques during one-step real-time RT-PCR (Applied
Biosystems, CA) eliminated the variability resulting from
quantification of end-point RT-PCR products. In addition, the
mitochondrial toxicity assay is an added tool to assess potential
side-effects for these chemotherapeutic agents.
[0104] This real time multiplex PCR system includes the
simultaneous measurements of cellular DNA (for example rDNA) or
cellular RNA (for example rRNA or .beta.-actin m-RNA), and viral
RNA or DNA. In one embodiment, the simultaneous real time analysis
of host and viral nucleic acid allows the calculation of a
sensitivity assay that indicates the comparative condition of the
host cell and the virus. In a separate aspect of the invention,
multiplex PCR is used to simultaneously measure the nuclear and the
mitochondrial nucleic acid of a cell to provide information on drug
toxicity, or to evaluate a cell (in vivo or in vitro) that may
exhibit a disease that involves mitochondrial toxicity, such as
peripheral neuropathy, peripheral lipodystrophy, or a genetic
disease that causes a disruption in mitochondrial DNA or RNA
synthesis.
[0105] The methods and processes are economic, non-radioactive,
rapid, accurate, reproducible, and amenable to large through-put.
It can provide a dynamic range of quantification with linearity of
over 5-7 logs. One way to express the antiviral effectiveness of a
compound is to subtract the threshold RT-PCR cycle of the test
compound with the average threshold RT-PCR cycle of the negative
control. This value is called DeltaCt (.DELTA.Ct). A .DELTA.Ct of
3.3 equals a 1-log reduction (equals EC.sub.90) in viral nucleic
acid production. Compounds that result in a reduction of viral
nucleic acid greater than 1.5, or more preferred, 2 Ct values (75%
reduction of viral nucleic acid) are typically useful compounds for
the inhibition of viral growth.
[0106] With the availability of both the viral .DELTA.Ct data and
the host .DELTA.Ct, a specificity parameter can be introduced. This
parameter is obtained by subtracting the host .DELTA.Ct value from
the viral .DELTA.Ct value. This results in .DELTA..DELTA.Ct values;
a value above 0 means that there is more inhibitory effect on the
viral nucleic acid, a .DELTA..DELTA.Ct value below 0 means that the
host nucleic acid is more affected. As a general rule,
.DELTA..DELTA.Ct values above 2 are considered as significantly
different from the no-drug treatment control, and hence, exhibits
useful antiviral activity. However, compounds with a
.DELTA..DELTA.Ct value of less than 2, but showing limited
molecular cytotoxicty data (rRNA .DELTA.CT between 0 and 2) may
also be desired for certain applications requiring compounds with
low toxicity.
[0107] As an example, a compound might reduce the host RNA
polymerase activity, but not the host DNA polymerase activity.
Therefore, quantification of rDNA or .beta.-actin DNA (or any other
host DNA fragment) and comparison with DNA levels of the no-drug
control is a relative measurement of the inhibitory effect of the
test compound on cellular DNA polymerases. With the availability of
both the HCV .DELTA.Ct data and the rDNA .DELTA.Ct, a specificity
parameter can be introduced. This parameter is obtained by
subtracting both .DELTA.Ct values from each other. This results in
.DELTA..DELTA.Ct values; a value above 0 means that there is more
inhibitory effect on the viral encoded polymerase, a
.DELTA..DELTA.Ct value below 0 means that the host rDNA levels are
more affected than the viral nucleic acid levels. As a general
rule, .DELTA..DELTA.Ct values above 2 are considered as
significantly different from the no-drug treatment control, and
hence, is an interested compound for further evaluation. However,
compounds with a .DELTA..DELTA.Ct value of less than 2, but with
limited molecular cytotoxicty (rDNA .DELTA.CT between 0 and 2) are
also possible active candidate compounds for further evaluation
[0108] In a first embodiment, a process for assessing a viral
disease is provided that includes contacting nucleic acid from a
viral infected host cell with an amplification reaction mixture
that contains at least two primers and/or probes that provide
detectable signals during a polymerase chain reaction, wherein
[0109] the first primer and/or probe provides a detectable signal
on the occurrence on the transcription of host nucleic acid;
and
[0110] the second primer and/or probe provides a second detectable
signal on the occurrence on the transcription of viral nucleic
acid.
[0111] In a particular embodiment, the level of transcription of
the viral and host nucleic acid is compared to that of a standard,
including but not limited to, a known viral infected host cell, or
alternatively, an internal standard can be established by comparing
the extent of transcription of the host and viral nucleic acid over
a number of samples from the host to monitor and measure the change
in infection. In another embodiment, the data can be assessed as
described above through the use of .DELTA.CT and .DELTA..DELTA.Ct
values.
[0112] In a preferred embodiment, the nucleic acid is a consensus
or non-coding sequence, which can be either 5' or 3' to the target
expressed sequence. In one embodiment, the non-coding sequence is
an intron or a part thereof. Non-limiting examples are non-coding
sequences from .beta.-actin or GAPDH.
[0113] The host nucleic acid can be nuclear or cytoplasmic, and in
particular, mitochondrial nucleic acid, and the viral nucleic acid
can be either DNA or RNA.
[0114] This process can be used to evaluate the ability of the
compound or substance to inhibit the replication of any virus,
including but not limited to a virus from the Retroviridae,
Flaviviridae, Orthomyxoviridae, Paramyxoviridae, Herpesviridae,
Hepadnaviridae, Picornaviridae, Reoviridae, Poxviridae,
Adenoviridae, Papoviridae, Parvoviridae, Bunyaviridae, Filoviridae,
Arenaviridae or Togaviridae family. In particular, the virus is
HIV, hepatitis (including but not limited to A, B, C, D and G),
BVDV (bovine diarrhea virus), herpes simplex, Adenovirus type 1,
influenza, including influenza A (HINI), influenza A (H3N2),
influenza B, influenza C and influenza D, measles, mumps,
parainfluenza type 3, RSV (respiratory syncytial virus), HSV
(herpes simplex virus), EBV (Epstein Barr virus), CMV
(cytomegalovirus) or West Nile Virus.
[0115] In a second embodiment, a process for assessing a disease
state that includes a disruption in mitochondrial DNA or RNA
synthesis is provided that includes contacting nucleic acid from a
host with an amplification reaction mixture that contains at least
two primers and/or probes that provide detectable signals during a
polymerase chain reaction, wherein
[0116] the first primer and/or probe provides a detectable signal
on the occurrence on the transcription of host mitochondrial
nucleic acid; and
[0117] the second primer and/or probe provides a second detectable
signal on the occurrence on the transcription of host nuclear
nucleic acid.
[0118] In a third embodiment, a process for identifying a compound
or substance that inhibits viral replication is provided that
includes (i) contacting nucleic acid from a virus infected host
that has been treated with the compound with (ii) an amplification
reaction mixture that contains at least two primers and/or probes
that provide detectable signals during a polymerase chain reaction,
wherein
[0119] the first primer and/or probe provides a detectable signal
on the occurrence of the transcription of viral nucleic acid;
and
[0120] the second primer and/or probe provides a second detectable
signal on the occurrence of the transcription of host nucleic
acid.
[0121] In a fourth embodiment, a process for assessing the
mitochondrial toxicity of a compound is provided that includes
contacting nucleic acid from a host that has been treated with the
compound with an amplification reaction mixture that contains at
least two primers and/or probes that provide detectable signals
during a polymerase chain reaction, wherein
[0122] the first primer and/or probe provides a detectable signal
on the occurrence on the transcription of host mitochondrial
nucleic acid; and
[0123] the second primer and/or probe provides a second detectable
signal on the occurrence on the transcription of host nuclear
nucleic acid.
[0124] In a fifth embodiment, a process for assessing the tendency
of a compound to induce peripheral neuropathy or peripheral
lipodystrophy is provided that includes contacting nucleic acid
from a host cell that has been treated with the compound with an
amplification reaction mixture that contains at least two primers
and/or probes that provide detectable signals during a polymerase
chain reaction, wherein
[0125] the first primer and/or probe provides a detectable signal
on the occurrence on the transcription of host mitochondrial
nucleic acid; and
[0126] the second primer and/or probe provides a second detectable
signal on the occurrence on the transcription of host nuclear
nucleic acid.
[0127] These processes and methods optimally utilize the conserved
regions in the genome of the virus and host to design unique
combinations of a PCR primer/probe-sets. In one embodiment, this
probe contains a detectable signal, so that upon exonucleic
degradation, the signal, indicating target nucleic acid, can be
detected in real-time. This technique has been found to be
sensitive and accurate; in addition, quantification using PCR cycle
threshold (Ct) detection during one-step real-time RT-PCR (Applied
Biosystems, CA) has eliminated the variability resulting from
quantification of end-point RT-PCR products.
[0128] In a particular embodiment of the present invention, a
method of simulatneous real-time PCR includes the following
steps:
[0129] a) contacting at least a portion of a target nucleic acid
sequence in a sample comprising:
[0130] i) a suitable amplification reaction mixture; and
[0131] ii) two or more independently labeled oligonucleotides or
probes that hybridizes to the target nucleic acid sequence, such
that the when the target nucleic acid sequence is amplified, each
independently labeled probe releases an unique detectable
signal;
[0132] iii) wherein at least one independently labeled
oligonucleotide or probe that hydrbiridizes to a target viral
nucleic acid sequence; and
[0133] iv) at least one independently labeled oligonucleotide or
probe that hydrbiridizes to a target host nucleic acid
sequence;
[0134] b) carrying out an amplification procedure on the
amplification mixture; and
[0135] c) detecting in real time the release of the unique
signals.
[0136] The presence of the amplicon, of course, indicates that the
target nucleic acid is present in the sample; the target RNA or DNA
in the sample can be quantitated based on signal intensity.
[0137] The current invention can also be applied to a new method
for sensitive and accurate determination of mitochondrial toxicity
of candidate chemotherapeutic compounds using real-time-PCR by
determining the ratio of nuclear (or endogenous control) DNA or RNA
to mitochondrial DNA or RNA. In a preferred embodiment, this
toxicity screening assay is used to determine toxicity of potential
anti-viral agents, and in particular anti-HIV, especially
anti-HIV-1, and anti-hepatitis viruses, especially HBV and HCV.
[0138] This method includes the following steps:
[0139] a) contacting at least a portion of a nuclear nucleic acid
sequence in a sample comprising:
[0140] i) an amplification reaction mixture; and
[0141] i) two or more independently labeled oligonucleotides or
probes that hybridizes to the target nucleic acid sequence, such
that the when the target nucleic acid sequence is amplified, each
independently labeled probe releases an unique detectable
signal;
[0142] ii) wherein at least one independently labeled
oligonucleotide or probe hybridizes to a target nuclear nucleic
acid sequence; and
[0143] iii) at least one independently labeled oligonucleotide or
probe that hybridizes to a target mitochondrial nucleic acid
sequence;
[0144] d) carrying out an amplification procedure on the
amplification mixture;
[0145] e) detecting in real time the release of the signal.
[0146] The quantity of the nuclear amplicon can be compared to the
quantity of mitochondrial amplicon based on differences in signal
intensity, thereby indicating the level of mitochondrial
toxicity.
[0147] I. Screening
[0148] These processes and methods can be used to evaluate the
ability of the compound or substance to inhibit the replication of
any virus, including but not limited to a virus from the
Retroviridae, Flaviviridae, Orthomyxoviridae, Paramyxoviridae,
Herpesviridae, Hepadnaviridae, Picornaviridae, Reoviridae,
Poxviridae, Adenoviridae, Papoviridae, Parvoviridae, Bunyaviridae,
Filoviridae, Arenaviridae or Togaviridae family. In particular, the
virus is HIV, hepatitis (including but not limited to A, B, C, D
and G), BVDV (bovine diarrhea virus), herpes simplex, Adenovirus
type 1, influenza, including influenza A (HINI), influenza A
(H3N2), influenza B, influenza C and influenza D, measles, mumps,
parainfluenza type 3, RSV (respiratory syncytial virus), HSV
(herpes simplex virus), EBV (Epstein Barr virus), CMV
(cytomegalovirus) or West Nile Virus.
