U.S. patent application number 15/251507 was filed with the patent office on 2016-12-15 for selective detection of neisseria meningitis.
The applicant listed for this patent is The United States of America, as represented by the Secretary, Department of Health and Human Serv, The United States of America, as represented by the Secretary, Department of Health and Human Serv. Invention is credited to Jennifer Thomas.
Application Number | 20160362728 15/251507 |
Document ID | / |
Family ID | 45928498 |
Filed Date | 2016-12-15 |
United States Patent
Application |
20160362728 |
Kind Code |
A1 |
Thomas; Jennifer |
December 15, 2016 |
SELECTIVE DETECTION OF NEISSERIA MENINGITIS
Abstract
Provided are reagents and methods for detecting and
distinguishing Neisseria meningitidis from other infectious agents.
A kit is provided for detecting and quantifying Neisseria
meningitidis in a sample.
Inventors: |
Thomas; Jennifer; (Atlanta,
GA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
The United States of America, as represented by the Secretary,
Department of Health and Human Serv |
Rockville |
MD |
US |
|
|
Family ID: |
45928498 |
Appl. No.: |
15/251507 |
Filed: |
August 30, 2016 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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13816903 |
Jun 13, 2013 |
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PCT/US11/55784 |
Oct 11, 2011 |
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15251507 |
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61391493 |
Oct 8, 2010 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C12Q 1/689 20130101 |
International
Class: |
C12Q 1/68 20060101
C12Q001/68 |
Goverment Interests
GOVERNMENT INTEREST
[0002] The invention described herein may be manufactured, used,
and licensed by or for the United States Government.
Claims
1. A kit for detecting Neisseria meningitidis infection comprising:
a forward primer consisting of sequence SEQ ID NO: 1; a reverse
primer consisting of SEQ ID NO: 2; and a probe comprising a
fluorescent label.
2. The kit of claim 1 wherein said probe comprises the sequence SEQ
ID NO: 3.
3. An isolated oligonucleotide comprising the sequence selected
from the group consisting of SEQ ID NO: 3 and a fluorescent label.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application is a divisional of U.S. patent application
Ser. No. 13/816,903 filed Feb. 13, 2013, which is a U.S. national
phase of PCT/US2011/055784 filed Oct. 11, 2011 and which depends
from and claims priority to U.S. Provisional Patent Application No.
61/391,493 filed Oct. 8, 2010, the entire contents of each of which
are incorporated herein by reference.
FIELD
[0003] This invention relates generally to processes for detection
of foreign organisms in fluid samples. More specifically, the
invention relates to selective detection of Neisseria meningitidis
in biological or other fluid media. Processes are described for
rapid and sensitive detection of N. meningitidis in biological
samples and quantification thereof. Diagnostic kits are provided
for detection of N. meningitidis in a clinical, laboratory, or
field setting.
BACKGROUND
[0004] Neisseria meningitidis (Nm) is the etiologic agent of
epidemic bacterial meningitis and rapidly fatal sepsis throughout
the world. Rapid detection of Nm infection and early treatment
initiation are essential to positive outcomes in patients.
Techniques commonly employed for the identification of Nm include
biochemical tests, slide agglutination serogrouping (SASG), and the
polymerase chain reaction (PCR) Coreless, C E, et al., J Clin
Microbiol, 2001; 39:1553-8; Jordens, J. Z., and J. E. Heckels, J
Med Microbiol, 2005; 54:463-6; Mothershed, E. A. et al., J Clin
Microbiol, 2004; 42:320-8; Taha, M. K., J Clin Microbiol, 2000;
38:855-7. The chromogenic tests and SASG can be subjective, which
often complicates species identification.
[0005] ctrA may be the most frequently targeted gene to detect Nm
using PCR. Taha, M. et al., J Clin Microbiol, 2005; 43:144-9.
However, the capsule locus, including ctrA, is subject to
rearrangement, and 16% or more of carried meningococci have been
shown to lack ctrA altogether. Invasive NG meningococci can undergo
similar rearrangements of the capsule region (J. Dolan Thomas,
unpublished data), although these events may be less common than in
carriage isolates.
