U.S. patent application number 09/805785 was filed with the patent office on 2002-03-28 for methods and reagents for preservation of dna in bodily fluids.
This patent application is currently assigned to Sierra Diagnostics, Inc.. Invention is credited to Baker, Tony.
Application Number | 20020037512 09/805785 |
Document ID | / |
Family ID | 26881112 |
Filed Date | 2002-03-28 |
United States Patent
Application |
20020037512 |
Kind Code |
A1 |
Baker, Tony |
March 28, 2002 |
Methods and reagents for preservation of DNA in bodily fluids
Abstract
A method and system are provided for preserving nucleic acids in
a bodily fluid, such as urine, blood, blood serum, and amniotic
fluid. The preservative includes an amount of a divalent metal
chelator selected from ethylenediaminetetraacetic acid (EDTA),
[ethylenebis(oxyethylenenitr- ilo)]tetraacetic acid (EGTA) and
1,2-bis(2-aminophenoxy) ethane-N,N,N',N'-tetraacetic acid (BAPTA),
or salts thereof in the range of from about 0.001M to 0.1M; and an
amount of at least one chelator enhancing component selected from
lithium chloride, guanidine, sodium salicylate, sodium perchlorate,
and sodium thiocyanate in the range of from about 0.1M to 2M.
Inventors: |
Baker, Tony; (Sonora,
CA) |
Correspondence
Address: |
LAHIVE & COCKFIELD
28 STATE STREET
BOSTON
MA
02109
US
|
Assignee: |
Sierra Diagnostics, Inc.
|
Family ID: |
26881112 |
Appl. No.: |
09/805785 |
Filed: |
March 13, 2001 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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09805785 |
Mar 13, 2001 |
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09185402 |
Nov 3, 1998 |
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09185402 |
Nov 3, 1998 |
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08988029 |
Dec 10, 1997 |
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Current U.S.
Class: |
435/6.12 ;
435/270; 536/23.1 |
Current CPC
Class: |
C12Q 1/6832 20130101;
C12Q 1/6806 20130101; C12N 15/1003 20130101 |
Class at
Publication: |
435/6 ; 435/270;
536/23.1 |
International
Class: |
C12Q 001/68; C07H
021/02; C07H 021/04; C12N 001/08 |
Claims
What is claimed is:
1. A method of preserving a nucleic acid in a bodily fluid,
comprising the steps of: a) providing a nucleic acid preservative
solution comprising i) an amount of a divalent metal chelator
selected from the group consisting of ethylenediaminetetraacetic
acid (EDTA), [ethylenebis(oxyethylenenitril- o)]tetraacetic acid
(EGTA) and 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tet- raacetic
acid (BAPTA), or salts thereof in the range of from about 0.001M to
0.1M; and ii) an amount of at least one chelator enhancing
component selected from the group consisting of lithium chloride,
guanidine, sodium salicylate, sodium perchlorate and sodium
thiocyanate in the range of from about 0.1M to 2M; and b) adding
said nucleic acid preservative to said bodily fluid.
2. The method of claim 1 wherein said nucleic acid preservative is
an aqueous solution comprising said divalent metal chelator and
said chelator enhancing component.
3. The method of claim 1 wherein said chelator enhancing component
is selected from the group consisting of sodium perchlorate, sodium
thiocyanate, and lithium chloride.
4. The method of claim 1 wherein said chelator enhancing component
is present in an amount of about 1M.
5. The method of claim 1 wherein said divalent metal chelator is
present in an amount of at least about 0.01M.
6. The method of claim 1 wherein said nucleic acid preservative
further comprises an amount of at least one enzyme inactivating
component selected from the group consisting of manganese chloride,
sarkosyl, and sodium dodecyl sulfate in the range of about 0-5%
molar concentration.
7. The method of claim 1 wherein said nucleic acid is selected from
the group consisting of DNA, RNA, mRNA, and cDNA.
8. The method of claim 7 wherein said DNA is eukaryotic DNA.
9. A nucleic acid preservative solution comprising: a) an amount of
a divalent metal chelator selected from the group consisting of
ethylenediaminetetraacetic acid (EDTA),
[ethylenebis(oxyethylenenitrilo)]- tetraacetic acid (EGTA) and
1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraa- cetic acid (BAPTA),
or salts thereof in the range of from about 0.001M to 0.1M; and b)
an amount of at least one chelator enhancing component selected
from the group consisting of lithium chloride, guanidine, sodium
salicylate, sodium perchlorate, and sodium thiocyanate in the range
of from about 0.1M to 2M.
10. The nucleic acid preservative of claim 9 wherein said nucleic
acid preservative is an aqueous solution comprising said divalent
metal chelator and said chelator enhancing component.
11. The nucleic acid preservative of claim 9 wherein said chelator
enhancing component is present in an amount of about 1M.
12. The nucleic acid preservative of claim 9 wherein said divalent
metal chelator is present in an amount of about 0.01M.
13. The nucleic acid preservative of claim 9 wherein further
comprising an amount of at least one enzyme inactivating component
selected from the group consisting of manganese chloride, sarkosyl,
and sodium dodecyl sulfate in the range of about 0-5% molar
concentration.
14. A preserved nucleic acid-containing fluid comprising: a) an
amount of a divalent metal chelator selected from the group
consisting of ethylenediaminetetraacetic acid (EDTA),
[ethylenebis(oxyethylenenitrilo)]- tetraacetic acid (EGTA) and
1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraa- cetic acid (BAPTA),
or salts thereof in the range of from about 0.001M to 0.1M; and b)
an amount of at least one chelator enhancing component selected
from the group consisting of lithium chloride, guanidine, sodium
salicylate, sodium perchlorate, and sodium thiocyanate in the range
of from about 0.1M to 2M.
15. A method of improving the signal response of a molecular assay
of a test sample, comprising the steps of: a) providing a nucleic
acid preservative solution comprising i) an amount of a divalent
metal chelator selected from the group consisting of
ethylenediaminetetraacetic acid (EDTA),
[ethylenebis(oxyethylenenitrilo)]tetraacetic acid (EGTA) and
1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA),
or salts thereof in the range of from about 0.001M to 0.1M; and ii)
an amount of at least one chelator enhancing component selected
from the group consisting of lithium chloride, guanidine, sodium
salicylate, sodium perchlorate, and sodium thiocyanate in the range
of from about 0.1M to 2M; b) adding said nucleic acid preservative
to a test sample to provide a preserved test sample; c) extracting
molecular analytes of interest from said preserved test sample; and
d) conducting a molecular assay on said extracted molecular
analytes of interest.
16. The method of claim 15 wherein said test sample is a bodily
fluid.