[0149] In particular, quantitative real-time PCR antiviral
screening can be combined with calibration for a host RNA targets
(in RT-PCR) in the following non-limiting examples:
[0150] a) anti-HCV compound screening can be combined with rRNA
calibration, mRNA calibration, and in particular -actin mRNA
calibration, mitochondrial RNA calibration and/or any other nuclear
or mitochondrial nucleic acid calibration;
[0151] b) anti-HIV compound screening can be combined with rRNA
calibration, mRNA calibration, and in particular -actin mRNA
calibration, mitochondrial RNA calibration and/or any other nuclear
or mitochondrial nucleic acid calibration;
[0152] c) anti-HBV compound screening can be combined with rRNA
calibration, mRNA calibration, and in particular -actin mRNA
calibration, mitochondrial RNA calibration and/or any other nuclear
or mitochondrial nucleic acid calibration;
[0153] d) anti-RSV compound screening can be combined with rRNA
calibration, mRNA calibration, and in particular -actin mRNA
calibration, mitochondrial RNA calibration and/or any other nuclear
or mitochondrial nucleic acid calibration;
[0154] e) anti-BVDV compound screening can be combined with rRNA
calibration, mRNA calibration, and in particular -actin mRNA
calibration, mitochondrial RNA calibration and/or any other nuclear
or mitochondrial nucleic acid calibration;
[0155] f) anti-lentivirus compound screening can be combined with
rRNA calibration, mRNA calibration, and in particular -actin mRNA
calibration, mitochondrial RNA calibration and/or any other nuclear
or mitochondrial nucleic acid calibration;
[0156] g) anti-flaviviridae (Flavivirus, Hepacivirus, Pestivirus)
compound screening can be combined with rRNA calibration, mRNA
calibration, and in particular -actin mRNA calibration,
mitochondrial RNA calibration and/or any other nuclear or
mitochondrial nucleic acid calibration;
[0157] h) anti-hepadnavirus compound screening can be combined with
rRNA calibration, mRNA calibration, and in particular -actin mRNA
calibration, mitochondrial RNA calibration and/or any other nuclear
or mitochondrial nucleic acid calibration;
[0158] i) anti-picornavirus compound screening can be combined with
rRNA calibration, mRNA calibration, and in particular -actin mRNA
calibration, mitochondrial RNA calibration and/or any other nuclear
or mitochondrial nucleic acid calibration;
[0159] j) anti-herpetoviridae (HSV, HCMV, EBV) compound screening
can be combined with rRNA calibration, mRNA calibration, and in
particular -actin mRNA calibration, mitochondrial RNA calibration
and/or any other nuclear or mitochondrial nucleic acid
calibration.
[0160] Quantitative real-time PCR antiviral screening can be
combined with calibration for a host DNA target (in PCR) in the
following non-limiting examples:
[0161] a) anti-HCV compound screening can be combined with rDNA
calibration, DNA calibration, and in particular -actin DNA
calibration, mitochondrial DNA calibration and/or any other nuclear
or mitochondrial nucleic acid calibration;
[0162] b) anti-HIV compound screening can be combined with rDNA
calibration, DNA calibration, and in particular -actin DNA
calibration, mitochondrial DNA calibration and/or any other nuclear
or mitochondrial nucleic acid calibration;
[0163] c) anti-HBV compound screening can be combined with rDNA
calibration, DNA calibration, and in particular -actin DNA
calibration, mitochondrial DNA calibration and/or any other nuclear
or mitochondrial nucleic acid calibration;
[0164] d) anti-RSV compound screening can be combined with rDNA
calibration, DNA calibration, and in particular -actin DNA
calibration, mitochondrial DNA calibration and/or any other nuclear
or mitochondrial nucleic acid calibration;
[0165] e) anti-BVDV compound screening can be combined with rDNA
calibration, DNA calibration, and in particular -actin DNA
calibration, mitochondrial DNA calibration and/or any other nuclear
or mitochondrial nucleic acid calibration;
[0166] f) anti-lentivirus compound screening can be combined with
rDNA calibration, DNA calibration, and in particular -actin DNA
calibration, mitochondrial DNA calibration and/or any other nuclear
or mitochondrial nucleic acid calibration;
[0167] g) anti-flaviviridae (Flavivirus, Hepacivirus, Pestivirus)
compound screening can be combined with rDNA calibration, DNA
calibration, and in particular -actin DNA calibration,
mitochondrial DNA calibration and/or any other nuclear or
mitochondrial nucleic acid calibration;
[0168] h) anti-hepadnavirus compound screening can be combined with
rDNA calibration, DNA calibration, and in particular -actin DNA
calibration, mitochondrial DNA calibration and/or any other nuclear
or mitochondrial nucleic acid calibration;
[0169] i) anti-picornavirus compound screening can be combined with
rDNA calibration, DNA calibration, and in particular -actin DNA
calibration, mitochondrial DNA calibration and/or any other nuclear
or mitochondrial nucleic acid calibration;
[0170] j) anti-herpetoviridae (HSV, HCMV, EBV) compound screening
can be combined with rDNA calibration, DNA calibration, and in
particular -actin DNA calibration, mitochondrial DNA calibration
and/or any other nuclear or mitochondrial nucleic acid
calibration.
[0171] The current invention also provides a new process and method
for sensitive and accurate determination of mitochondrial toxicity
of chemotherapeutic or other pharmaceutical agents by determining
the ratio of mitochondrial DNA or RNA to nuclear DNA or RNA. The
rationale behind this methodology is driven by the fact that DNA
polymerase .gamma. inhibition eventual leads to lower amounts of
mitochondrial DNA or RNA, while the amounts of nuclear DNA or RNA
(for which replication is dependent on DNA polymerase .alpha.
and/or .beta.) remains constant.
[0172] In order to quantify the total amount of mitochondrial DNA
or RNA, amplification of an endogenous control needs to be
performed to standardize the amount of such mitochondrial DNA or
RNA. This endogenous control is an RNA or DNA that is present in
each experimental sample and is representative of the total amount
of nuclear DNA or RNA. By using this endogenous control as an
active reference, quantities of mitochondrial DNA or RNA can be
normalized for differences in the amount of total DNA or RNA added
to each reaction. Endogenous controls can be any human gene, but
often .beta.-actin, glyceraldehyde-3-phosphate dehydrogenase, or
ribosomal RNA have been used. An effective process to quantify the
total amount of endogenous control in a reaction by real-time PCR
is provided
[0173] II. Definitions
[0174] As used herein, "sample" or "clinical sample" relates to any
sample obtained from a host for use in carrying out the procedures
of the present invention. In one aspect, the host is suffering from
a disease or syndrome that is at least partially caused by a virus.
The host may also be an asymptomatic considered to be at risk of
viral infection. The sample may be a cellular sample such as a
tissue sample, for example of lung tissue obtained as a biopsy or
post-mortem, a fluid sample, such as blood, saliva, sputum, urine,
cerebrospinal fluid, or a swabbed sample obtained by swabbing a
mucus membrane surface such as nasal surface, a pharyngeal surface,
a buccal surface, and the like, or it may be obtained from an
excretion such as feces, or it may be obtained from other bodily
tissues or body fluids commonly used in diagnostic testing.
[0175] The term "purified" in reference to RNA or DNA, as used
herein, relates to released RNA or DNA from latent or inaccessible
form in a virion or a cell and allowing the RNA or DNA to become
freely available. In such a state, it is suitable for effective
amplification by use of the polymerase chain reaction. Releasing
RNA or DNA may include steps that achieve the disruption of virions
containing viral RNA or DNA, as well as disruption of cells that
may harbor such virions. Purification of RNA or DNA is generally
carried out under conditions that rigorously and effectively
exclude or inhibit any nuclease activity that may be present.
Additionally, purification may include steps that achieve at least
a partial separation of the RNA or DNA dissolved in an aqueous
medium from other cellular or viral components, wherein such
components may be either particulate or dissolved.
[0176] As used herein, "reverse transcription" or "RT" relates to a
procedure catalyzed by an enzyme, reverse transcriptase, that
synthesizes a cDNA from a single stranded RNA molecule, with the
use of oligonucleotide primers having free 3'-hydroxyl groups. As
used herein, the term "polymerase chain reaction" or "PCR" relates
to a procedure whereby a limited segment of a nucleic acid
molecule, which frequently is a desired or targeted segment, is
amplified repetitively to produce a large amount of DNA molecules
which consist only of that segment. The procedure depends on
repetition of a large number of priming and transcription cycles.
In each cycle, two oligonucleotide primers bind to the segment, and
define the limits of the segment. A primer-dependant DNA polymerase
then transcribes, or replicates, the strands to which the primers
have bound. Thus, in each cycle, the number of DNA duplexes is
doubled.
[0177] The term "primer" or "oligonucleotide primer," as used
herein, relates to an oligonucleotide having a specific or desired
nucleotide sequence that is complementary to a particular sequence
on one of the strands of a DNA duplex. When the primer is caused to
hybridize to the specific sequence in a DNA duplex to which it is
complimentary, it may serve as the priming position, or the
initiation position, for the action of a primer-dependent DNA
polymerase activity. The primer, once hybridized, acts to define
the 5'-end of the operation of the transcription activity of the
polymerase on the duplex. Commonly in PCR, a specific pair of
primers is employed, wherein one of the primers hybridizes to one
of the strands and the second primer hybridizes to the
complementary strand. The primers hybridize in such an orientation
that transcription, which proceeds in the direction from 5' to 3',
is in the direction leading from each primer toward the site of
hybridization of the other primer. After several rounds of
hybridization and transcription the amplified DNA produced is a
segment having a defined length whose ends are defined by the sites
to which the primers hybridize.
[0178] The term "probe" or "labeled oligonucleotide," as used
herein, relates to an oligonucleotide having a specific or desired
nucleotide sequence that is complementary to a particular sequence
on one of the strands of a DNA duplex, as well as a detectable
signal, such as a fluorescent dye. When the primer is caused to
hybridize to the specific sequence in a DNA duplex to which it is
complimentary, the signal is inactive, for example due to a
covalently linked quenching dye. However, upon amplification and
subsequent analysis, the signal is activated by exonucleic
degradation and thus can be detected in real time. In particular,
the probe can contain a fluorescent dye and a quenching dye, such
that at the time of hybridization, the fluorescent dye in quenched
by the quenching dye. After amplification and exonucleic
degradation, the fluorescent dye is released from the quenching dye
and a fluorescent signal can be detected in real time.
[0179] The term "amplification reaction mixture," as used herein
refers to any reaction substance, or combination of substances that
promotes the amplification of a target nucleic acid sequence,
including enzymes such as polymerase, or polymerases with
exonuclease activity, substrates such as nucleic acids and
oligonucleotide primers, as defined herein.
[0180] As used herein, the term "specific to" or "specific for" a
target sequence, in relation to a nucleic acid sequence such as an
oligonucleotide sequence, relate to a nucleotide sequence that
hybridizes, under conditions used in given experimental
circumstances, to the target but does not hybridize under those
circumstances to sequences that are not target sequences.
Nucleotide sequences that are specific for a particular target,
such as the HIV target sequences that are included in the subject
matter of the present invention, are those that include bases all
of which are complementary to the corresponding base on the
target.
[0181] Further, the term "specificity," as used herein, of a
nucleic acid sequence for a target sequence also encompasses
nucleic acids and oligonucleotides having a small number of
nucleotides that may not be complementary to the corresponding
nucleotides of the target sequence. Such sequences are still
"specific" for the target sequence, as long as the extent of the
deviation from complementarity remains functionally of no
consequence. In particular, such a sequence is "specific" for the
target sequence as long as it hybridized effectively to the target
sequence but does not hybridize to any sequence that is not a
target sequence in the sample, under the conditions used in given
experimental circumstances.
[0182] The term "amplicon" as used herein refers to a double
stranded nucleic acid segment having a defined size and sequence
that results from an amplification procedure, such as a PCR
procedure. The size of the amplicon is limited by the sites on the
two strands of a nucleic acid duplex to which the primers bind.
That segment of the product nucleic acid becomes the prevalent
product of the amplification procedure after a small number of
cycles of amplification.
[0183] The term "host," as used herein, refers to a unicellular or
multicellular organism in which the virus can replicate, including
cell lines and animals, and preferably a human. Alternatively, the
host can be carrying a part of the viral genome, whose replication
or function can be altered by the compounds of the present
invention. The term host specifically refers to infected cells,
cells transfected with all or part of the viral genome and animals,
in particular, primates (including chimpanzees) and humans. In most
animal applications of the present invention, the host is a human
patient. Veterinary applications, in certain indications, however,
are clearly anticipated by the present invention (such as bovine
viral diarrhea virus in cattle, hog cholera virus in pigs, and
border disease virus in sheep).
[0184] III. Host Primers and Probes
[0185] For the detection of host nucleic acids, any suitable primer
and/or probe known in the art may be used. These primers and/or
probes may be purchase or made by any means known in the art. There
are several primers and/or probe combinations commercially
available, for example the primer probe set for rRNA gene (Perkin
Elmer/Applied Biosystems). The latter set is often used as
calibrator PCR in this invention. Alternatively, suitable probes
and primers can be designed by using the Primer Express software
(Applied Biosystems, CA), and in particular new primers and probes
for the .beta.-actin gene, and for the mitochondrial cytochrome
oxidase subunit II (COXII) gene.
[0186] .beta.-Actin
[0187] In one embodiment, the nuclear DNA or RNA used to derive a
set of oligonucleotides for the endogenous control is the DNA for
.beta.-actin. Any suitable primers and/or probes can be used. In a
specific embodiment of the present invention, the primers and/or
probes are complementary to sequences from the third exon of the
human -actin gene (GenBandk accession number E01094). The probe
comprises a reporter and quencher that provides a detectable signal
upon amplification. Any reporter/quencher probe set can be used,
including, but not limited to TaqMan, molecular beacons, single dye
probe, SYBR green, Amplifluor probes and dual labeled probe
sets.
[0188] In a preferred embodiment of the invention, the
oligonucleotides used to amplify .beta.-actin (primers) are sense
sense 5'-GCGCGGCTACAGCTTCA-3' (Sequence ID No. 1) and antisense
5'-TCTCCTTAATGTCACGCACGAT-3' (Sequence ID No. 2). The labeled
oligonulceotide (probe) used to detect host nucleic acid has a
sequence of 5'-CACCACGGCCGAGCGGGA-3' (Sequence ID No. 3). In one
emobidment, the probe is labeled with a reporter at the 5'-end and
a quencher molecule at the 3'-end, and in particular, the reporter,
FAM, at the 5' end, and the quencher molecule, TAMRA, at the 3'
end.