[0006] Thus, there is a need for compositions and methods to detect
of meningococci, especially of carriage isolates that may be
ctrA-negative and NG.
SUMMARY
[0007] The following summary of the invention is provided to
facilitate an understanding of some of the innovative features
unique to the present invention and is not intended to be a full
description. A full appreciation of the various aspects of the
invention can be gained by taking the entire specification, claims,
drawings, and abstract as a whole.
[0008] Processes and materials are provided for the detection of
the presence or absence of Neisseria meningitidis in a sample. A
process illustratively includes producing an amplification product
by amplifying a Neisseria meningitidis nucleotide sequence using a
forward primer that hybridizes to a region within the sodC gene of
Neisseria meningitidis, a reverse primer that hybridizes to a
region within the sodC gene of Neisseria meningitidis, and
optionally a probe that hybridizes to a region within the sodC gene
of Neisseria meningitidis under conditions suitable for a
polymerase chain reaction; and detecting the amplification product
to detect the Neisseria meningitidis in said sample. A forward
primer optionally includes SEQ ID NO: 1. A reverse primer
optionally includes SEQ ID NO: 2. A probe is optionally labeled. A
probe optionally includes SEQ ID NO: 3.
[0009] The probe is hybridized to an amplification product under
conditions suitable for a polymerase chain reaction so as to
produce a first detection signal. The detection of an amplification
optionally diagnoses Neisseria meningitidis infection in a subject
from which the sample is derived. The absence of an amplification
product or a first amplification signal optionally diagnoses the
absence of Neisseria meningitidis infection in a subject from which
the sample is derived.
[0010] One or more controls are optionally analyzed. Optionally,
the first detection signal is compared to a second detection
signal, wherein the second detection signal results from detection
of a complementary amplification product produced from a control
sample. Optionally, the complementary amplification product is
generated by PCR amplification of a purified Neisseria
meningitidis, or portion thereof, or from a nucleic acid
calibrator. A second detection signal, or a third detection signal
derived from a nucleic acid calibrator are optionally generated in
parallel with the first detection signal. A nucleic acid calibrator
is optionally extracted in parallel to said sample. A nucleic acid
calibrator is optionally a known amount of Neisseria meningitidis
sodC nucleic acid sequence and a known amount of a medium similar
to the sample.
[0011] Also provided are kits for detecting Neisseria meningitidis
infection including a first forward primer with sequence SEQ ID NO:
1, a first reverse primer with SEQ ID NO: 2, and a probe. The probe
optionally has the sequence SEQ ID NO: 3.
[0012] Also provided are isolated oligonucleotides suitable for use
in detecting the presence or absence of a sodC nucleic acid
sequence in a sample. An oligonucleotide optionally is or includes
the sequence of SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3.
DETAILED DESCRIPTION
[0013] The following description is merely exemplary in nature and
is in no way intended to limit the scope of the invention, its
application, or uses, which may, of course, vary. The description
is presented with relation to the non-limiting definitions and
terminology included herein. These definitions and terminology are
not designed to function as a limitation on the scope or practice
of the invention but are presented for illustrative and descriptive
purposes only. While the process is described as an order of
individual steps or using specific materials, it is appreciated
that described steps or materials may be interchangeable such that
the description of the invention includes multiple parts or steps
arranged in many ways as is readily appreciated by one of skill in
the art.
[0014] The invention has utility for the detection of Neisseria
meningitidis (Nm) in a sample. As it is necessary to detect small
numbers of Nm in clinical specimens due to bacterial loads in
cerebrospinal fluid (CSF) of patients ranging from 3.times.10.sup.1
to 4.times.10.sup.9 CFU/mL, sensitive techniques such as PCR may
provide a more reliable diagnostic than other currently employed
assay systems. Unlike chromogenic tests and SASG, PCR does not
require viable bacteria and can be used to identify and
characterize even nongroupable (NG) meningococci. Real-time PCR
(rt-PCR) of the ctrA gene is capable of detecting as few as 8
meningococcal genomes per reaction (17, 26) and results are
obtained within 2.5 hours. Unfortunately, prior attempts at
detection of Nm using PCR techniques are incapable of detecting all
strains due to selection pressure resulting in false negatives in
samples. Among the thousands of possible candidates, the inventors
discovered that the sodC gene is less susceptible to mutation due
to bacterial selection pressures and is present in all strains
tested. Further, sodC presents a selective target as it is not
present in other Neisseria spp. Thus, the invention has superior
utility over prior methods of detection of Nm in samples and
diagnosis of bacterial meningitis in a subject.