17. The method of claim 16 wherein said bodily fluid is selected
from the group consisting of urine, blood, blood serum, amniotic
fluid, salivary fluid, vaginal fluid, conjunctival fluid, stool,
seminal fluid, and sweat.
18. The method of claim 17 wherein said nucleic acid is selected
from the group consisting of DNA, RNA, mRNA, and cDNA.
19. The method of claim 18 wherein said DNA is eukaryotic DNA.
20. The method of claim 15 wherein said molecular assay is selected
from the group consisting of the polymerase chain reaction, ligase
chain technology test, and a genetic transformation test.
21. A method of improving hybridization of nucleic acids,
comprising the steps of: a) contacting a test nucleic acid with a
nucleic acid preservative solution comprising i) an amount of a
divalent metal chelator selected from the group consisting of
ethylenediaminetetraacetic acid (EDTA),
[ethylenebis(oxyethylenenitrilo)]tetraacetic acid (EGTA) and
1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA),
or salts thereof in the range of from about 0.001M to 0.1M; and ii)
an amount of at least one chelator enhancing component selected
from the group consisting of lithium chloride, guanidine, sodium
salicylate, sodium perchlorate, and sodium thiocyanate in the range
of from about 0.1M to 2M, such that a test solution is formed; b)
contacting said test solution with a target nucleic acid under
conditions favorable for hybridization, such that hybridization
occurs.
Description
RELATED APPLICATIONS
[0001] This application is a continuation-in-part of copending
application Ser. No. 08/988,029, filed Dec. 10, 1997, the entire
contents of which are incorporated herein by reference.
BACKGROUND OF THE INVENTION
[0002] The present disclosure relates generally to the field of DNA
analysis. More particularly, the present disclosure relates to
methods, preservative reagents and systems for preserving DNA in
bodily fluids for subsequent testing and analysis.
[0003] Modern testing and treatment procedures have successfully
reduced the prevalence and severity of many infectious diseases.
For example, sexually-transmitted disease (STD) clinics regularly
screen and treat patients for such diseases as gonorrhea and
syphilis. It is now well-known to identify infectious agents such
as gonococci by analyzing a DNA sample. A genetic transformation
test (GTT), such as Gonostat.RTM. (Sierra Diagnostics, Inc.,
Sonora, Calif.), can be used to detect gonococcal DNA in specimens
taken from the urethra of men, and the cervix and anus of women,
according to Jaffe H W, Kraus S J, Edwards T A, Zubrzycki L.
Diagnosis of gonorrhea using a genetic transformation test on
mailed clinical specimens, J Inf Dis 1982; 146:275-279. A similar
finding was also published in Whittington W L, Miller M, Lewis J,
Parker J, Biddle J, Kraus S. Evaluation of the genetic
transformation test,. Abstr Ann Meeting Am Soc Microbiol 1983; p.
315.
[0004] The GTT is a test for biologically active or native DNA. For
example, the Gonostat(3) GTT can be used to detect DNA such as
gonococcal DNA in urine specimens. The Gonostat.RTM. assay uses a
test strain, N. Gonorrhoeae, ATCC 31953. This test strain is a
mutant that is unable to grow into visible colonies on chocolate
agar at 37.degree. C. in 5% CO.sub.2. Gonococcal DNA extracted from
clinical material can restore colony growth ability to this test
strain. The Gonostat.RTM. assay is discussed in Zubrzycki L,
Weinberger S S, Laboratory diagnosis of gonorrhea by a simple
transformation test with a temperature-sensitive mutant of
Neisseria gonorrhoeae. Sex Transm Dis 1980; 7:183-187.
[0005] It is not always possible to immediately test a patient for
the presence of such an infectious agent. For example, clinical
laboratories are not readily found in many rural or underdeveloped
areas. In such circumstances, it is necessary to transport patient
test specimens to a laboratory for analysis. It is therefore
desirable to preserve such specimens for subsequent analysis with a
GTT or other testing procedure.
[0006] Urine specimens are frequently practical and convenient for
use in diagnoses of an infection, such as gonorrhea. A urine
specimen can be collected by a patient, therefore avoiding the
invasion of privacy and discomfort accompanying collection of other
specimens, such as blood specimens, urethral cultures, or cervical
cultures. Collection of a urine specimen by the patient also
reduces the work load of the staff in the clinic or office.
[0007] DNA culture results of urine from males are quite sensitive
when the urine is cultured within two hours of collection. Such
results can approach 92% to 94%, or even 100%, as described in
Schachter J. Urine as a specimen for diagnosis of sexually
transmitted diseases. Am J Med 1983; 75:93-97. However, the culture
results of urine from females are not very reliable, even when
cultured within two hours. According to Schachter, only 47% to 73%
of female urine cultures are positive relative to the culture
results of cervical and anal specimens. Furthermore, it is known
that culture results from any anatomic site are not 100% sensitive.
(See, for example, Johnson D W, Holmes K K, Kvale P A, Halverson C
W, Hirsch W P. An evaluation of gonorrhea case finding in the
chronically infected male. Am J Epidemiol 1969; 90:438-448; Schmale
J D, Martin J E, Domescik G. Observations on the culture diagnosis
of gonorrhea in women. JAMA 1969; 210:213-314; Caldwell J G, Price
E V, Pazin G J, Cornelius E C. Sensitivity and reproducibility of
Thayer-Martin culture medium in diagnosing gonorrhea in women. Am J
Gynecol 1971; 109:463-468; Kieth L, Moss W, Berger G S. Gonorrhea
detection in a family planning clinic: A cost-benefit analysis of
2,000 triplicate cultures. Am J Obstet Gynecol 1975; 121:399-403;
Luciano A A, Grubin L. Gonorrhea screening JAMA 1980; 243:680-681;
Goh B T, Varia K B, Ayliffe P F, Lim F K. Diagnosis of gonorrhea by
gram-stained smears and cultures in men and women: Role of the
urethral smear. Sex Trans Dis 1985; 12:135-139.
[0008] Currently, urine specimens must be tested quickly for the
presence of naked gonococcal DNA. Naked DNA is intact double
stranded DNA which is released from viable gonococci. Such naked
DNA can be found in the urine of an infected patient. However,
enzymes in urine rapidly destroy any DNA present in the specimen.
The DNA is either denatured, broken into single strands or totally
destroyed by the enzymatic activity. This destruction of the DNA
can effectively inactivate the naked gonococcal DNA for purposes of
testing.