[0189] Mitochondiral Nucleic Acid
[0190] In one embodiment, the mitochondrial nucleic acids can be
specifically derived from mitochondrial DNA. In an alternate
embodiment, the mitochondiral nucleic acids can be specifically
derived from mitochondrial RNA. In an alternate embodiment, the
mitochondiral nucleic acids are complementary to sequences from
themitochondrial COXII gene. Any suitable primers and/or probes can
be used. The probe comprises a reporter and quencher that provides
a detectable signal upon amplification. Any reporter/quencher probe
set can be used, including, but not limited to TaqMan, molecular
beacons, single dye probe, SYBR green, Amplifluor probes and dual
labeled probe sets.
[0191] In a preferred embodiment of the invention, the
oligonucleotides used to amplify mitochondrial nucleic acids
primers) are sense sense sense 5'-TGCCCGCCATCATCCTA-3' (Sequence ID
No. 19) and 5'-TCGTCTGTTATGTAAAGGATGCGT-3' (Sequence ID No. 20).
The labeled oligonulceotide (probe) used to detect host nucleic
acid has a sequence of 5'-TCCTCATCGCCCTCCCATCCC-3' (Sequence ID No.
21). In one emobidment, the probe is labeled with a reporter at the
5'-end and a quencher molecule at the 3'-end, and in particular,
the reporter, TET, at the 5' end, and the quencher molecule, TAMRA,
at the 3' end.
[0192] IV. Viral Primers and Probes
[0193] For viral targets, any suitable primer and/or probe known in
the art may be used. These primers and/or probes may be purchase or
made by any means known in the art. Alternatively, suitable probes
and primers can be designed by using the Primer Express software
(Applied Biosystems, CA), and in particular, primers and probes
designed to be complementary to highly conserved areas. This is
particularly important for viruses with a high genetic variability,
like for example HCV, HBV, and HIV, BVDV and RSV.
[0194] Ideally, the viral primer/probe set should fulfill to the
following criteria: (i) be able to detect the huge variability of
clades or genotypes with the same efficiency; ii) have a dynamic
range of at least five logs or higher; and iii) the lower limit of
detection should be as low as a few viral copies/mL. Although
variability at the PCR-primer binding sites is often problematic,
RT-PCR based assays are some of the most sensitive
technologies.
[0195] In one embodiment of the present invention, complementary
viral sequences were designed based on conserved regions of the
viral genome to obtain a unique combination of PCR primers and/or
probe-set. In an alternative embodiment of the present invention,
the primers/probes are designed based on predicted sequence
conservation over the different genotypes. In a preferred
embodiment, the primers/probes are designed based on both the
conserved region of the viral genome and predicted sequence
conservation over the different genotypes.
[0196] HIV
[0197] In one embodiment of the invention, the target viral nucleic
acid is from HIV, and in particular, HIV-1. Any suitable primers
and/or probes can be used. In a specific embodiment of the present
invention, the primers and/or probes are complementary to the
reverse transcriptase domain between codons 200 and 280. The probe
comprises a reporter and quencher that provides a detectable signal
upon amplification. Any reporter/quencher probe set can be used,
including, but not limited to TaqMan, molecular beacons, single dye
probe, SYBR green, Amplifluor probes and dual labeled probe
sets.
[0198] In a preferred embodiment of the invention, the
oligonucleotides used to amplify HIV-1 (primers) are sense
5'-TGGGTTATGAACTCCATCCTGAT-3' (Sequence ID No. 4) and antisense
5'-TGTCATTGACAGTCCAGCTGTCT-3' (Sequence ID No. 5). The labeled
oligonulceotide (probe) used to detect HIV-1 viral load has a
sequence of 5'-TTTCTGGCAGCTCTCGGCTGTACTGTCCATT-3' (Sequence ID No.
6). In one emobidment, the probe is labeled with a reporter at the
5'-end and a quencher molecule at the 3'-end, and in particular,
the reporter, FAM, at the 5' end, and the quencher molecule, TAMRA,
at the 3' end.
[0199] HCV
[0200] In another embodiment of the invention, the target viral
nucleic acid is from HCV. Any suitable primers and/or probes can be
used. In a specific embodiment of the present invention, the
primers and/or probes are derived from thighly conserved sequences
complementary to the RNA sequences present in HCV, such as the HCV
5' non-coding region. The probe comprises a reporter and quencher
that provides a detectable signal upon amplification. Any
reporter/quencher probe set can be used, including, but not limited
to TaqMan, molecular beacons, single dye probe, SYBR green,
Amplifluor probes and dual labeled probe sets.
[0201] In a preferred embodiment of the invention, the
oligonucleotides used to amplify HCV (primers) are sense
5'-AGCCATGGCGTTAGTA(T/A)GAGTGT-3' (Sequence ID No. 7) and antisense
5'-TTCCGCAGACCACTATGG-3' (Sequence ID No. 8). The labeled
oligonulceotide (probe) used to detect HCV viral load has a
sequence of 5'-CCTCCAGGACCCCCCCTCCC-3' (Sequence ID No. 9). In one
emobidment, the probe is labeled with a reporter at the 5 '-end and
a quencher molecule at the 3 '-end, and in particular, the
reporter, FAM, at the 5' end, and the quencher molecule, TAMRA, at
the 3' end.
[0202] BVDV
[0203] In another embodiment of the invention, the target viral
nucleic acid is from BVDV. Any suitable primers and/or probes can
be used. In a specific embodiment of the present invention, the
primers and/or probes are derived from thighly conserved sequences
complementary, such as sequences complementary to nucleotides 1611
to 1751 of the NS5B gene. The probe comprises a reporter and
quencher that provides a detectable signal upon amplification. Any
reporter/quencher probe set can be used, including, but not limited
to TaqMan, molecular beacons, single dye probe, SYBR green,
Amplifluor probes and dual labeled probe sets.
[0204] In a preferred embodiment of the invention, the
oligonucleotides used to amplify BVDV (primers) are sense sense
5'-AGTCTTCAGTTTCTTGCTGATGT- -3' (Sequence ID No. 10) and antisense
5'-TGTTGCGAAAGGACCAACAG-3' (Sequence ID No. 11). The labeled
oligonulceotide (probe) used to detect BVDV viral load has a
sequence of 5'-AAATCCTCCTAACAAGCGGGTTCCAGG-3' (Sequence ID No. 12).
In one emobidment, the probe is labeled with a reporter at the
5'-end and a quencher molecule at the 3'-end, and in particular,
the reporter, FAM, at the 5' end, and the quencher molecule, TAMRA,
at the 3' end.
[0205] HBV
[0206] In another embodiment of the invention, the target viral
nucleic acid is from HBV. Any suitable primers and/or probes can be
used. In a specific embodiment of the present invention, the
primers and/or probes are derived from thighly conserved sequences
complementary to the DNA sequences present in HBV, such as the
amino-terminal region of the HBV surface antigen gene. The probe
comprises a reporter and quencher that provides a detectable signal
upon amplification. Any reporter/quencher probe set can be used,
including, but not limited to TaqMan, molecular beacons, single dye
probe, SYBR green, Amplifluor probes and dual labeled probe
sets.
[0207] In a preferred embodiment of the invention, the
oligonucleotides used to amplify HBV (primers) are sense sense
5'-GGACCCCTGCTCGTGTTACA-3' (Sequence ID No. 13) and antisense
5'-GAGAGAAGTCCACCACGAGTCTAG-3' (Sequence ID No. 14). The labeled
oligonulceotide (probe) used to detect HBV viral load has a
sequence of 5'-TGTTGACAA(A/G)TCCTCACAATACC(A/G)CAGA-- 3' (Sequence
ID No. 15). In one emobidment, the probe is labeled with a reporter
at the 5'-end and a quencher molecule at the 3'-end, and in
particular, the reporter, FAM, at the 5' end, and the quencher
molecule, TAMRA, at the 3' end.
[0208] RSV
[0209] In another embodiment of the invention, the target viral
nucleic acid is from RSV. Any suitable primers and/or probes can be
used. In a specific embodiment of the present invention, the
primers and/or probes are derived from thighly conserved sequences
complementary, such as sequences complementary to nucleotides that
encode for the RNA polymerase large subunit (L). The probe
comprises a reporter and quencher that provides a detectable signal
upon amplification. Any reporter/quencher probe set can be used,
including, but not limited to TaqMan, molecular beacons, single dye
probe, SYBR green, Amplifluor probes and dual labeled probe
sets.
[0210] In a preferred embodiment of the invention, the
oligonucleotides used to amplify RSV (primers) are sense sense
sense 5'-CAACAACCCTAATCATGTGGTATCA-3' (Sequence ID No. 16) and
antisense 5'-CCGGTTGCATTGCAAACA-3' (Sequence ID No. 17). The
labeled oligonulceotide (probe) used to detect RSV viral load has a
sequence of 5'-TGACAGGCAAAGAAAGAGAACTCAGTGTAGGTAGA-3' (Sequence ID
No. 18). In one emobidment, the probe is labeled with a reporter at
the 5'-end and a quencher molecule at the 3'-end, and in
particular, the reporter, FAM, at the 5' end, and the quencher
molecule, TAMRA, at the 3' end.
[0211] V. Methods
[0212] Amplification Procedure
[0213] The process for amplification of a desired nucleic acid
sequence can be achieve by any means necessary to achieve
amplification of the desired amplicon. The amplification can be
achieved using any known means in the art, including polymerase
chain reaction techniques. The primers and probes can be purchased
or prepared by any means known in the art, including automated
processes. In a preferred embodiment, the primers and probes are
designed for specificity for the target nucleic acid sequence, as
disclosed herein. The enzymes used to promote amplification can be
purchased or can be prepared by any means known in the art,
including cellular extraction. Substrates to aid in the
amplification can also be purchased or can be prepared by any means
known in the art, including any synthetic methodology to synthesis
natural and unnatural nucleic acids. The enzyme and substrates can
be added to the amplification mixture at any time and order that
allows for the amplification of the desired amplicon. In a
preferred embodiment, the polymerase and substrates follow TaqMan
7700 chemistry provided by Applied Biosystems in California.
[0214] Additionally, amplification conditions vary depending on the
choice of primers and probes, due to differences in their melting
temperatures.TM.. Preferred temperatures are from 50.degree. C. to
95.degree. C. for incubation and 60.degree. C. to 95.degree. C. for
amplification. The temperature for amplification can be done at any
temperature that allows for replication of the desired amplicon at
a suitable rate. As an exemplary embodiment, reverse-transcriptase
polymerase chain reaction ("RT-PCT") can be used to amplify the
desired amplicon. After reverse transcription incubation, an
amplification cycle can be performed. The incubation cycle can be
performed at one temperature or on a multi-temperature basis; for
example, the incubation cycle can be performed on a two-step
temperature gradient, preferably, first a moderate time at moderate
temperature followed by an extended period at higher temperatures.
The amplification cycle can be performed at one temperature or on a
multi-temperature basis; for example, the amplification cycle can
be performed on a two-step gradient, preferably, first a short
phase of higher temperatures followed by a longer period of
moderate temperatures. The amplification procedure can be repeated
as many times as necessary, but preferably repeated around 40
times.
[0215] As a non-limiting example, HIV-1, .beta.-actin and
mitochondrial nucleic acid sequences can be amplified using the
following procedure. First the amplification reaction mixture is
incubated for two minutes at 50.degree. C., then ten minutes at
95.degree. C. This is then followed by forty cycles of a two-step
amplification reaction at 95.degree. C. for fifteen seconds then
sixty seconds at 60.degree. C.
[0216] Detection systems
[0217] The presence of the amplicon can be detected in real time
based on the labeled oligonucleotide, which is labeled with a
variety of substances, termed reporting dyes, and quenching dye,
which upon amplification, are capable of emitting a detectable
signal. Any combination of reporting dyes and quenching dyes can be
used. Some non-limiting examples of reporting dyes are FAM, VIC,
PAT and JOE. A non-limiting example of quenching dyes is TAMRA.
These reporting dyes and quenching dyes can be purchased or can be
prepared by any means known in the art, including radical and
organometallic chemistry.
[0218] In one embodiment, the detectable signal is a fluorescent
dye that can be detected in a spectrometer that is covalently bound
to a quenching dye through the oligonucleotide. This renders the
fluorescent dye inactive while bound to the oligonucleotide.
However, upon exonuclease degradation of the oligonucleotide, the
fluorescent dye can be released from the quenching dye, thus
emitting a detectable signal.
[0219] Many of the new DNA tags and labels depend on two phenomena
that are extensions of fluorescence: quenching and energy transfer.
In general, anything that reduces the lifetime of the excited state
decreases the quantum yield of the fluorophore; anything that
decreases the quantum yield is called quenching. There are three
main mechanisms for determining these phenomena: collisional, in
which the excited state of the fluorophore loses its energy by
bumping into a nonfluorescent molecule; static, in which the
excited state reacts with the quencher, forming a nonfluorescent
complex; and energy transfer, which involves the nonradiative
transfer of energy from a donor to an acceptor.