[0015] The [Cu, Zn]-cofactored superoxide dismutase gene, sodC, is
located 1.23 Mb from the capsule locus in the 2.27-Mb Nm serogroup
B strain MC58 genome and encodes the virulence factor Cu, Zn Sod.
Cu, Zn Sod is a periplasmic enzyme, making it theoretically less
susceptible to antigenic variation due to selective pressure than a
cell-surface exposed molecule. sodC is believed to have been
acquired by Nm via horizontal transfer from Haemophilus influenzae
(Hi), but the inventors find no cross-reactivity between Nm and Hi
using the inventive sodC assay.
[0016] Compositions and methods are provided for the sensitive
detection of Nm in samples, such as biological or environmental
samples, using techniques involving PCR. Primers are provided that
amplify regions of sodC from Nm with high specificity and broad Nm
recognition that are subsequently detectable, optionally by
sensitive detection systems.
[0017] In some aspects, sodC is used to define a consensus sequence
for Neisseria meningitidis sodC obtained from meningococcal strains
Z2491 (nts 1521721-1522258), FAM18, and MC58 (respective GenBank
accession numbers AL157959.1, AM421808.1, and AE002098.2).
[0018] The following definitional terms are used throughout the
specification without regard to placement relative to these
terms.
[0019] As used herein, the term "variant" defines either a
naturally occurring genetic mutant of the sodC gene or gene
products of Nm, or a recombinantly prepared variation of the sodC
gene or gene products of Nm, each of which contain one or more
mutations in its sodC gene compared to the sequence of one or more
of Genbank accession nos. AL157959.1, AM421808.1, or AE002098.2.
The term "variant" may also refer to either a naturally occurring
variation of a given peptide or a recombinantly prepared variation
of a given peptide or protein in which one or more amino acid
residues have been modified by amino acid substitution, addition,
or deletion.
[0020] As used herein, the term "analog" in the context of a
non-proteinaceous analog defines a second organic or inorganic
molecule that possesses a similar or identical function as a first
organic or inorganic molecule and is structurally similar to the
first organic or inorganic molecule.
[0021] As used herein, the term "derivative" in the context of a
non-proteinaceous derivative defines a second organic or inorganic
molecule that is formed based upon the structure of a first organic
or inorganic molecule. A derivative of an organic molecule
includes, but is not limited to, a molecule modified, e.g., by the
addition or deletion of a hydroxyl, methyl, ethyl, carboxyl or
amine group. An organic molecule may also be esterified, alkylated
and/or phosphorylated. A derivative also defined as a degenerate
base mimicking a C/T mix such as that from Glen Research
Corporation, Sterling, Va., illustratively LNA-dA or LNA-dT, or
other nucleotide modification known in the art or otherwise.
[0022] As used herein, the term "mutant" defines the presence of
mutations in the nucleotide sequence of an organism as compared to
a wild-type organism. A mutant is a variant.
[0023] The description of a target nucleic acid molecule is
presented herein as sodC. In some aspects, sodC specifically
includes variants, analogues, derivatives, and mutants of sodC. In
some aspects, sodC specifically excludes variants, analogues,
derivatives, and mutants of sodC and is, therefore, directed to
only sequences of sodC found in nature.
[0024] A "purified" nucleic acid molecule is one that is separated
from other nucleic acid molecules that are present in the natural
source of the nucleic acid molecule and is often substantially free
of other cellular material, or culture medium when produced by
recombinant techniques, or substantially free of chemical
precursors or other chemicals when chemically synthesized. This
term is exclusive of a nucleic acid that is a member of a library
that has not been purified away from other library clones
containing other nucleic acid molecules.