[0009] In a test such as the GTT, inactivation beyond the limits of
detection is determined by the inherent genetic needs for select
gene sequences of the Gonostat mutant strain used in the Gonostat
test. For example, the Gonostat transformation assay is a very
sensitive measurement tool for nucleic acid protection. In the GTT,
the Gonostat organism must have approximately 1 picogram of native
DNA to transform. This amount is equal to the presence of
approximately 30 gonorrhea bacteria in an inoculum. The average
clinical infection has 10.sup.3-10.sup.5 such organisms.
[0010] The destruction of DNA by enzyme activity in a urine
specimen increases with time. For example, naked gonococcal DNA in
a urine specimen that is stored in excess of two hours is
inactivated beyond the limits of detection of the GTT. As a result,
the testing of urine specimens for DNA is very time-sensitive. For
example, DNA-based tests such as the polymerase chain reaction
(PCR), the ligase chain technology (LC.sub.x) test of Abbott
Laboratories, Abbott Park, Ill., and the GTT all must be performed
on a urine specimen within approximately two hours. FIG. 1 is a
graph of DNA concentration in unpreserved urine according to the
prior art, demonstrating DNA destruction over time. The gonococcal
DNA concentrations of ten different types of urine specimens were
tested using a GTT at hourly intervals, commencing one hour from
time of inoculation. Approximately 200 transformants were counted
at the one hour measurement. However, for all specimens, the number
of transformants declined by more than 100% within one hour of this
initial measurement. The number of transformants approached zero
within the two hours of the ipitial measurement. FIG. 2 is a graph
of eight day serial data on unpreserved urine according to the
prior art, further illustrating DNA destruction in unpreserved
samples. Approximately seven transformants were counted at the one
day measurement. However, by the second day, testing indicated that
the biologically active DNA in the unpreserved urine had been
totally destroyed by enzyme activity.
[0011] Tests such as the GTT can also be used to detect DNA in such
bodily fluids and excretions as blood, blood serum, amniotic fluid,
spinal fluid, conjunctival fluid, salivary fluid, vaginal fluid,
stool, seminal fluid, and sweat. FIG. 3 is a graph of DNA
concentration in unpreserved serum according to the prior art,
demonstrating DNA destruction over time. The gonococcal DNA
concentrations of normal and abnormal serum of both male and female
were tested at hourly intervals, commencing from the time of
inoculation. Approximately 100 transformants were counted at the
one hour measurement. However, for all specimens, the number of
transformants declined by more than 100% within three hours of this
initial measurement. The number of transformants approached zero
within the eight hours of the initial measurement.
[0012] Another test that can be used to identify DNA in a bodily
fluid specimen is the PCR test. PCR testing uses discrete nucleic
acid sequences and therefore can be effective even in the absence
of intact DNA. FIG. 4 is a graph of PCR detection of MOMP Chlamydia
in unpreserved urine according to the prior art, demonstrating DNA
destruction over time. In PCR testing of an unpreserved urine
specimen, four PCR absorbances were observed one hour after the
addition of the MOMP Chlamydia. However, the number of PCR
absorbances declined 100%, to two, when tested at two hours, and to
zero by the third hour. This testing indicates that, even though
PCR testing doesn't require intact DNA, the enzymatic activity of
urine rapidly destroys even discrete nucleic acid sequences 45
within approximately three hours.
[0013] Unfortunately, practical and effective techniques for
preserving DNA in certain bodily fluids have not been readily
available. For example, one method used to deactivate urine enzymes
is heating. In an experiment, urine was heated for five minutes in
a boiling water bath (100.degree. C.) and then cooled. Naked DNA
and DNA released from gonococcal cells that were subsequently added
to this urine were not deactivated. This suggests that the
deoxyribonuclease component in urine is a protein(s).
[0014] However, heating can denature DNA that is already present in
the urine specimen, including gonococcal DNA, as well as the DNA of
Haemophilus influenzoe and Bacillus subtilis. Thus, heating is not
an appropriate method for preserving a patient urine specimen to
test for the presence of such DNA.
[0015] In other known DNA assay systems, it is known to add
detergents or other chemicals to assist in the detection of DNA.
For example, in the DNA assay system described in Virtanen M,
Syvanen A C, Oram J, Sodurlund H, Ranki M. Cytomegalovirus in
urine: Detection of viral DNA by sandwich hybridization. J Clin
Microbiol. 1984; 20:1083-1088, sarkosyl was used to detect
cytomegalovirus (CMV) in urine by hybridization. In Boom R, Sol C J
A, Salimans M M M, Jansen C L, Wertheim-van Dillen PME, van der
Noordaa J. Rapid and simple method for purification of nucleic
acids. J Clin Microbiol 1990; 28:495-503, guanidinium chloride in
urine was used to purify nucleic acids as assayed by gel
electrophoresis. Although the reason for their use in these studies
was not stated, the chemicals inactivated the deoxyribonuclease
activity in urine that would have interfered with those assay
systems.
[0016] It would therefore be advantageous to provide a method and
system for preserving DNA in a bodily fluid such as urine, blood,
blood serum, amniotic fluid, spinal fluid, conjunctival fluid,
salivary fluid, vaginal fluid, stool, seminal fluid, and sweat,
such that the efficacy of the DNA assays, e.g., the PCR, LC.sub.x,
and the GTT is optimized.
SUMMARY OF THE INVENTION
[0017] The present disclosure relates to methods, systems and
reagents for preserving nucleic acids, e.g., DNA and RNA, in bodily
fluids. In one advantageous embodiment, the invention is directed
to the preservation of nucleic acids in urine. In another
advantageous embodiment, the invention enables the molecular assay
of nucleic acids in other bodily fluids and excretions, such as
blood, blood serum, amniotic fluid, spinal fluid, conjunctival
fluid, salivary fluid, vaginal fluid, stool, seminal fluid, and
sweat to be carried out with greater sensitivity, as the methods
and preservatives of the invention have been found to surprisingly
increase the signal obtained with such nucleic acid testing methods
as polymerase chain reaction (PCR), LC.sub.x, and genetic
transformation testing (GTT). An unexpected advantage of the
invention is that hybridization in such nucleic acid testing
methods is improved compared to when such nucleic acid testing
methods are carried out without employing the present
invention.
[0018] In an embodiment, the invention relates to methods of
preserving a nucleic acid in a fluid such as a bodily fluid,
including providing a nucleic acid preservative solution comprising
an amount of a divalent metal chelator selected from
ethylenediaminetetraacetic acid (EDTA),
[ethylenebis(oxyethylenenitrilo)]tetraacetic acid (EGTA) and
1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA),
or salts thereof; and an amount of at least one chelator enhancing
component selected from lithium chloride, guanidine, sodium
salicylate, sodium perchlorate, and sodium thiocyanate; and adding
the nucleic acid preservative to the fluid, e.g., a bodily fluid.