[0220] The brightness of a fluorescent dye depends on many
parameters. The parameters can be divided between the physical and
chemical properties of the dyes and the excitation system. The
important physical properties of the dyes are quantum yield and
extinction coefficient. The quantum yield is an expression of the
number of photons emitted divided by the number of photons
absorbed. A quantum yield of 0 indicates a nonfluorescent molecule,
and a quantum yield of 1 indicates that 100 percent of the
excitation photons result in lower-wavelength emitted photons. The
extinction coefficient is an expression of the probability that a
photon of a given wavelength will be absorbed by the fluorophore. A
high extinction coefficient combined with a high quantum yield
generally leads to a "bright" fluorophore; fluorescein, for
example, is a relatively "bright" dye, having an extinction
coefficient of about 80,000 at its absorption maximum and a quantum
yield of .about.0.9.
[0221] For fluorescence resonant energy transfer (FRET) to occur,
there must be a precise overlap in quantum energy levels between
the donor and the acceptor, the energy being transferred by dipolar
coupling rather than emission and reabsorption of a photon. FRET
has been used very productively to create dyes for DNA sequencing,
where a common donor eliminates the need for multiple excitation
wavelengths but instead transfers its energy to four separate dyes
that have easily discernable emission spectra. FRET and
fluorescence quenching are very distance dependant, allowing their
exploitation in several novel assays that alter donor-acceptor
geometries.
[0222] Many of the methods described depend on a variety of
modified oligonucleotides. Many fluorescent dyes are available as
dye-phosphoramidites (or as dye-CPG derivatives), which are
compatible with automated oligonucleotide synthesis methods. Using
this approach, dyes can be incorporated at the 5' or 3' end or at
any internal position during routine synthesis. Similarly,
amino-modified bases can be incorporated into an oligo at any
position, enabling a wider variety of labeling, because many
additional dyes are available in an NHS-ester form that can be
conjugated to an amino-modified oligonucleotide after synthesis.
Different applications call for different modifications, including
such esoterica as variable-length spacers, universal bases and
branched backbones.
[0223] Reagent kits that support quantitative amplification and
detection in multiplex are commercially available. The QPCR kits
are used with DNA templates, either to detect DNA mutations or to
measure gene or viral copy number. The QRT-PCR kits are used with
RNA templates, typically for measuring RNA levels. Mutations can
also be detected in expressed RNA with these kits. These kits have
the capability of high performance with various fluorescent
detection systems, including, the AmpliFluor system, molecular
beacons, TaqMan.TM. probes, dual fluorophore approach, single-dye
primers and DNA bynding dyes.
[0224] (i) Amplifluor Universal Amplification and Detection System,
Intergen Co., Purchase, N.Y.
[0225] In this system, PCR amplification and detection steps take
place in the same reaction vessel. Resultant PCR products fluoresce
and can be monitored with real-time or endpoint fluorescence
detection instruments. The Amplifluor system is based on an
innovative adaptation of the molecular beacon technology. Molecular
beacons are hairpin-shaped oligonucleotides that contain
fluorophore and quencher moieties. Molecular beacons act like
switches that are normally closed to bring the fluorophore/quencher
pair together to turn fluorescence "off." When prompted to undergo
conformational changes that open the hairpin structure, the
fluorophore and quencher are separated, and fluorescence is turned
"on." Similarly, the Amplifluor system uses a primer that contains
a hairpin-shaped end in which fluorescein is paired up with the
quencher 4-(dimethylamine)azo benzene sulfonic acid (DABSYL).
However, Intergen points out that there is an important difference
between the Amplifluor system and other currently available energy
transfer-based PCR methods (e.g., molecular beacons or
Perkin-Elmer's Taqman.TM.). In Amplifluor, the fluorescent
oligonucleotides are actually incorporated into the reaction
products. This enables the direct detection of PCR products,
reducing the number of false positive reactions, which can be
caused by even the most minimal carry-over contamination. Three
primers are used to amplify products with Intergen's Amplifluor
system. Forward and reverse primers specific for the gene of
interest are generated by the user. Additionally, reactions contain
the UniPrimer.TM. Energy-Transfer-labeled Primer--the key component
of the Amplifluor system. The 5' end of UniPrimer consists of a
hairpin structure labeled with fluorescein and DABSYL. A tail
sequence (Z) is at the primer's 3' end. The Z sequence acts as a
universal PCR primer; it is specifically designed to reduce PCR
background due to heterodimer formation. Any PCR reaction can be
adapted to the Amplifluor system by synthesizing a modified version
of one of the target-specific primers (the Z sequence is simply
added to the 5' end of the modified primer). Conventional post-PCR
detection methods such as gel electrophoresis or dot blot
techniques are not required.
[0226] (ii) Molecular Beacon
[0227] The molecular beacon is a hairpin-shaped oligo with a loop
sequence complementary to part of the target sequence and flanked
by two arms that anneal to form a short (5-7 base pair) stem. At
the end of one arm is a fluorophore and at the other a quencher
that prevents fluorescence when the stem is intact. However, with
careful consideration given to the relative stability of the stem
versus that of the beacon-target hybrid, the oligo is designed to
remain folded in free solution but to readily hybridize to any
available target; once hybridized, the quencher is moved away from
the fluorophore, which then fluoresces to signal that target is
present. Molecular beacons thus can be used to monitor real-time
PCR by using a target sequence in the middle of the amplicon and
measuring fluorescence during the annealing step of PCR.
[0228] In order to detect multiple targets in the same solution,
molecular beacons can be made in many different colors utilizing a
broad range of fluorophores. Dabcyl, a non-fluorescent chromophore,
serves as the universal quencher for any fluorophore in molecular
beacons. Owing to their stem, the recognition of targets by
molecular beacons is so specific that single-nucleotide differences
can be readily detected. Because of these properties, molecular
beacons have been used for the detection of RNAs within living
cells, for monitoring the synthesis of specific nucleic acids in
sealed reaction vessels, for homogenous one-tube assays for
genotyping single-nucleotide variations in DNA and for multiplex
PCRs for the detection of four different pathogenic retroviruses
(Vet et al., 1999).
[0229] When fully optimized, molecular beacons make for efficient
detection systems, but occasionally some pitfalls are encountered.
False positives or low signal-to-background can result from impure
preparations that contain free fluorophores or from oligos with a
fluorophore but no quencher, or from design problems such as a stem
that is too strong at low temperatures. Care must be taken with
design as well as with the necessary control experiments to ensure
that molecular beacons operate as intended.
[0230] (iii) TagMan probe.
[0231] A cousin of the molecular beacon is the TaqMan probe from
Applied Biosystems of Foster City, Calif. This system exploits the
5' exonuclease activity of Taq DNA polymerase. During the PCR
extension an annealed oligonucleotide that has a reporter
fluorophore at the 5' exonuclease and a quencher at the 3'
exonuclease is chewed up by a polymerase 5'-3' exonuclease
activity, releasing the fluorophore from its quencher (the presence
of the TaqMan probe doesn't significantly inhibit PCR product
synthesis). The resulting fluorescence is proportional to the
amount of PCR product.
[0232] (iv) The Dual Fluorophore
[0233] An alternative to the fluorophore-quencher system is a dual
fluorophore approach that exploits FRET. This is the principle
behind the LightCycler hybridization probes from Roche Molecular
Biosystems of Indianapolis. Two oligo probes, rather than TaqMan's
one, anneal to the amplicon; one carries a fluorescein label (the
FRET donor) at its 3' end and the second is labeled with LC red 640
(the FRET acceptor) at its 5' end. The oligos are designed to
hybridize in a head-to-tail orientation with the fluorophores
separated at a distance that is compatible with efficient energy
transfer.
[0234] (v) Fluorescent Oligonucleotides for Homogeneous Detection,
Life Technologies, Inc.
[0235] A novel fluorescent detection system that does not require a
quenching moiety for homogeneous detection was developed. The
technology is based on oligonucleotides labeled with a single
fluorophore with significant increase in fluorescence intensity
upon hybridization or incorporation into double stranded DNA. This
detection technology is a platform for fluorescent detection of
nucleic acids in real time as well as in closed tube endpoint
formats. This detection methodology has been used as hybridization
probes and as amplification primers in homogenous PCR amplification
assays.
[0236] (vi) SYBR Green I Dye
[0237] The fluorescent dye SYBR Green I binds to the minor groove
of the DNA double helix. In solution, the unbound dye exhibits very
little fluorescence, however, fluorescence is greatly enhanced upon
DNA-binding. SYBR Green I dye is very stable (only 6% of the
activity is lost during 30 amplification cycles).
[0238] At the beginning of amplification, the reaction mixture
contains the denatured DNA, the primers and the dye. The unbound
dye molecules weakly fluoresce, producing a minimal background
fluorescence signal which is subtracted during computer
analysis.
[0239] After annealing of the primers, a few dye molecules can bind
to the double strand. DNA binding results in a dramatic increase of
the SYBR Green I molecules to emit light upon excitation.
[0240] During elongation, more and more dye molecules bind to the
newly synthesized DNA. If the reaction is monitored continuously,
an increase in fluorescence is viewed in real-time. Upon
denaturation of the DNA for the next heating cycle, the dye
molecules are released and the fluorescence signal falls.
[0241] Fluorescence measurement at the end of the elongation step
of every PCR cycle is performed to monitor the increasing amount of
amplified DNA To separate specific from unspecific signals
fluorescence can be measured at high temperature. The unspecific
products usually melt at a much lower temperature than the specific
product. Therefore, the specificity of the signal can be
significantly enhanced if the temperature is raised near to the
melting point of the specific fragment
[0242] (vii) Other DNA Binding dyes/intercalators:
[0243] DNA binding dyes, some of which are intercalators, bind
double-stranded DNA and to a lesser extent single-stranded DNA and
RNA. With some of these dyes, binding to DNA substantially
increases the intensity of their fluorescence. Dimeric dyes are
noteworthy for their higher affinity. RNA and single-stranded DNA
stains can be used to detect RNA and single stranded DNA.
[0244] Other methods of detection are described in J. Ju et al.,
"Fluorescent energy transfer dye-labeled primers for DNA sequence
analysis, " Proceedings of the National Academy of Sciences,
92:4347-51, 1995; S. Tyagi, F. R. Kramer, "Molecular beacons:
probes that fluoresce upon hybridization," Nature Biotechnology,
14:303-8, 1996; A. J.-C. Eun, S.-M. Wong, "Molecular beacons: A new
approach to plant virus detection," Phytopathology, 90:269-75,
March 2000; L. G. Kostrikis et al., "Spectral genotyping of human
alleles," Science, 279:1228-19, 1998; G. Bonnet et al.,
"Thermodynamic basis of the enhanced specificity of structured DNA
probes," Proceedings of the National Academy of Sciences,
96:6171-6, 1999; R. D. Oberst et al., "PCR-based DNA amplification
and presumptive detection of Escherichia coli O157:H7 with an
internal fluorogenic probe and the 5' nuclease (TaqMan) assay,"
Applied and Environmental Microbiology, 64:3389-96, 1998; I. Tapp
et al., "Homogenous scoring of single-nucleotide polymorphisms:
comparison of the 5'-nuclease TaqMan assay and Molecular Beacon
probes," Biotechniques, 28:732-8, April 2000; I. A. Nazarenko et
al., "A closed tube format for amplification and detection of DNA
based on energy transfer," Nucleic Acids Research, 25:2516-21,
1997; G. J. Nuovo et al., "In situ amplification using universal
energy transfer-labeled primers," The Journal of Histochemistry and
Cytochemistry, 47:273-9, 1999; D. Schuster, "Novel fluorescent
oligonucleotides for homogenous detection and quantitation of
nucleic acids," Abstracts from the Cambridge Healthcare Institute's
fifth annual conference on Gene Quantification; Tyagi S and Kramer
F R (1996) Molecular beacons: probes that fluoresce upon
hybridization. Nat Biotechnol 14, 303-308; Tyagi S, Bratu D P, and
Kramer F R (1998) Multicolor molecular beacons for allele
discrimination. Nat Biotechnol 16, 49-53; Matsuo T (1998) In situ
visualization of mRNA for basic fibroblast growth factor in living
cells. Biochimica Biophysica Acta 1379, 178-184; Sokol D L, Zhang
X, Lu P, and Gewirtz A M (1998) Real time detection of DNA-RNA
hybridization in living cells. Proc Natl Acad Sci USA 95,
11538-11543; Leone G, van Schijndel H, van Gemen B, Kramer F R, and
Schoen C D (1998) Molecular beacon probes combined with
amplification by NASBA enable homogeneous, real-time detection of
RNA. Nucleic Acids Res 26, 2150-2155; Piatek A S, Tyagi S, Pol A C,
Telenti A, Miller L P, Kramer F R, and Alland D (1998) Molecular
beacon sequence analysis for detecting drug resistance in
Mycobacterium tuberculosis. Nat Biotechnol 16, 359-363; Kostrikis L
G, Tyagi S, Mhlanga M M, Ho D D, and Kramer F R (1998) Spectral
genotyping of human alleles. Science 279, 1228-1229; Giesendorf B
A, Vet J A, Tyagi S, Mensink E J, Trijbels F J, and Blom H J (1998)
Molecular beacons: a new approach for semiautomated mutation
analysis. Clin Chem 44, 482-486; Marras S A, Kramer F R, and Tyagi
S (1999) Multiplex detection of single-nucleotide variations using
molecular beacons. Genet Anal 14, 151-156; and Vet J A, Majithia A
R, Marras S A, Tyagi S, Dube S, Poiesz B J, and Kramer F R (1999)
Multiplex detection of four pathogenic retroviruses using molecular
beacons. Proc Natl Acad Sci USA 96, 6394-6399.
[0245] VI. Quantitative Real-Time Polymerase Chain Reaction Using
TagMan
[0246] Quantitative real-time polymerase chain reaction using
TaqMan and the Perkin-Elmer/Applied Biosystems division 7700
sequence detector (PE/ABD 7700) provides an accurate method for
determination of levels of specific DNA and RNA sequences in
samples. It is based on detection of a fluorescent signal produced
proportionally during amplification of a PCR product.
[0247] Quantitative real-time PCR using the PE/ABD 7700 is based on
detection of a fluorescent signal produced proportionally during
the amplification of a PCR product. The chemistry is the key to the
detection system. A probe is designed to anneal to the target
sequence between the traditional forward and reverse primers. The
probe is labeled at the 5' end with a reporter fluorochrome
(usually 6-carboxyfluorescein [6-FAM]) and a quencher fluorochrome
(6-carboxy-tetramethyl-rhodamine [TAMRA]) added at any T position
or at the 3' end. The probe is designed to have a higher Tm than
the primers, and during the extension phase, the probe must be 100%
hybridized for success of the assay. As long as both fluorochromes
are on the probe, the quencher molecule stops all fluorescence by
the reporter. However, as Taq polymerase extends the primer, the
intrinsic 5' to 3' nuclease activity of Taq degrades the probe,
releasing the reporter fluorochrome. The amount of fluorescence
released during the amplification cycle is proportional to the
amount of product generated in each cycle.
[0248] The 7700 detection system consists of a 96-well thermal
cycler connected to a laser and charge-coupled device (CCD) optics
system. An optical fiber inserted through a lens is positioned over
each well, and laser light is directed through the fiber to excite
the fluorochrome in the PCR solution. Emissions are sent through
the fiber to the CCD camera, where they are analyzed by the
software's algorithms. Collected data are subsequently sent to the
computer.
[0249] The sensitivity of detection allows acquisition of data when
PCR amplification is still in the exponential phase. This is
determined by identifying the cycle number at which the reporter
dye emission intensities rises above background noise; this cycle
number is called the threshold cycle (Ct). The Ct is determined at
the most exponential phase of the reaction and is more reliable
than end-point measurements of accumulated PCR products used by
traditional PCR methods. The Ct is inversely proportional to the
copy number of the target template; the higher the template
concentration, the lower the threshold cycle measured.
[0250] There are many advantages to quantifying gene sequences
using this technology, foremost being sensitivity and precision.
This precision exists because quantification of the gene sequence
is determined by the Ct, which is calculated during the exponential
phase of the reaction. High specificity is conferred by the
requirement of three oligos to anneal to the DNA before any data
are collected.
[0251] Competitive PCR is another technique often used to quantify
DNA or RNA. Optimization of competitive PCR is laborious and time
consuming. Several dilutions of target sequences must be tested to
achieve a suitable ratio of target to competitor, and efficiencies
of target and competitor must be similar. This assay is linear only
over a very short range compared with quantification with the 7700.
The number of samples that can be processed is also a limiting
factor.
[0252] The applications for quantitative real-time PCR are
innumerable. Detection of genomic or viral DNA in tissues can be a
valuable diagnostic tool. Gene expression can be measured after
extraction of total RNA and preparation of cDNA by a reverse
transcription (RT) step. Setup and analysis are simple and can more
easily be extended to the clinical environment than traditional PCR
techniques.
[0253] Optimization of the PCR reaction is required for each primer
and probe set. The optimal Mg2+ concentration is usually between 4
and 6 mM but sometimes can be as low as 2 mM. Optimal primer
concentrations are usually between 100 and 800 nM. Optimization
requires varying the concentration of one primer relative to the
other, because the optimal concentration may not be the same for
both. The optimal probe concentration may be as low as 50 nM or as
high as 200 nM. The optimal Mg2+ concentration and reverse primer
concentration must also be validated for the RT step.
[0254] The detection system is so sensitive that fewer than 10
copies of DNA can be detected. Aerosol contamination of primers and
probes is a potential problem if samples are prepared in the
laboratory where DNA is being extracted.
[0255] For determination of pathogens, total nucleic acids are
isolated. A specific cDNA can be produced by using the same reverse
primer used in the PCR reaction or by using random hexamer primers
to produce a range of cDNA products. RNA can easily be prepared
using kits such as RNAEasy from Qiagen (Valencia, Calif., USA) and
Triazol from Life Technologies (Gaithersburg, Md., USA).
[0256] Multiplexing quantitative PCR reactions by using more than
one fluorescent dye per tube became available for internal tube
controls. Kits are available for 18S ribosomal RNA or for
glyceraldehyde-3-phosphat- e dehydrogenase (GAPDH) as a control.
These two fluorochromes are preferred for use with FAM, the
reporter used on the probe.
[0257] If copy number is required, standard curves of plasmid DNA
can be constructed and assayed each time with samples containing
the target gene sequence. If the starting molecule is RNA, cRNA can
be prepared and used as a standard. Kits are available to prepare
RNA from plasmids containing the gene sequence. T7, T3, or SP6
primers typically are used to prepare the cRNA. The cRNA produced
must be validated in the RT and PCR reactions to determine if it is
transcribed and amplified at the same efficiency as the sample RNA
present in a mixture of extracted RNAs.
[0258] Other important controls are no-amplification controls
(NACs) and no-template controls (NTCs). NACs test for contamination
of RNA by genomic DNA. NTCs test for the contamination of assay
reagents.
[0259] Several types of reaction mixes are available. The TaqMan
Universal PCR Master Mix, contains the core reagents in an easy to
use 2.times. solution. The TaqMan Gold RT-PCR kit allows one-step
or two-step RT-PCR. The one-step option allows an investigator to
set up the RT and PCR steps without opening the tube, whereas the
two-step option separates the RT step from the PCR. Master mixes
can also be assembled by purchasing the various components, such as
NTPs, buffer, Mg2+, and Taq polymerase, from many other companies
offering molecular biology reagents.
[0260] Primers and probes must be carefully designed. The Primer
Express software, which is specifically designed to select the
primers and probes takes into account the required parameters for
well-designed primers and probe. These parameters include a Tm for
the probe that is 10.degree. C. higher than the primers, primer Tms
between 58.degree. C. and 60.degree. C., amplicon size between 50
and 150 bases, absence of 5' Gs, and primer length.
[0261] The best design for primers and probes to use for the
quantification of RNA expression requires positioning of a primer
or the probe in a conserved region of the virus, or in case of
genetic testing, over an intron.
[0262] VII. Kits
[0263] In addition, the present invention also provides for a kit
for use in conducting viral assays for efficacy that includes a
mixture of oligonucleotides, the mixture containing at least one
the first primer and/or probe set that provides a detectable signal
on the occurrence on the transcription of viral nucleic acid; and
at least one primer and/or probe set provides a second detectable
signal on the occurrence on the transcription of host nucleic
acid.
[0264] The present invention also provides for a kit for use in
conducting toxicity assays for efficacy that includes a mixture of
oligonucleotides, the mixture containing at least one the first
primer and/or probe that provides a detectable signal on the
occurrence on the transcription of host mitochondrial nucleic acid;
and at least one primer and/or probe provides a second detectable
signal on the occurrence on the transcription of host nuclear
nucleic acid.
[0265] In particular, the kit comprises a primer/probe set for host
nucleic acid wherein the primers are given by Sequence ID No. 1 and
2, and the probe is a sequence given by Sequence ID No. 3 along
with a fluorescent dye and quenching dye.
[0266] In particular, the kit comprises a primer/probe set for
viral nucleic acid for HIV-1 wherein the primers are given by
Sequence ID No. 4 and 5, and the probe is a sequence given by
Sequence ID No. 6 along with a fluorescent dye and quenching
dye.
[0267] In particular, the kit comprises a primer/probe set for
viral nucleic acid for HCV wherein the primers are given by
Sequence ID No. 7 and 8, and the probe is a sequence given by
Sequence ID No. 9 along with a fluorescent dye and quenching
dye.
[0268] In particular, the kit comprises a primer/probe set for
viral nucleic acid for BVDV wherein the primers are given by
Sequence ID No. 10 and 11, and the probe is a sequence given by
Sequence ID No. 12 along with a fluorescent dye and quenching
dye.
[0269] In particular, the kit comprises a primer/probe set for
viral nucleic acid for HBV wherein the primers are given by
Sequence ID No. 13 and 14, and the probe is a sequence given by
Sequence ID No. 5 along with a fluorescent dye and quenching
dye.
[0270] In particular, the kit comprises a primer/probe set for
viral nucleic acid for RSV wherein the primers are given by
Sequence ID No. 16 and 17, and the probe is a sequence given by
Sequence ID No. 18 along with a fluorescent dye and quenching
dye.
[0271] In particular, the kit comprises a primer/probe set for host
mitochondiral nucleic acid wherein the primers are given by
Sequence ID No. 19 and 20, and the probe is a sequence given by
Sequence ID No. 21 along with a fluorescent dye and quenching
dye.
[0272] This invention is further illustrated in the following
sections. The examples contained therein are set forth to aid in an
understanding of the invention. The following examples are
illustrative of the processes and products of the present
invention; but this section is not intended to, and should not be
interpreted to, limit in any way the invention set forth in the
claims that follow thereafter. Equivalent, similar, or suitable
solvents, reagents or reaction conditions may be substituted for
those particular solvents, reagents or reaction conditions
described herein without departing from the general scope of the
method.
EXAMPLES
Example 1
[0273] HIV-1 Cell Culture
[0274] Human PBMC (1.times.10.sup.6 cells/T25 flask) were PHA
stimulated for 2 days, and infected with either a sensitive (xxBRU)
or a 3TC-resistant (184V) HIV-1 strain at 100 TCID.sub.50. The
culture was kept for 5 days in presence of test antiviral compounds
at serial 1-log dilutions. Subsequently, human PBMC were removed
from the culture supernatant by centrifugation (10 min,
400.times.g, 4.degree. C.). This clarified supernatant was tested
either in the RT-assay, or in the real-time RT-PCR assay.
Example 2
[0275] Reverse Transcriptase (RT) Assay
[0276] Virus particles present in a 1 mL aliquot of culture
supernatant were concentrated by centrifugation (2 hr,
20,000.times.g, 4.degree. C.). After the 2 hour spin, supernatant
fluid was removed completely and the virus pellet was dispensed
into a 100 .mu.L Virus Solubilization Buffer (VSB: 0.5% Triton
X-100; 0.8 M NaCl, 0.5 mM phenylmethylsulfonyl, 20% glycerol, 50 mM
Tris.HCl pH 7.8). A 10 .mu.L aliquot of RT-VSB was mixed with 75
.mu.L RT cocktail (60 mM Tris.HCl pH 7.8, 12 MM MgCl.sub.2, 6 mM
DTT, 6 .mu.g/mL Poly (rA)-Poly (dT), 1.2 mM dATP, and 80 .mu.Ci/mL
H.sup.3-TTP) and incubated for 2 hr at 37.degree. C. Subsequently
100 .mu.L of 10% TCA was added, and the total amount of
incorporated H.sup.3-TTP was counted.
Example 3
[0277] RT-PCR Primer and Probe Assessment
[0278] The TaqMan probe and primers were designed by using the
Primer Express software (Applied Biosystems, CA) and are covering
highly conserved sequences complementary to the DNA sequences
present in HIV-1 RNA. By scanning the different genotypes of group
M for regions containing only minor variability, the conserved
domain was discovered. As a result, the region in the HIV-1 RT
domain between codon 200 and 280 fulfilled the required criteria;
thus this region was used to design an appropriate set of primers
and probe that could work in real time PCR ("RT-PCR"). Primer
sequences are as follows: sense 5'-TGGGTTATGAACTCCATCCTGAT-3'
(Sequence ID No. ) and 5'-TGTCATTGACAGTCCAGCTGTCT-3' (Sequence ID
No. ); the probe sequence is 5'-fluoresent
dye-TTTCTGGCAGCACTATAGGCTGTACTGTCCATT-quenching dye-3' (Sequence ID
No.). In this particular case, the probe was labeled with FAM at
the 5' end, and the quencher molecule is TAMARA, provided at the 3'
end. Any other combination of reporter and quencher dyes can be
used as well.
[0279] The primer and probe set gave a linear range over 6 logs
when tested on serial 1-log dilutions of cultured virus. In order
to evaluate this primer/probe set with an FDA approved methodology
for viral load measurement, a 1-log dilution series of a clinical
HIV-1 genotype B isolate (attenuated in vitro to obtain a high
viral load) was tested by real time RT-PCR and by Roche Amplicor
HIV-1 Monitor (FIG. 1). In this experiment, the 10.sup.-6 diluted
sample became positive at threshold cycle (Ct=35.52), which
corresponded with a 1410 copies/mL in the Roche monitor HIV-1
version II assay. When validated over a dynamic range of 3 logs of
virus, there was perfect correlation between the two methodologies
(FIG. 1) with a lower limit of detection for the real-time RT-PCR
assay of 141 copies/mL (Ct=38.85).
Example 4
[0280] Real-Time RT-PCR Assay
[0281] The real-time RT-PCR technology was evaluated against the
NASBA HIV-1 viral load assay. HIV-1 nucleic acid sequences was
amplified using the designed probes and primers as described above.