The amount of the divalert metal chelator is generally in the range
of from about 0.001M to 0.1M, and the amount of the chelator
enhancing component is generally in the range of from about 0.1M to
2M. The amount of chelator enhancing component is more desirably at
least 1M in the preservative solution, and the divalent metal
chelator is desirably present in an amount of at least about
0.1M.
[0019] In another embodiment, the invention relates to nucleic acid
preservative solutions comprising an amount of a divalent metal
chelator selected from EDTA, EGTA and BAPTA, and salts thereof; and
an amount of at least one chelator enhancing component selected
from lithium chloride, guanidine, sodium salicylate, sodium
perchlorate, and sodium thiocyanate. The amount of the divalent
metal chelator is generally in the range of from about 0.001M to
0.1M, and the amount of the chelator enhancing component is
generally in the range of from about 0.1M to 2M. The amount of
chelator enhancing component is more desirably at least 1M in the
preservative solution, and the divalent metal chelator is desirably
present in an amount of at least about 0.1M.
[0020] The methods and preservatives of the invention may further
include an amount of at least one enzyme inactivating component
such as manganese chloride, sarkosyl, or sodium dodecyl sulfate,
generally in the range of about 0-5% molar concentration.
[0021] In yet another aspect the invention relates to a method of
improving the signal response of a molecular assay of a test
sample, including providing a nucleic acid preservative solution
comprising an amount of a divalent metal chelator selected from
EDTA, EGTA and BAPTA, and salts thereof; and an amount of at least
one chelator enhancing component selected from lithium chloride,
guanidine, sodium salicylate, sodium perchlorate, and sodium
thiocyanate; adding the nucleic acid preservative to a test sample
to provide a preserved test sample; extracting molecular analytes
of interest, e.g., DNA, from the preserved test sample, and
conducting a molecular assay on the extracted molecular analytes of
interest. The amount of the divalent metal chelator is generally in
the range of from about 0.001M to 0.1M, and the amount of the
chelator enhancing component is generally in the range of from
about 0.1M to 2M. The chelator enhancing component is more
advantageously one or more of sodium perchlorate, sodium
thiocyanate, guanidine, and lithium chloride. The amount of
chelator enhancing component is more desirably at least 1M in the
preservative solution, and the divalent metal chelator is desirably
present in an amount of at least about 0.01M. Signal response is
believed to be enhanced in part due to enhanced hybridization as a
result of the use of the reagents of the present invention.
[0022] Use of the methods and preservatives disclosed herein
eliminate enzymatic destruction of the nucleic acid of interest in
the bodily fluid. The preservative can optionally be provided in
solid or gaseous forms. While the methods and preservatives of the
invention are useful in preserving all types of nucleic acids,
e.g., RNA and DNA, including human DNA, and bacterial, fungal, and
viral DNA, the invention is especially advantageous for use in
preserving prokaryotic DNA, e.g., gonococcal DNA, DNA of
Haemophilus influenzoe and Bacillus subtilis. Nucleic acids in a
bodily fluid are preserved for testing for a significantly longer
period of time than that permitted by the prior art. While the
maximum time between collecting, mailing, and testing patient
specimens is expected to be approximately six days, the invention
is effective beyond that period of time.
[0023] A further aspect of the invention relates to methods of
improving hybridization of nucleic acids, including contacting a
test nucleic acid with a nucleic acid preservative solution
comprising an amount of a divalent metal chelator selected from
ethylenediaminetetraacetic acid (EDTA),
ethylenebis(oxyethylenenitrilo)] tetraacetic acid (EGTA) and
1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA),
or salts thereof in the range of from about 0.001M to 0.1M; and an
amount of at least one chelator enhancing component selected from
lithium chloride, guanidine, sodium salicylate, sodium perchlorate,
and sodium thiocyanate in the range of from about 0.1M to 2M, such
that a test solution is formed; and contacting the test solution
with a target nucleic acid under conditions favorable for
hybridization, such that hybridization occurs.
BRIEF DESCRIPTION OF THE DRAWING
[0024] FIG. 1 is a graph of DNA concentration in unpreserved urine
according to the prior art;
[0025] FIG. 2 is a graph of eight day serial data on unpreserved
urine according to the prior art;
[0026] FIG. 3 is a graph of DNA concentration in unpreserved serum
according to the prior art;
[0027] FIG. 4 is a graph of PCR detection of MOMP Chlamydia in
unpreserved urine according to the prior art;
[0028] FIG. 5 is a bar graph of DNA concentration in preserved
urine according to the invention;
[0029] FIG. 6 is a graph of eight day serial data on preserved
urine according to the invention;
[0030] FIG. 7 is a graph comparing PCR results in unpreserved and
preserved normal urine according to the invention;
[0031] FIG. 8 is a graph of eight day serial data on preserved
serum according to the invention;
[0032] FIG. 9 is a graph of DNA concentration in preserved serum
according to the invention;
[0033] FIG. 10 is a flow chart of the method for preserving DNA
according to one embodiment of the invention;
[0034] FIG. 11 is a diagram of the system for preserving DNA
according to one embodiment of the invention;
[0035] FIG. 12 graphically illustrates a comparison of signal
response in PCR assays wherein the DNA has been treated with a
preservative of the invention, and one which has not;
[0036] FIG. 13 illustrates the efficacy of reagents of the present
invention to enhance signal response of a branched DNA assay of
blood plasma samples subjected to various storage conditions;
[0037] FIG. 14 illustrates the efficacy of reagents of the present
invention to enhance signal response of a branched DNA assay of
blood serum and plasma samples;
[0038] FIG. 15 is a graph showing the interference of methemoglobin
on PCR absorbance in a PCR amplification assay on hepatitis B
sequences MD03/06 in unprotected serum;
[0039] FIG. 16 is a graph showing the improvement in attenuating
the interference of methemoglobin on PCR absorbance in a PCR
amplification assay on hepatitis B sequences MD03/06 in serum which
has been treated with a preservative of the invention;
[0040] FIG. 17 illustrates the synergistic effect provided by the
components of the inventive reagents in protecting hepatitis B
sequences in serum stored at room temperature and subsequently
subjected to MD03/06 PCR detection;
[0041] FIGS. 18A-18G are graphs showing the absence of preservative
effect on gonococcal DNA in urine stored at room temperature and
subsequently subjected to PCR detection offered by the individual
addition of certain components which are included in the reagents
of the invention; and
[0042] FIG. 19 illustrates the synergistic effect obtained by the
combination of the components of the inventive reagents in
protecting gonococcal DNA in urine stored at room temperature and
subsequently subjected to PCR detection.