Viral RNA present in the culture supernatant was prepared using
commercially available columns (QIAamp Viral RNA mini Kit, Qiagen,
CA). The amplification reaction mixture was incubated for two
minutes at 50.degree. C., then ten minutes at 95.degree. C. Then,
the mixture was amplified using forty cycles of a two-step
amplification reaction at 95.degree. C. for fifteen seconds then
sixty seconds at 60.degree. C. Real-time RT-PCR-amplified RNA was
detected in real-time by monitoring increases in fluorescence
signal that resulted from degradation of a quenched fluorescent
probe molecule following to the hybridization of the probe to the
amplified viral DNA (TaqMan 7700 chemistry, Applied Biosystems,
CA).
[0282] A total of 5 .mu.L RNA was RT-amplified using reagents and
conditions as described by the manufacturer (Applied Biosystems,
CA). The standard curve ranged from 1.41.times.10.sup.2 copies/mL
to over 1.41.times.10.sup.8 copies/mL. Copy numbers were calibrated
using the Roche Amplicor HIV-1 Monitor test.TM. (Roche Diagnostics,
Branchburg, N.J.), or the NASBA HIV-1 viral load assay (Organon
Technika).
[0283] Samples containing HIV-1 (genotype B) over a range of 3 logs
(5.times.10.sup.3 to 5.times.10.sup.6 copies/mL) were tested in
both methodologies. The correlation between the two methodologies
is shown in FIG. 2. All samples tested felt within the 95%
confidence interval, and only 2 samples were outside the 99%
confidence interval. It can be concluded that the currently
designed primer and probe set allowed reliable quantification of
the both clinical samples and HIV-1 in vitro virus preparations.
The real-time-RT-PCR has a lower limit of detection of 141
copies/mL and showed linearity over 6-logs of virus dilution.
Example 5
[0284] Optimization
[0285] Optimization of the PCR reaction is required for each primer
and probe set. The optimal Mg2+ concentration is usually between 4
and 6 mM but sometimes can be as low as 2 mM. Optimal primer
concentrations are usually between 100 and 800 nM. Optimization
requires varying the concentration of one primer relative to the
other, because the optimal concentration may not be the same for
both. The optimal probe concentration may be as low as 50 nM or as
high as 200 nM. The optimal Mg2+ concentration and reverse primer
concentration must also be validated for the RT step.
[0286] Potential Contamination
[0287] The detection system is so sensitive that fewer than 10
copies of DNA can be detected. Aerosol contamination of primers and
probes is a potential problem if samples are prepared in the
laboratory where DNA is being extracted.
[0288] Sample Preparation
[0289] For determination of pathogens, total nucleic acids are
isolated. A specific cDNA can be produced by using the same reverse
primer used in the PCR reaction or by using random hexamer primers
to produce a range of cDNA products. RNA can easily be prepared
using kits such as RNAEasy from Qiagen (Valencia, Calif., USA) and
Triazol from Life Technologies (Gaithersburg, Md., USA).
[0290] Controls
[0291] Multiplexing quantitative PCR reactions by using more than
one fluorescent dye per tube became available for internal tube
controls. Kits are available for 18S ribosomal RNA or for
glyceraldehyde-3-phosphat- e dehydrogenase (GAPDH) as a control.
These two fluorochromes are preferred for use with FAM, the
reporter used on the probe.
[0292] If copy number is required, standard curves of plasmid DNA
can be constructed and assayed each time with samples containing
the target gene sequence. If the starting molecule is RNA, cRNA can
be prepared and used as a standard. Kits are available to prepare
RNA from plasmids containing the gene sequence. T7, T3, or SP6
primers typically are used to prepare the cRNA. The cRNA produced
must be validated in the RT and PCR reactions to determine if it is
transcribed and amplified at the same efficiency as the sample RNA
present in a mixture of extracted RNAs.
[0293] Other important controls are no-amplification controls
(NACs) and no-template controls (NTCs). NACs test for contamination
of RNA by genomic DNA. NTCs test for the contamination of assay
reagents.
[0294] Reaction Mix
[0295] Several types of reaction mixes are available. The TaqMan
Universal PCR Master Mix, contains the core reagents in an easy to
use 2.times. solution. The TaqMan Gold RT-PCR kit allows one-step
or two-step RT-PCR. The one-step option allows an investigator to
set up the RT and PCR steps without opening the tube, whereas the
two-step option separates the RT step from the PCR. Master mixes
can also be assembled by purchasing the various components, such as
NTPs, buffer, Mg2+, and Taq polymerase, from many other companies
offering molecular biology reagents.
[0296] Primer and Probe Design
[0297] Primers and probes must be carefully designed. The Primer
Express software, which is specifically designed to select the
primers and probes takes into account the required parameters for
well-designed primers and probe. These parameters include a Tm for
the probe that is 10.degree. C. higher than the primers, primer Tms
between 58.degree. C. and 60.degree. C., amplicon size between 50
and 150 bases, absence of 5' Gs, and primer length.
[0298] The best design for primers and probes to use for the
quantification of RNA expression requires positioning of a primer
or the probe in a conserved region of the virus, or in case of
genetic testing, over an intron.
[0299] The protocol for Real-Time PCR can be achieved by any means
known in the art. See, for example; Gibson U E M, Heid C A,
Williams P M. A novel method for real-time quantitative RT-PCR.
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Williams P M. Real-time quantitative PCR. Genome Res 1996;
6:986-994; Livak K J, Flood S J A, Marmaro J, Giusti W, Deetz K.
Oligonucleotides with fluorescent dyes at opposite ends provide a
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Abramson R D, Watson R, Gelfand D H. Detection of specific
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quantitation of human papillomavirus by using the fluorescent 5'
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and evaluation of a real-time quantitative PCR for the detection of
human cytomegalovirus. J Virol Methods. 95:121-131; Kessler, H. H.,
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Mullah, and A. Andrus 1997. Structural analogues of TaqMan probes
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Lewin, S. R., M. Vesanen, L. Kostrikis, A. Hurley, M. Duran, L.
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molecular beacons to detect virus replication in human
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G., F. Santoro, F. Veglia, A. Gobbi, P. Lusso, and M. S. Malnati
2000. Real-time quantitative PCR for human herpesvirus 6 DNA. J
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High-throughput real-time reverse transcription-PCR quantitation of
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Ohyashiki, J. H., A. Suzuki, K. Aritaki, A. Nagate, N. Shoji, K.
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S. R., R. K. Williams, D. McChesney, E. K. Mont, J. E. Smialek, and
S. E. Straus 1999. Quantitation of latent varicella-zoster virus
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Example 6
[0300] Real-Time RT-PCR Assay for HIV-1
[0301] HIV-1 particles were brought into culture using human PBM
cells. Viral RNA present in the culture supernatant was prepared
using commercially available columns (QIAamp Viral RNA mini Kit,
Qiagen, CA). RT-PCR-amplified RNA was detected in real-time by
monitoring increases in fluorescence signal. A total of 5 L RNA was
RT-amplified using reagents and conditions as described by the
manufacturer (Applied Biosystems, CA). The standard curve ranged
from 1.41.times.10.sup.2 copies/mL to over 1.41.times.10.sup.8
copies/mL. Copy numbers were calibrated using the Roche Amplicor
HIV-1 Monitor test.TM. (Roche Diagnostics, Branchburg, N.J.), or
the NASBA HIV-1 viral load assay (Organon Technika). Correlation
coefficient is in all experiments greater than 0.99. (FIG. 1).
Example 7
[0302] Real-Time RT-PCR Assay for HCV.
[0303] As of today, the only reliable and available system for HCV
RNA replication is the replicon system in Huh7 cells. The cells
were brought into culture for several days and total RNA present in
the culture was prepared using commercially available columns
(QIAamp Viral RNA mini Kit, Qiagen, CA). RT-PCR-amplified RNA was
detected in real-time by monitoring increases in fluorescence
signal. A total of 5 L RNA was RT-amplified using reagents and
conditions as described by the manufacturer (Applied Biosystems,
CA). The standard curve ranged from 45 IU/mL to over
4.7.times.10.sup.7 IU/mL. Copy numbers were calibrated using the
Roche Amplicor HCV Monitor test.TM. (Roche Diagnostics, Branchburg,
N.J.). Correlation coefficient is in all experiments greater than
0.99. (FIG. 2).
Example 8
[0304] Real-Time RT-PCR Assay for HBV.
[0305] HBV viral particles are released from at leasts three
different cell lines: HepG2.2.1.5, HEPAD38 and HepAD79 cell lines.
The cells were brought into culture for several days and total
nucleic acids present in the culture supernatant, or in the cells,
was prepared using commercially available columns (QIAamp Viral RNA
mini Kit, Qiagen, CA). PCR-amplified DNA was detected in real-time
by monitoring increases in fluorescence signal. A total of 5 L DNA
was RT-amplified using reagents and conditions as described by the
manufacturer (Applied Biosystems, CA). The standard curve ranged
from 2 copies to over 2.times.10.sup.7 copies per reaction mix.
Copy numbers were calculated form OD260 values obtained from an HBV
standard. Correlation coefficient is in all experiments greater
than 0.99.
Example 9
[0306] Real-Time RT-PCR Assay for BVDV.
[0307] BVDV viral particles are released from infection experiments
using the strain NADL on MDBK cells (both available form ATTC).
After infection, the cell were brought into culture for several
days and total nucleic acids present in the culture supernatant, or
in the cells, was prepared using commercially available columns
(QIAamp Viral RNA mini Kit, Qiagen, CA). RT-PCR-amplified RNA was
detected in real-time by monitoring increases in fluorescence
signal. A total of 5 L DNA was RT-amplified using reagents and
conditions as described by the manufacturer (Applied Biosystems,
CA). The standard curve ranged from 0.6 plaque forming units to
over 6.times.10.sup.3 plaque forming units per reaction mix. Plaque
forming units were calculated form traditional plaque assays.
Correlation coefficient is in all experiments greater than
0.99.
Example 10
[0308] Real-Time RT-PCR Assay for RSV.
[0309] RSV viral particles are released from infection experiments
using the available virus strain derived from a clinical sample on
A549 or Hep2 cells. After infection, the cell were brought into
culture for several days and total nucleic acids present in the
culture supernatant, or in the cells, was prepared using
commercially available columns (QIAamp Viral RNA mini Kit, Qiagen,
CA). RT-PCR-amplified RNA was detected in real-time by monitoring
increases in fluorescence signal. A total of 5 L DNA was
RT-amplified using reagents and conditions as described by the
manufacturer (Applied Biosystems, CA). The standard curve ranged
from 70 plaque forming units to over 7.times.10.sup.3 plaque
forming units/mL. Plaque forming units were calculated form
traditional plaque assays. Correlation coefficient is in all
experiments greater than 0.99. Hep2 cells gave the highest virus
titer after 72 hours of incubation, the amount of cells used varied
between 10,000 and 50,000 cells per well, but there were no
differences observed in total amount of virus production at 72
hours.
Example 11
[0310] Real-time PCR Assay for Human Mitochondrial DNA and
.beta.-actin DNA
[0311] The current inventions involve the amplification of these
two genetic targets for mitochondrial toxicity testing. Therefore,
a set of primers and fluorescent probes for both mitochondrial and
nuclear DNA or RNA was designed.
[0312] As one illustration of this method, in a first step, HepG2
cells are kept in culture in presence of 10 microMolar of a set of
candidate antiviral agents. Subsequently, total DNA is isolated
from cultured HepG2 cells by means of a commercially available
columns (QIAamp DNA Blood Mini Kit, Qiagen, CA). Total DNA was
eluted from columns in 200 L water. The mitochondrial gene and
nuclear gene are then amplified with a quantitative real-time PCR
protocol using the suitable primers and probes. Reagents and
conditions used in quantitative PCR were purchased from PE-Applied
Biosystems.
[0313] In a separate experiment, the amplification efficiencies of
both targets were evaluated. The standard curve that was created
using the diluted total cell DNA showed linearity over 4 logs [FIG.
3]. Furthermore, FIG. 3 demonstrates that efficiencies of target
and reference amplification are approximately equal, because the
value of the slope of input amount versus DeltaCt (Ct
.beta.-actin-Ct mitochondrial; Ct=PCR cycle threshold where a
sample becomes detectable) is less than 0.1.
[0314] There are at least two methods to obtain accurate quantity
measurements, one method is using standard curves, the other method
is known as the comparative cycle threshold method. The basics of
the two methods are explained in the User Bulletin # 2 of PE
Applied Biosystems. Since both target mitochondrial and the nuclear
endogenous control gene are amplified with almost identical
efficiencies using the described primer-probe sets, either method
can be used to measure the mitochondrial toxicities induced by
antiviral agents. Preferably, the comparative Ct method is used.
This method uses arithmetic formulas to achieve the same result for
relative quantification as obtained by standard curve methods (see
User Bulletin #2; PE Applied Biosystems). In this arithmetic
formula, the amount of target (mitochondrial DNA) is normalized to
a calibrator (nuclear gene) and is relative to an endogenous
reference (no drug control at day 7 or 14, depending on the setup
of the experiment). This arithmetic formula is given by
2.sup.-Ct.
[0315] In order to find out whether antiviral compounds should have
any inhibitory effect on the mitochondrial DNA polymerase .gamma.,
the mitochondrial COXII gene and the nuclear -actin gene were
amplified simultaneously. The relative mitochondrial DNA polymerase
.gamma. toxicity of two antiviral compounds (-)-FTC and D-DDC were
compared with some candidate new antiviral compounds. FIG. 4
demonstrates the results obtained for these antiviral agents. It is
clear from this figure that (-)-FTC does not induce any significant
mitochondrial DNA reduction as compared to the no-drug control.
Instead, important differences were observed for D-DDC at 1 and 10
microM concentration. The observed reduction in mitochondrial DNA
in the DDC settings illustrates the usefulness of the simultaneous
amplification of two or more different targets in molecular
toxicology.
[0316] Similar results were also obtained if this technology was
carried out in a quantitative reverse-transcriptase-PCR protocol.
This approach measures the potential inhibition of antiviral
compounds for the mitochondrial RNA polymerase, in comparison with
the nuclear RNA polymerases I (generating mainly rRNA transcripts),
RNA polymerase II (generating mainly mRNA transcripts), or of
lesser importance, RNA polymerase III (generating mainly tRNA
transcripts). To obtain such results, amplification of either rRNA,
or -actin mRNA as calibrator is required. In these experiments and
after calibration against the relevant nuclear RNA polymerase
transcripts and normalization for no treatment, DDC also showed a
significant reduction in mitochondrial RNA levels, while (-) FTC
did not affect the COXII RNA levels.
[0317] This approach can be used to evaluate the molecular toxicity
levels of any candidate antiviral compounds tested in any cell
type.
[0318] Total DNA is isolated from cultured HepG2 cells by
commercially available columns (QIAamp DNA Blood Mini Kit, Qiagen,
CA). Total DNA was eluted from columns in 200 .mu.L of water. The
mitochondrial gene and nuclear gene are then amplified with a
quantitative real-time PCR protocol using suitable primers and
probes. A set of primers and fluorescent probes for both nuclear
and mitochondrial DNA or RNA was designed; the endogenous control
DNA primer set is given by 5'-GCG CGG CTA CAG CTT CA-3' (Sequence
ID No. ) and 5'-TCT CCT TAA TGT CAC GCA CGA T-3' (Sequence ID No.
); the mitochondrial DNA primer set is given by 5'-TGC CCG CCA TCA
TCC TA-3' (Sequence ID No. ) and 5'-TCG TCT GTT ATG TAA AGG ATG
CGT-3' (Sequence ID No. ). The probe for nuclear gene is given by
5'-fluorescent Dye-CAC CAC GGC CGA GCG GGA-fluorescent quencher-3'
(Sequence ID No. ); fluorescent labeled probes for mitochondrial
genome is given by 5'-fluorescent Dye-TCC TCA TCG CCC TCC CAT
CCC-fluorescent quencher-3' (Sequence ID No. ). Reagents and
conditions used in quantitative PCR were purchased from PE-Applied
Biosystems.
[0319] The standard curve created using the diluted total cell DNA
showed linearity over 4 logs [FIG. 4]. Furthermore, FIG. 4
demonstrates that efficiencies of target and reference
amplification are approximately equal, because the value of the
slope of input amount versus .DELTA.Ct (Ct .beta.-actin-Ct
mitochondrial; Ct=PCR cycle threshold where a sample becomes
detectable) is less than 0.1.
[0320] There are at least two methods to obtain accurate quantity
measurements, one method is using standard curves, and the other
method is known as the comparative cycle threshold method. The
basics of the two methods are explained in the User Bulletin # 2 of
PE Applied Biosystems. Since both target mitochondrial and the
nuclear endogenous control gene are amplified with almost identical
efficiencies using the described primer-probe sets, either method
can be used to measure the mitochondrial toxicities induced by
antiviral agents. Preferably, the comparative Ct method is used.
This method uses arithmetic formulas to achieve the same result for
relative quantification as obtained by standard curve methods (see
User Bulletin #2; PE Applied Biosystems). In this arithmetic
formula, the amount of target (mitochondrial DNA) is normalized to
an endogenous reference (nuclear gene) and is relative to a
calibrator (no drug control at day 7 or 14, depending on the setup
of the experiment). This arithmetic formula is given by
2.sup.-.DELTA..DELTA.Ct.
Example 12
[0321] Simultaneous Amplification of HCV RNA and Cellular
Targets.
[0322] Huh7 cells harboring the HCV replicon can be cultivated in
DMEM media (high glucose, no pyruvate) containing 10% fetal bovine
serum, 1.times. non-essential Amino Acids, Pen-Strep-Glu (100
units/liter, 100 microgram/liter, and 2.92 mg/liter, respectively)
and 500 to 1000 microgram/milliliter G418. Antiviral screening
assays can be done in the same media without G418 as follows: in
order to keep cells in logarithmic growth phase, seed cells in a
96-well plate at low density, for example 1000 cells per well. Add
the test compound immediate after seeding the cells and incubate
for a period of 3 to 7 days at 37.degree. C. in an incubator. Media
is then removed, and the cells are prepared for total nucleic acid
extraction (including replicon RNA and host RNA). Replicon RNA can
then be amplified in a Q-RT-PCR protocol, and quantified
accordingly. The observed differences in quantification of replicon
RNA is one way to express the antiviral potency of the test
compound. A typical experiment demonstrates that in the negative
control and in the non-active compounds-settings a comparable
amount of replicon is produced. This can be concluded because the
measured threshold-cycle for HCV RT-PCR in both setting is close to
each other. In such experiments, one way to express the antiviral
effectiveness of a compound is to subtract the threshold RT-PCR
cycle of the test compound with the average threshold RT-PCR cycle
of the negative control. This value is called DeltaCt (Ct). A Ct of
3.3 equals a 1-log reduction (equals EC.sub.90) in replicon
production. Compounds that result in a reduction of HCV replicon
RNA levels of greater than 2 Ct values (75% reduction of replicon
RNA) are candidate compounds for antiviral therapy. However, this
HCV Ct value does not include any specificity parameter for the
replicon encoded viral RNA-dependent RNA polymerase. In a typical
setting, a compound might reduce both the host RNA polymerase
activity and the replicon-encoded polymerase activity. Therefore,
quantification of rRNA (or any other host RNA polymerase I product)
or beta-actin mRNA (or any other host RNA polymerase II) and
comparison with RNA levels of the no-drug control is a relative
measurement of the effect of the test compound on host RNA
polymerases.
[0323] With the availability of both the HCV .DELTA.Ct data and the
rRNA .DELTA.Ct, a specificity parameter can be introduced. This
parameter is obtained by subtracting both .DELTA.Ct values from
each other. This results in .DELTA..DELTA.Ct values; a value above
0 means that there is more inhibitory effect on the replicon
encoded polymerase, a .DELTA..DELTA.Ct value below 0 means that the
host rRNA levels are more affected than the replicon levels. As an
illustration of this technology, the antiviral activity of tested
compounds, expressed as .DELTA..DELTA.Ct values, is given in FIG.
5. As a general rule, .DELTA..DELTA.Ct values above 2 are
considered as significantly different from the no-drug treatment
control, and hence, is an interested compound for further
evaluation. However, compounds with a .DELTA..DELTA.Ct value of
less than 2, but showing limited molecular cytotoxicty data (rRNA
.DELTA.CT between 0 and 2) are also possible active candidate
compounds for further evaluation
[0324] In another typical setting, a compound might reduce the host
RNA polymerase activity, but not the host DNA polymerase activity.
Therefore, quantification of rDNA or beta-actin DNA (or any other
host DNA fragment) and comparison with DNA levels of the no-drug
control is a relative measurement of the inhibitory effect of the
test compound on cellular DNA polymerases. With the availability of
both the HCV .DELTA.Ct data and the rDNA .DELTA.Ct, a specificity
parameter can be introduced. This parameter is obtained by
subtracting both .DELTA.Ct values from each other. This results in
.DELTA..DELTA.Ct values; a value above 0 means that there is more
inhibitory effect on the replicon encoded polymerase, a
.DELTA..DELTA.Ct value below 0 means that the host rDNA levels are
more affected than the replicon levels. As a general rule,
.DELTA..DELTA.Ct values above 2 are considered as significantly
different from the no-drug treatment control, and hence, is an
interested compound for further evaluation. However, compounds with
a .DELTA..DELTA.Ct value of less than 2, but with limited molecular
cytotoxicty (rDNA .DELTA.CT between 0 and 2) are also possible
active candidate compounds for further evaluation
[0325] Quantitative real-time PCR antiviral screening can be
combined with calibration for a nuclear RNA targets (in RT-PCR) in
the following settings: anti-HCV compound screening can be combined
with rRNA calibration, or -actin mRNA calibration, or any other
nuclear or mitochondrial gene calibration. Anti-HIV compound
screening can be combined with rRNA calibration, -actin mRNA
calibration or any other nuclear or mitochondrial gene calibration.
Anti-HBV compound screening can be combined with rRNA calibration,
-actin mRNA calibration, or any other nuclear or mitochondrial gene
calibration. Anti-RSV compound screening can be combined with rRNA
calibration, -actin mRNA calibration, or any other nuclear or
mitochondrial gene calibration. Anti-BVDV compound screening can be
combined with rRNA calibration, -actin mRNA calibration or any
other nuclear or mitochondrial gene calibration. Anti-lentivirus
compound screening can be combined with rRNA calibration, -actin
mRNA calibration or any other nuclear or mitochondrial gene
calibration. Anti-flaviviridae (Flavivirus, Hepacivirus,
Pestivirus) compound screening can be combined with rRNA
calibration, -actin mRNA calibration or any other nuclear or
mitochondrial gene calibration. Anti-hepadnavirus compound
screening can be combined with rRNA calibration, -actin mRNA
calibration or any other nuclear or mitochondrial gene calibration.
Anti-Picornavirus compound screening can be combined with rRNA
calibration, -actin mRNA calibration or any other nuclear or
mitochondrial gene calibration. Anti-Herpetoviridae (HSV, HCMV,
EBV) compound screening can be combined with rRNA calibration,
-actin mRNA calibration or any other nuclear or mitochondrial gene
calibration.
[0326] Quantitative real-time PCR antiviral screening can be
combined with calibration for a nuclear DNA target (in PCR) in the
following conditions: anti-HCV compound screening can be combined
with rDNA calibration, or -actin DNA calibration, or any other
nuclear or mitochondrial gene calibration. Anti-HIV compound
screening can be combined with rDNA calibration, -actin DNA
calibration or any other nuclear or mitochondrial gene calibration.
Anti-HBV compound screening can be combined with rDNA calibration,
-actin DNA calibration or any other nuclear or mitochondrial gene
calibration. Anti-RSV compound screening can be combined with rDNA
calibration, -actin DNA calibration or any other nuclear or
mitochondrial gene calibration. Anti-BVDV compound screening can be
combined with rDNA calibration, -actin DNA calibration, or any
other nuclear or mitochondrial gene calibration. Anti-lentivirus
compound screening can be combined with rDNA calibration, -actin
DNA calibration or any other nuclear or mitochondrial gene
calibration. Anti-flaviviridae (Flavivirus, Hepacivirus,
Pestivirus) compound screening can be combined with rDNA
calibration, -actin DNA calibration or any other nuclear or
mitochondrial gene calibration. Anti-hepadnavirus compound
screening can be combined with rDNA calibration, -actin DNA
calibration or any other nuclear or mitochondrial gene calibration.
Anti-Picornavirus compound screening can be combined with rDNA
calibration, -actin DNA calibration or any other nuclear or
mitochondrial gene calibration. Anti-Herpetoviridae (HSV, HCMV,
EBV) compound screening can be combined with rDNA calibration,
-actin DNA calibration or any other nuclear or mitochondrial gene
calibration.
Example 13
[0327] Toxicity Assays
[0328] HepG2, VERO (5.times.10.sup.3 cells per well), CEM
(2.5.times.10.sup.3 per well), and PBMC (5.times.10.sup.4 per well)
were seeded in 96-well plates at in the presence of increasing
concentrations of the test compound and incubated in a 37.degree.
C., 5% CO.sub.2 incubator. After a three day-incubation, or 4 for
CEM, or 5 days for PBMC, cell viability and mitochondrial activity
were measured in a colorimetric assay using the MTS- or MTT dye
(Promega, WI).
Example 14
[0329] Antiviral RT-PCR Versus RT Assay
[0330] .beta.-L and .beta.-D analogues of
2',3'-didehydro-2',3'-dideoxy-2'- -fluoro-4'-thio-cytidine
["d4-2'-F-(4S-pentenyl)-C"] were compared with a selection of
antivirals that are currently FDA-approved, or in clinical trial
such as AZT, 3TC, d4T, and (-)-FTC against a two HIV-1 viral
strains--a sensitive strain, xxBRU, and a 3TC-resistant viral
strain with the 184V mutation. Human PBMC were PHA stimulated for 2
days, HIV-1 infected, and kept in culture for 5 days in presence of
test compounds at different concentrations. Subsequently, culture
supernatant was clarified, and tested for reverse transcription
activity by two separate methods. The first method is the standard
endogenous viral RT assay with read-out in log counts per minute/mL
(CPM/mL) by incorporating tritium-labeled TTP; the second is the
RT-PCR method disclosed herein, a quantification method of HIV-1
viral load using real-time PCR quantification assay with read-out
in log copies/mL. FIG. 3 shows the result for some of the tested
compound on both viral strains. Although the two methodologies are
measuring for different items (viral RNA versus active RT enzyme)
results were not-significantly different from each other (FIG. 3,
Table 1). The median 50% (EC.sub.50) and 90% (EC.sub.90) effective
antiviral concentrations were in concordance for the two
methodologies used.