DETAILED DESCRIPTION OF THE INVENTION
[0043] Improved methods, systems and reagents for preserving
nucleic acids, e.g., DNA and RNA, in bodily fluids are disclosed
herein. In one advantageous embodiment, the invention is may be
used for preservation of nucleic acids in urine. In another
advantageous embodiment, the invention enables the molecular assay
of nucleic acids in other bodily fluids and excretions, such as
blood, blood serum, amniotic fluid, spinal fluid, conjunctival
fluid, salivary fluid, vaginal fluid, stool, seminal fluid, and
sweat to be carried out with greater sensitivity, as the methods
and preservatives of the invention have been found to surprisingly
increase the signal obtained with such nucleic acid testing methods
as the polymerase chain reaction (PCR), LC.sub.X, and genetic
transformation testing (GTT). In particular, the invention has also
been found to surprisingly modulate the effect of hemoglobin, e.g.,
methemoglobin, interference on nucleic acid assays such as PCR on
serum samples. Additionally, hybridization in such nucleic acid
testing methods is unexpectedly improved.
[0044] In an embodiment, the invention relates to methods of
preserving a nucleic acid in a fluid such as a bodily fluid,
including providing a nucleic acid preservative solution comprising
an amount of a divalent metal chelator selected from
ethylenediaminetetraacetic acid (EDTA),
[ethylenebis(oxyethylenenitrilo)]tetraacetic acid (EGTA) and
1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA),
or salts thereof; and an amount of at least one chelator enhancing
component selected from lithium chloride, guanidine, sodium
salicylate, sodium perchlorate, and sodium thiocyanate; and adding
the nucleic acid preservative to the fluid, e.g., a bodily fluid.
The amount of the divalent metal chelator is generally in the range
of from about 0.001M to 0.1M, and the amount of the chelator
enhancing component is generally in the range of from about 0.1M to
2M. The amount of chelator enhancing component is more desirably at
least 1M in the preservative solution, and the divalent metal
chelator is desirably present in an amount of at least about
0.1M.
[0045] In another embodiment, the invention relates to nucleic acid
preservative solutions comprising an amount of a divalent metal
chelator selected from EDTA, EGTA and BAPTA, and salts thereof; and
an amount of at least one chelator enhancing component selected
from lithium chloride, guanidine, sodium salicylate, sodium
perchlorate, and sodium thiocyanate. The amount of the divalent
metal chelator is generally in the range of from about 0.001M to
0.1M, and the amount of the chelator enhancing component is
generally in the range of from about 0.1M to 2M. The amount of
chelator enhancing component is more desirably at least 1M in the
preservative solution, and the divalent metal chelator is desirably
present in an amount of at least about 0.01M.
[0046] The methods and preservatives of the invention may further
include an amount of at least one enzyme inactivating component
such as manganese chloride, sarkosyl, or sodium dodecyl sulfate,
generally in the range of about 0-5% molar concentration.
[0047] In yet another aspect the invention relates to a method of
improving the signal response of a molecular assay of a test
sample, including providing a nucleic acid preservative solution
comprising an amount of a divalent metal chelator selected from
EDTA, EGTA and BAPTA, and salts thereof; and an amount of at least
one chelator enhancing component selected from lithium chloride,
guanidine, sodium salicylate, sodium perchlorate, and sodium
thiocyanate; adding the nucleic acid preservative to a test sample
to provide a preserved test sample; extracting molecular analytes
of interest, e.g., DNA, from the preserved test sample, and
conducting a molecular assay on the extracted molecular analytes of
interest. The amount of the divalent metal chelator is generally in
the range of from about 0.001M to 0.1M, and the amount of the
chelator enhancing component is generally in the range of from
about 0.1M to 2M. The chelator enhancing component is more
advantageously one or more of sodium perchlorate, sodium
thiocyanate, sodium perchlorate, guanidine, and lithium chloride.
The amount of chelator enhancing component is more desirably at
least 1M in the preservative solution, and the divalent metal
chelator is desirably present in an amount of at least about 0.01M.
Signal response is believed to be enhanced in part due to enhanced
hybridization as a result of the use of the reagents of the present
invention.
[0048] Use of the methods and preservatives disclosed herein
eliminate enzymatic destruction of the nucleic acid of interest in
the bodily fluid. The preservative can optionally be provided in
solid or gaseous forms. While the methods and preservatives of the
invention are useful in preserving all types of nucleic acids,
e.g., RNA and DNA, including human DNA, and bacterial, fungal, and
viral DNA, the invention is especially advantageous for use in
preserving prokaryotic DNA, e.g., gonococcal DNA, DNA of
Haemophilus influenzoe and Bacillus subtilis. Nucleic acids in a
bodily fluid are preserved for testing for a significantly longer
period of time than that permitted by the prior art. While the
maximum time between collecting, mailing, and testing patient
specimens is expected to be approximately six days, the invention
is effective beyond that period of time.
[0049] The preservatives of the invention may be used
advantageously to preserve prokaryotic, e.g., gonococcal DNA, as
shown below, although the teachings of the invention may be readily
applied to the preservation of other types of DNA, including human,
bacterial, fungal, and viral DNA, as well as to RNA. The reagents
of the invention are believed to function by inactivating two
classes of enzymes present in bodily fluids such as blood or urine
which the inventor has recognized as destructive to DNA integrity,
metal-dependent and metal independent enzymes. The divalent metal
chelator removes, e.g., magnesium and calcium cation (Mg.sup.+2,
Ca.sup.+2) so as to effectively inactivate metal dependent enzymes
such as deoxyribonucleases, a component of which has been found to
inactivate gonococcal DNA in unpreserved urine. The divalent metal
chelator may be ethylenediaminetetraacetic acid (EDTA),
[ethylenebis(oxyethylenenitrilo)]- tetraacetic acid (EGTA), or 1
,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetra- acetic acid (BAPTA),
or salts thereof. The amount of the divalent metal chelator is
generally in the range of from about 0.001M to 0.1M. More
desirably, the amount of the divalent metal chelator in the
preservative solution is at least 0.01M.