[0331] Wild type xxBRU virus production in this system was very
high, with a total of up to 3.times.10.sup.8 copies/mL in the
untreated samples. Upon addition of antiviral compounds to the
culture media, a dose-related decrease in virus production was
observed. Maximal effect of suppression of viral
1 TABLE 1 xxBRU 184 V real-time real-time RT assay RT-PCR RT assay
RT-PCR EC.sub.50 EC.sub.90 EC.sub.50 EC.sub.90 EC.sub.50 EC.sub.90
EC.sub.50 EC.sub.90 AZT 0.0034 0.034 0.0039 0.036 0.013 0.12 ND ND
3TC 0.018 0.077 0.03 0.12 >100 >100 ND ND d4T 0.0034 0.13
0.0027 0.12 0.032 0.19 0.00078 0.19 (-) FTC 0.011 0.05 0.0059 0.088
65.1 160 0.34 >100 .beta.-L-d4-2'F-(4-S-pentenyl)-C 1.61 11.6
0.4 2.68 >100 >100 0.091 34.9
.beta.-D-d4-2'F-(4-S-pentenyl)-C 5.98 23.2 2.68 18.1 >100
>100 11.8 >100 ND: not done
Example 15
[0332] MTS/MTT Toxicity and Real-time PCR Mitochondrial DNA
Polymerase Toxicity
[0333] Mitochondrial toxicity (.gamma.-DNA polymerase inhibition)
was evaluated by real-time PCR, using the comparative cycle
threshold (Ct) method. .beta.-Actin served as an endogenous
reference. All compounds were tested in routine MTT or MTS toxicity
assays (material and methods). In order to find out whether these
compounds should have any inhibitory effect on the mitochondrial
DNA polymerase .gamma., a real time PCR technology for
mitochondrial DNA polymerase toxicity was designed. In a first
step, standard curves using 1-log diluted total HepG2 DNA were
created, and showed linearity over at least 4 logs (only 4-logs
were tested for these targets). FIG. 4 demonstrates that
efficiencies of target and reference amplification are
approximately equal, because the value of the slope of input amount
versus .DELTA.Ct (Ct .beta.-actin minus Ct mitochondrial, wherein
Ct is the PCR cycle threshold where a sample becomes detectable) is
less than 0.1.
[0334] Furthermore, total DNA was isolated from HepG2 cells
cultured in presence of the antiviral compound. The mitochondrial
gene and the .beta.-actin gene were then amplified. There are at
least two methods to obtain accurate quantity measurements, one
method is using standard curves, the other method is known as the
comparative cycle threshold method (User Bulletin # 2; Applied
Biosystems, CA). Since both targets (mitochondrial and
.beta.-actin) are amplified with almost identical efficiencies
using the described primer-probe sets, either method can be used to
measure the mitochondrial toxicities induced by antiviral agents.
In our experiments, the comparative Ct method was used. This method
uses arithmetic formulas in which the amount of target
(mitochondrial DNA) is normalized to an endogenous reference
(.beta.-actin gene) and is relative to a calibrator (no drug
control at day 7). This arithmetic formula is given by
2.sup.-.DELTA..DELTA.Ct.
[0335] The relative mitochondrial DNA polymerase 7 toxicity of two
antiviral compounds (-)-FTC and D-DDC were compared alongside. FIG.
5 demonstrates the results obtained for each antiviral agent. It is
clear from this figure that (-)-FTC does not induce any significant
mitochondrial DNA reduction as compared to the no-drug control.
Instead, important differences were observed for D-DDC at 1 and 10
.mu.M concentration. D-DDC demonstrated dose-dependent reduction in
mitochondrial DNA synthesis as compared to the no-drug control. The
.beta.-L and .beta.-D analogues of
2',3'-didehydro-2',3'-dideoxy-2'-fluor- o-4'-thio-cytidine
["d4-2'-F-(4S-pentenyl)-C"] both showed no toxicity after a 7-day
incubation with up to 10 .mu.M of the compounds using this
approach. Similarly, in an MTS-dye assay (Promega), no cytotoxicity
was observed for these compounds in human PBMC, Vero and CEM cells
when evaluated up to 100 .mu.M; its CC.sub.50 values were higher
than 100 .mu.M on all cell-types tested (HepG2, VERO, PBMC, and
CEM).
Example 16
[0336] Cell Culture Assays Were Used to Determine the
anti-Flaviviridae Activity of Unmodified or Modified
Ribonucleosides.
[0337] (a) RNA Isolation and Quantitative RT-PCR Analysis
[0338] An effective process to quantify the viral load in a host,
termed real-time polymerase chain reaction ("RT-PCR") is provided.
The process involves using a quenched fluorescent probe molecule
that can be hybridized to viral DNA or RNA. Therefore, upon
exonucleolytic degradation, a detectable fluorescent signal can be
monitored. Therefore, the RT-PCR amplified DNA or RNA is detected
in real time by monitoring the presence of fluorescence
signals.
[0339] As one illustration of this method, in the case of BVDV in
MDBK cells, in a first step, viral RNA is isolated from 140 .mu.L
of the cell culture supernatant by means of a commercially
available column (Viral RNA extraction kit, QiaGen, CA). The viral
RNA is then eluted from the column to yield a total volume of 60
.mu.L, and subsequently amplified with a quantitative RT-PCR
protocol using a suitable primer for the BVDV NADL strain. A
quenched fluorescent probe molecule is hybridized to the BVDV DNA,
which then undergoes exonucleolytic degradation resulting in a
detectable fluorescent signal. Therefore, the RT-PCR amplified DNA
was detected in real time by monitoring the presence of
fluorescence signals. The TaqMan probe molecule
(5'-6-FAM-AAATCCTCCTAACAAGCGGGTTCCAGG-TAMRA 3' [Sequence ID No ]
and primers (sense: 5'-AGCCTTCAGTTTCTTGCTGATGT-3' [Sequence ID No
]; and antisense: 5'-TGTTGCGAAAGCACCAACAG-3' [Sequence ID No ])
were designed with the aid of the Primer Express software
(PE-Applied Biosystems) to be complementary to the BVDV NADL NS5B
region. A total of 10 .mu.L of RNA was analyzed in a 50 .mu.L
RT-PCR mixture. Reagents and conditions used in quantitative PCR
were purchased from PE-Applied Biosystems. The standard curve that
was created using the undiluted inoculum virus ranged from 6000
plaque forming units (PFU) to 0.6 PFU per RT-PCR mixture. A linear
range of over 4-logs was routinely obtained.
[0340] A comparable approach can be taken to measure the amount of
other Flaviviridae (more importantly HCV, YFV, Dengue, West Nile
Virus and others) in a clinical sample or in a tissue culture
sample. For example, the combination of HCV RNA purification with
real-time RT-PCR using the following primers
(5'-TTCCGCAGACCACTATGG-3' [Sequence ID No. ] and
5'-AGCCATGGCGTTAGTATGAGTGT-3' [Sequence ID No. ]) and probe
(5'-6-FAM-CCTCCAGGACCCCCCCTCCC-TAMRA-3' [Sequence ID No. ])
resulted in a 7-log linear range of viral load detection.
[0341] (b) Cell/Viral Materials
[0342] One of the best characterized members of the Pestivirus
genus is BVDV. BVDV and HCV share at least three common features,
which are the following: (1) they the both undergo IRES-mediated
translation; (2) NS4A cofactor is required by their NS3 serine
protease; and (3) they undergo similar polyprotein processing
within the non-structural region, especially at the NS5A and NS5B
junction site.
[0343] The BVDV replication system was used for the discovery of
anti-Flaviviridae compounds. The compounds described herein are
active against Pestiviruses, Hepaciviruses and/or Flaviviruses.
[0344] Maldin-Darby bovine kidney (MDBK) cells were grown and
maintained in a modified eagle medium (DMEM/F12; GibcoBRL),
supplemented with 10% heat inactivated horse serum at 37.degree. C.
in a humidified, 5% CO.sub.2, incubator.
[0345] Bovine viral diarrhea virus (BVDV), strain NADL, causes a
cytopathogenic effect (CPE) after infection of these cells.
[0346] (c) Antiviral Assay
[0347] MDBK-cells, grown in DMEM/F12--10% horse serum (HS), were
isolated in standard techniques using trypsin-EDTA. Cells were
seeded in a 96-well plate at 5.times.10.sup.4 cells/well, with test
compound (20 micromolar (.mu.M) concentration) to give a total
volume of 100 microliters (.mu.L). After one hour, the media was
removed and the cells were infected at a multiplicity of infection
(MOI) of 0.02 or 0.002 in a total volume of 50 .mu.L for 45
minutes. Thereafter, the virus was removed and the cells were
washed twice with 100 .mu.L of assay media. Finally, the infected
cells were incubated in a total volume of 100 .mu.L containing the
test compound at 10, 40 or 100 .mu.M concentration. After 22 hours,
the cell supernatant was collected by removing the cellular debris
by low-speed centrifugation, and subsequently tested for the
presence of virus in a quantitative manner.
[0348] (d) Cytotoxicity Testing of Candidate Anti-Flaviviridae
Compounds
[0349] The cytotoxicity testing as performed here is a standard
technique. Briefly, cells are seeded in 96-well plates at various
concentrations (dependent on cell type, duration of assay),
typically at 5.times.10.sup.3 cells per well, in the presence of
increasing concentrations of the test compound (0, 1, 3, 10, 33,
and 100 .mu.M). After a three day-incubation, cell viability and
mitochondrial activity are measured by adding the MTS-dye
(Promega), followed by a 3 hours incubation. Afterwards the plates
containing the dye are read at 490 nm. Such methodologies are well
described and available from the manufacturer (Promega).
Example 17
[0350] The BVDV RT-PCR Quantification Standard Curve
[0351] The standard BVDV virus stock contained 2.times.10.sup.6
PFU/mL, as determined by routine plaque assay (Mendez, E. et al. J.
Virol. 1998, 72, 4737). Viral RNA was extracted from 140 .mu.L of
this inoculum material and eluted from a column using 60 .mu.L of
an elution buffer. This purified RNA material then was diluted
stepwise from 10.sup.-1 to 10.sup.-5. Using the real-time RT-PCR
amplification technique, 10 .mu.L of each dilution was tested. The
results of this dilution series are plotted in FIG. 1, relating PFU
to concentration of standard. From this experiment, it is clear
that this technology allows for reliable quantification over 4-logs
of virus (from 6000 to 0.6 PFU/input in amplification mix). The
lower limit of detection in this experiment is 0.6 PFU or -0.22 log
PFU. Therefore, the real-time RT-PCR quantification values of test
samples below this detection limit were considered
non-reliable.
Example 18
[0352] The BVDV Replication Cycle in MDBK Cells
[0353] In order to measure the BVDV production in MDBK cells and to
determine the optimal harvesting time over a certain period of
time, cells were seeded at 5.times.10.sup.4 cells/well and infected
either with MOI=0.02 or MOI=0.002. After infection, the inoculum
was removed and the cells were washed twice with culture medium. At
different time points, the cell supernatant was harvested; and, the
amount of virus was measured and compared to the original inoculum
and the cell wash. At least 2 wash-steps were needed to remove the
inoculum virus, as shown in FIG. 2. The amount of virus produced 22
hours after infection approximately equals the amount of virus used
to inoculate the cells. Based on these results, the time required
for one replication cycle of BVDV in MDBK cells was 22 hours. Note
that the detection level set in these experiments was based on the
lower limit of detection as determined by the standard curve.
Example 19
[0354] Evaluation of Candidate Antiviral Compounds Using RT-PCR
[0355] MDBK cells were seeded at 5.times.10.sup.4 cells/well,
infected with BVDV with a multiplicity of infection (MOI) equal to
0.02 and grown for 22 hours in the presence of a test compound.
Cells that were not treated with a test compound were considered a
negative control, while ribavirin served as a positive control.
Viral RNA was extracted and analyzed by real time RT-PCR. A typical
experiment, shown in FIG. 3, demonstrates that the negative control
and the majority of the treated cells produced comparable amounts
of virus (between 1.5 and 2 log PFU/input), effectively showing the
test compounds as non-active. However, the cells treated with the
positive control, ribavirin (RIB) or with 5-hydroxyuridine (I-a-45)
show an almost complete absence of viral RNA. RIB and I-a-45 reduce
viral production by approximately 2 log PFU, or 99%, in the 22 hour
reproduction period. The exact potency of these compounds cannot be
deduced from this kind of experiment, since the detection limit in
this experiment is set at -0.22 log PFU and only one cycle of viral
replication occurs under the stated experimental conditions.
[0356] Potencies, or the effect concentration of compounds that
inhibits virus production by 50% or 90% (EC.sub.50 or EC.sub.90
values, respectively), of anti-BVDV compounds were determined in a
similar set of experiments, but over a broad range of test compound
concentrations (0, 1, 3, 10, 33, 100 .mu.M). The EC.sub.90 value
refers to the concentration necessary to obtain a 1-log reduction
in viral production within a 22 hour period.
[0357] The invention has been described with reference to various
specific and preferred embodiments and techniques. However, it
should be understood that many variations and modifications will be
obvious to those skilled in the art from the foregoing detailed
description of the invention and may be made while remaining within
the spirit and scope of the invention.
* * * * *