[0050] The second component of the reagents disclosed herein
include a chelator enhancing component which assists the divalent
metal chelator in protecting the nucleic acids in the fluid. These
chelator enhancing components are believed to inactivate metal
independent enzymes found in bodily fluids such as DNA ligases,
e.g., D4 DNA ligase; DNA polymerases, e.g., T7 DNA polymerase;
exonucleases, e.g., exonuclease 2, .lambda.-exonuclease; kinases,
e.g., T4 polynucleotide kinase; phosphotases, e.g., BAP and CIP
phosphotase; nucleases, e.g., BL31 nuclease, and XO nuclease; and
RNA-modifying enzymes such as E coli RNA polymerase, SP6, T7, T3
RNA polymerase, and T4 RNA ligase. Lithium chloride, guanidine,
sodium salicylate, sodium perchlorate, and sodium thiocyanate have
been found to be particularly effective. The amount of the chelator
enhancing component is generally in the range of from about 0.1M to
2M, and more desirably the amount of chelator enhancing component
in the preservative solution is at least 1M.
[0051] The methods and preservatives of the invention have been
found to surprisingly increase the signal obtained with such
nucleic acid testing methods as the polymerase chain reaction
(PCR), LC.sub.X, and genetic transformation testing (GTT). The
invention has been found to surprisingly and unexpectedly enhance
hybridization in such nucleic acid testing methods such as the PCR.
FIG. 12 illustrates the improvement in hybridization obtained by
use of a preservative disclosed herein on the hybridization of
penicillinase-producing Neisseria gonorrhea (PPNG) DNA and PPNG-C
probe.
[0052] A further aspect of the invention relates to methods of
improving hybridization of nucleic acids, including contacting a
test nucleic acid with a nucleic acid preservative solution
comprising an amount of a divalent metal chelator selected from
ethylenediaminetetraacetic acid (EDTA),
ethylenebis(oxyethylenenitrilo)] tetraacetic acid (EGTA) and
1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA),
or salts thereof in the range of from about 0.001M to 0.1M; and an
amount of at least one chelator enhancing component selected from
lithium chloride, guanidine, sodium salicylate, sodium perchlorate,
and sodium thiocyanate in the range of from about 0.1M to 2M, such
that a test solution is formed; and contacting the test solution
with a target nucleic acid under conditions favorable for
hybridization, such that hybridization occurs.
[0053] FIGS. 13 and 14 further illustrate the efficacy of the
methods and preservatives of the invention in improving the results
obtained with nucleic acid testing methods, in this case, a
branched DNA assay (Chiron). In the tests run in FIG. 13, the bDNA
assay was used to assess the protective effect of the DNA/RNA
protect reagents. DNA sequences from the hepatitis C virus were
spiked into serum and plasma. The protected serum and plasma were
mixed with 9 ml of serum or plasma and 1 ml of preservative. The
following formulations were used: 1) 1M guanidine HCL/0.01M EDTA,
2) 1M sodium perchlorate/0.01M BAPTA, 3) 1M sodium
thiocyanate/0.01M EGTA, and 4) 1M lithium chloride/0.01M EGTA. The
formulations were stored for seven days at 4.degree. C. bDNA assay
relies on hybridization; it can clearly be seen from the absorbance
results that the target sequences were not only protected against
degradation, but the more than doubling of the absorbance results
indicates an enhancement of hybridization/annealing of the target
sequences.
[0054] FIG. 14 illustrates a serum v. plasma study. 50 .mu.l
samples of fresh human plasma, and 1 ml samples of fresh human
serum were protected with 1M guanidine HCL/0.01M EDTA and the bDNA
assay was run on these samples after the samples were stored at
20.degree. F. for 48 hours. Results were compared to unprotected
samples. It can clearly be seen from the absorbance results that
the target sequences were not only protected against degradation,
but the more than doubling of the absorbance results indicates an
enhancement of hybridization/annealing of the target sequences.
[0055] The preservative reagents of the invention have also
surprisingly been found to remove the interference with heme
compounds, e.g., methemoglobin, on PCR assays run on blood serum.
FIGS. 15 and 16 illustrate the improvement obtained by use of the
preservatives disclosed herein. Increasing amounts of methemoglobin
were spiked into unprotected fresh human serum, to a concentration
of 10 dl/ml. Serial PCR assays were run over a four hour
period.
[0056] FIG. 17 illustrates the surprising and synergistic effect
obtained by the combination of divalent metal chelators and
chelator enhancing components in the inventive reagent (i.e., 1M
sodium perchlorate/0.01M EGTA) in protecting hepatitis B sequences
in serum stored at room temperature and subsequently subjected to
MD03/06 PCR detection. The protocol run was as above (i.e., as
illustrated in FIG. 16.) It can be seen from the figures that
compared to the addition of EGTA or sodium perchlorate
individually, but protection of Hep B sequences is dramatically
increased when preservative solutions of the present invention are
used.
[0057] FIG. 18 illustrates the relatively weak preservative effect
on gonococcal DNA in urine stored at room temperature and
subsequently subjected to PCR detection offered by the individual
addition of components of the reagents of the present invention,
i.e., divalent metal chelators 0.01M BAPTA (18A), 0.01M EDTA (18B),
0.01M EGTA (18C); and chelator enhancing components 1M sodium
perchlorate (18D), 1M salicylic acid (18E), 1M guanidine HCl (18F),
1M sodium thiocyanate (18G), and lithium chloride (18H). The number
of transformants in ten types of urine specimens were tested using
a GTT, counted hourly, and then summarized. The standard Gonostat
protocol (see Example 2, infra) was employed. FIG. 19 illustrates
the synergistic effect obtained by the combination of divalent
metal chelators and chelator enhancing components in protecting
gonococcal DNA in urine stored at room temperature and subsequently
subjected to PCR detection.
[0058] Another embodiment of the invention, a method 10 for
preserving DNA, is illustrated diagrammatically in FIG. 11. This
embodiment uses an exemplary protocol to preserve and test the
urine specimens. The protocol is described in Table 1, below. This
system produces high yields of DNA/RNA suitable for such testing
methods as PCR, restriction fragment length polymorphisms assay
(RFLP), and nucleic acid probes from urine specimens.
1TABLE 1 1. 10 ml of clean catch urine 16 is added to a specimen
test tube 18 containing divalent metal chelator 12 and chelator
enhancing component 14. Test tube is inverted two or three times to
mix the urine. 2. Test tube is transported to laboratory. No
refrigeration is necessary. Note: The test tube should be stored in
a cool place and not in direct sunlight. 3. At the laboratory, the
test tube is centrifuged 20 at 3200 rpm for 10 minutes. 4. Using a
sterile transfer pipette, the pellet 22 at the bottom of the test
tube is transferred to another test tube containing buffer 24. (As
little urine as possible should be transferred with the pellet
material.) 5. The buffered material is stored 26 at between
2-8.degree. C. until ready to test 28. 6. The specimen size
necessary to run the assay needs to be validated on the individual
test methodology and individual testing protocol being used.
[0059] A test kit embodiment can advantageously be provided. For
example, a specimen test tube containing the preservative reagent
of the invention and a buffer test tube can be provided together
for laboratory use. Alternatively, the specimen test tube
containing the preservative reagent of the invention can be
provided to an individual patient with instructions for use. The
individual can then mail or bring the preserved sample to a
laboratory for testing.
[0060] Other aspects of the invention are further demonstrated and
illuminated by reference to the following examples, which are
intended to be non-limiting.
EXAMPLE 1
[0061] FIG. 5 is a bar graph of DNA concentration in preserved
urine in accordance with the invention. The number of transformants
in ten types of urine specimens were tested using a GTT, counted
hourly, and then summarized. The standard Gonostat protocol (see
Example 2, infra) was employed, and the preservative used was 1M
guanidine HCl/0.01M EDTA. A count of two hundred colonies
demonstrates total preservation of a specimen. The number of
gonococcal transformants in the preserved urine remained relatively
constant, approaching two hundred, throughout the four hours of the
test. No significant difference in level of preservation was
observed among the different types of urine specimens. Therefore,
it can be seen that the invention provides nearly total protection
for DNA in urine.
EXAMPLE 2
[0062] FIG. 6 is a graph of eight day GTT serial data on preserved
urine according to the invention. 1 pg of gonococcal DNA was spiked
into 9 ml of fresh human urine and 1 ml of aqueous preservative
containing 1M sodium perchlorate and 0.01M EGTA. 300 .mu.l was
spotted onto a lawn of the Gonostat organism at 24 hour intervals
for eight days. The plates contained BBL Chocolate II agar and were
incubated at 37.degree. C. for 24 hours before readings were taken.
The number of colonies observed throughout the eight-day testing
period ranged from a low count of one hundred eighty-eight to a
high count of one hundred ninety-seven. Thus, it can be seen that
the invention preserves DNA in urine for a significantly longer
period of time than previously provided.
EXAMPLE 3
[0063] FIG. 7 is a graph comparing PCR results in unpreserved and
preserved normal urine according to the invention. A MOMP template
to Chlamydia trachomatis was used and amplified using a standard
PCR protocol. 200 copies of the MOMP target were spiked into 9 ml
of fresh human urine containing 1 M sodium perchlorate and 0.01M
BAPTA. PCR was done each hour for eight hours total. In the
unprotected urine, approximately three PCR absorbances were
measured one hour after the addition of DNA to the urine. The
number of PCR absorbances approached zero by the sixth hour. By
contrast, in the preserved specimen, in excess of three PCR
absorbances were measured at the one hour testing. However,
approximately three PCR absorbances were still observed by the
sixth hour. Therefore, the invention preserves sufficient DNA and
nucleic acid sequences to permit PCR testing well beyond the
testing limits of unpreserved urine. The results shown in the
Figure are consistent for all types of DNA in a urine specimen.
EXAMPLE 4
[0064] The reagents and methods of the invention may be used for
preserving other bodily fluids and excretions, such as blood serum.
FIG. 8 is a graph of eight day serial data on preserved serum
according to the invention. The protocol used was similar to
Example 3, except fresh human serum was used. The number of
transformant colonies observed throughout the eight-day testing
period ranged from a high count of one hundred ten at the one day
measurement to a low count of approximately ninety-two at the seven
day measurement. In fact, the test results actually showed an
increase in transformant colonies between days seven and eight.
Thus, it can be seen that the invention preserves DNA in serum for
a significantly longer period of time than previously
attainable.
EXAMPLE 5
[0065] FIG. 9 is a graph of DNA concentration in preserved serum
according to the invention. The serum was preserved with
preservative solution comprising 1M guanidine HCl/0.01M EDTA. The
protocol used was similar to Example 3, except fresh human serum
was used, and the duration time of the study was ten hours. In
excess of 120 transformants were measured at the time gonococcal
DNA was added to the serum. Approximately 100 transformants were
counted at the six hour measurement. However, by the tenth hour,
testing indicated that the concentration of biologically active DNA
in the preserved serum had increased to approximately 110
transformant colonies.
EXAMPLE 6
[0066] Preservation of DNA in Simulated Clinical Specimens
[0067] In the following experiment, simulated clinical urine
specimens were produced and tested for the presence of gonococcal
DNA. The chemicals listed in Table 2, below, were added, at the
concentrations previously described, to urine specimens from
healthy adults, as was EDTA.
[0068] A suspension of gonococci was immediately added to each
urine specimen. The added gonococci were an ordinary strain of N.
Gonorrhoeae, 49191, which was grown overnight on GC agar medium at
37.degree. C. in a 5% CO.sub.2 atmosphere. The N. Gonorrhoeae
colonies were picked and suspended in GC buffer. A {fraction
(1/10)} volume of a suspension containing approximately 10 Colony
forming units (cfu) per ml was added to the urine. As a positive
control, the suspension of gonococci was also added to Hepes
buffer.
[0069] All simulated clinical specimens and the Hepes controls were
tested at time zero, i.e., when the chemicals and gonococci were
added. The specimens and controls were also tested after storage at
room temperature for six days. This six day period was selected to
approximate the maximum time expected between collecting, mailing,
and testing patient specimens.
[0070] With the exception of urine samples containing SDS and
sarkosyl, the simulated specimens and Hepes controls were processed
as follows:
[0071] 1. A 10 ml quantity was centrifuged at 4000 rpm for 30
minutes.
[0072] 2. The supernatant was decanted, and the pellet was
suspended in 1 ml PO.sub.4 buffer.
[0073] 3. The suspension was heated for 10 minutes in a water bath
at 60.degree. C.
[0074] 4. After cooling, the suspension was used in the GTT.
[0075] The simulated urine specimens containing SDS-EDTA or
sarkosyl-EDTA were processed as follows:
[0076] 1. Approximately a 21/2 volume (approximately 25 ml) of 95%
ethyl alcohol was added to the tube with the urine and
preservative. The contents were mixed by inverting the tube several
times.
[0077] 2. The mixture was centrifuged at 4000 rpm for 30
minutes.
[0078] 3. The pellet was suspended in 10 ml of 70% alcohol and
centrifuged.
[0079] 4. The pellet was then suspended in 1 ml PO.sub.4
buffer.
[0080] 5. The suspension was heated for 10 minutes in a water bath
at 60.degree. C.
[0081] 6. After cooling, the suspension was used in the GTT.
[0082] The inoculated urine was stored at room temperature for 6
days prior to testing.
[0083] The formulations that preserved (+) or did not preserve (-)
gonococcal DNA in the inoculated urine for six days to
approximately the same degree as in the Hepes buffer control are
indicated. Although the results of the Gonostat.RTM. assay can be
semi-quantitated, the tests were not designed to rank the relative
efficacy of the chemical preservatives. Thus, the results given in
Table 2 indicate whether or not the particular chemical preserved
DNA in urine over a six day period to same degree as in the Hepes
buffer.
2TABLE 2 Preservative + - 0.01 M EDTA + Guanidine hydrochloride
(1M) Sodium periodate (1M) 0.01 M EDTA + Guanidine thiocyanate (1M)
Trichloroacetic acid (1M) 0.01 M EDTA + Lithium chloride (1M) Urea
(1M) 0.01 M EDTA + Manganese chloride (1M) 0.01 M EDTA + Sarkosyl
(1%) 0.01 M EDTA + Sodium dodecyl sulfate (SDS) (1%) 0.01 M EDTA +
Sodium perchlorate (1M) 0.01 M EDTA + Sodium salicylate (1M) 0.01 M
EDTA + Sodium thiocyanate (1M)
[0084] The 92% sensitivity exhibited with male urine specimens is
comparable to the culture results reported in the literature. In
addition, the 88% sensitivity exhibited with female urine specimens
exceeds the previously-reported levels.
[0085] While a preferred embodiment of the invention is directed to
the preservation of gonococcal DNA, it will be readily apparent to
one skilled in the art that the invention is adaptable for use in
preserving other types of DNA, such as that of Haemophilus
influenzae and Bacillus subtilis. The invention can also be used to
preserve RNA contained in bodily fluid samples. Such preserved RNA
can be used for RNA transcriptase and reverse transcriptase assays
for viral segments and human gene sequence testing.
[0086] Furthermore, although in the preferred embodiment the
preservatives are added to a bodily fluid, e.g., a urine specimen,
the urine specimen can also be added to the preservatives without
detriment to the efficacy of the invention. Optimal preservation of
the DNA is typically and conveniently achieved by adding a single
reagent of the invention to the specimen.
EXAMPLE 7
[0087] PCR Detection of Penicillinase-Producing Neisseria
gonorrhea
[0088] The PCR signal-enhancing effect of the preservative reagents
of the disclosure is demonstrated by the following example. Four
varieties of TEM-encoding plasmids are found in PPNG. These are the
6.7 kb (4.4 Mda) Asian type, the 5.1 kb (3.2 Mda) African type, the
4.9 kb (3.05-Mda) Toronto type and the 4.8 kb (2.9-Mda) Rio Type.
This PCR assay for PPNG takes advantage of the fact that the TEM-1
gene is located close to the end of the transposon Tn2; by the use
of one primer in the TEM-1 gene and the other in a sequence beyond
the end of Tn2, and common to all four plasmids, a PCR product only
from plasmids and not from TEM-1 encoding plasmids was obtained.
(Table 3, below) The conditions associated with this protocol were
modified to include the DNA/RNA protect reagent in the
hybridization and the treated probe was mixed with the 761-bp
amplification product per standard PCR protocol. The results were
read at A.sub.450nm.
[0089] Materials and Reagents:
[0090] BBL chocolate II agar plates
[0091] Sterile Tris Buffer 10 mM Tris (pH 7.4), 1 mM EDTA
[0092] 0.5-ml Gene Amp reaction tubes
[0093] Sterile disposable pasteur pipette tips
[0094] Aerosol-resistant tips
[0095] PCR master mix:
[0096] 50 mM KCL
[0097] 2 mM MgCl
[0098] 50 .mu.M each of
[0099] Deoxyribonucleoside triphosphate;
[0100] 2.5 U of taq Polymerase (Perkin Elmer);
[0101] 5% glycerol;
[0102] 50 pmol each of primers PPNG-L and PNG-R (per 100 .mu.l
reaction)
[0103] Denaturation solution
[0104] 1M Na 5.times.Denhardt's solution
[0105] Prehybridization Solution
[0106] 5.times.SSC(1.times.SSc is 0.015M NaCl plus 0.015M sodium
citrate);
[0107] 5.times.Denhardt's solution;
[0108] 0.05% SDS;
[0109] 0.1% Sodium Ppi, and
[0110] 100 .mu.g of sonicated salmon sperm DNA per ml.
[0111] Hybridization Solution
[0112] Same as prehybridization solution but without Denhardt's
solution and including 200 .mu.l of DNA/RNA protect reagent 1.
[0113] 1 ml DNA/RNA preservative (1M guanidine HCl/0.01M EDTA)
[0114] Avidin-HRP peroxidase complex (Zymed)
[0115] Magnetic microparticles (Seradyne)
3TABLE 3 Function Name Nucleotide sequence 5' to 3' Primer PPNG-L
AGT TAT CTA CAC GAC GG Primer PPNG-R GGC GTA CTA TTC ACT CT Probe
PPNG-C GCG TCA GAC CCC TAT CTA TAA ACT C
[0116] Methods
[0117] Sample preparation: 2 colonies were picked from a chocolate
agar plate. Colonies were suspended in DI water just prior to
setting up PCR. The master mix was prepared according to the recipe
above. 5 .mu.l of the freshly prepared bacterial suspension was
added to 95 .mu.l of master mix. The DNA was liberated and
denatured in a thermocycler using three cycles of 3 min at
94.degree. and 3 min at 55.degree.. The DNA was amplified in the
thermal cycler by using a two step profile: a 25 s denaturation at
95.degree. C. and a 25 s annealing at 55.degree. C. for a total of
thirty cycles. The time was set between the two temperature
plateaus to enable the fastest possible anealing between the two
temperatures. 15 pmol of labeled (avidin-HRP complex) detection
probe PPNG-C was added to the hybridization solution bound to
magnetic micro particles with and without the preservative reagent
at 37.degree. C. for 1 hour. The control and treated probes were
then added to the amplification product and the reaction was
colorimetrically detected at A.sub.450nm. The signal obtained from
the hybridization probes treated with a reagent of the invention
was found to be significantly higher than the untreated probes.
[0118] EQUIVALENTS
[0119] Those skilled in the art will recognize, or be able to
ascertain using no more than routine experimentation, numerous
equivalents to the specific procedures described herein. Such
equivalents are considered to be within the scope of this invention
and are covered by the following claims. The contents of all
references, issued patents, and published patent applications cited
throughout this application are hereby incorporated by reference.
Sequence CWU 1
1
3 1 17 DNA Homo sapiens 1 agttatctac acgacgg 17 2 17 DNA Homo
sapiens 2 ggcgtactat tcactct 17 3 25 DNA Homo sapiens 3 gcgtcagacc
cctatctata aactc 25
* * * * *