[0025] As used herein, the term "hybridizes under stringent
conditions" describes conditions for hybridization and washing
under which nucleotide sequences having at least 30%, 35%, 40%,
45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or more,
base pair matches to each other typically remain hybridized to each
other. Illustrative hybridization conditions are described in, for
example but not limited to, Current Protocols in Molecular Biology,
John Wiley & Sons, N.Y. (1989), 6.3.1 6.3.6.; Basic Methods in
Molecular Biology, Elsevier Science Publishing Co., Inc., N.Y.
(1986), pp. 75 78, and 84 87; and Molecular Cloning, Cold Spring
Harbor Laboratory, N.Y. (1982), pp. 387 389, and are well known to
those skilled in the art. A non-limiting example of stringent
hybridization conditions is hybridization in 6.times. sodium
chloride/sodium citrate (SSC), 0.5% SDS at about 60.degree. C.
followed by one or more washes in 2.times.SSC, 0.5% SDS at room
temperature. Another non-limiting example of stringent
hybridization conditions is hybridization in 6.times.SSC at about
45.degree. C. followed by one or more washes in 0.2.times.SSC, 0.1%
SDS at 50 to 65.degree. C. Other stringent hybridization conditions
will be evident to one of ordinary skill in the art based on
general knowledge in the art as well as this specification.
[0026] An "isolated" or "purified" nucleotide or oligonucleotide
sequence is substantially free of cellular material or other
contaminating proteins from the cell or tissue source from which
the nucleotide is derived, or is substantially free of chemical
precursors or other chemicals when chemically synthesized. The
language "substantially free of cellular material" includes
preparations of a nucleotide/oligonucleotide in which the
nucleotide/oligonucleotide is separated from cellular components of
the cells from which it is isolated or produced. Thus, a
nucleotide/oligonucleotide that is substantially free of cellular
material includes preparations of the nucleotide having less than
about 30%, 20%, 10%, 5%, 2.5%, or 1%, (by dry weight) of
contaminating material. When nucleotide/oligonucleotide is produced
by chemical synthesis, it is optionally substantially free of
chemical precursors or other chemicals, i.e., it is separated from
chemical precursors or other chemicals which are involved in the
synthesis of the molecule. Accordingly, such preparations of the
nucleotide/oligonucleotide have less than about 30%, 20%, 10%, 5%
(by dry weight) of chemical precursors or compounds other than the
nucleotide/oligonucleotide of interest. In some aspects of the
present invention, a nucleotide/oligonucleotide is isolated or
purified.
[0027] As used herein, the term "sample" is a portion of a larger
source. A sample is optionally a solid, gaseous, or fluidic sample.
A sample is illustratively an environmental or biological sample.
An environmental sample is illustratively, but not limited to,
water, sewage, soil, or air. A "biological sample" is as sample
obtained from a biological organism, a tissue, cell, cell culture
medium, or any medium suitable for mimicking biological conditions.
Non-limiting examples include, saliva, gingival secretions,
cerebrospinal fluid, gastrointestinal fluid, mucous, urogenital
secretions, synovial fluid, blood, serum, plasma, urine, cystic
fluid, lymph fluid, ascites, pleural effusion, interstitial fluid,
intracellular fluid, ocular fluids, seminal fluid, mammary
secretions, and vitreal fluid, and nasal secretions, throat or
nasal materials. In some aspects, target agents are contained in:
CSF; serum; whole blood; throat fluid; nasopharyngeal fluid; or
other respiratory fluid.
[0028] As used herein, the term "medium" refers to any liquid or
fluid sample in the presence or absence of a bacterium. A medium is
illustratively a solid sample that has been suspended, solubilized,
or otherwise combined with fluid to form a fluidic sample.
Non-limiting examples include buffered saline solution, cell
culture medium, acetonitrile, trifluoroacetic acid, combinations
thereof, or any other fluid recognized in the art as suitable for
combination with bacteria or other cells, or for dilution of a
biological sample or amplification product for analysis.
[0029] To determine the percent identity of two nucleic acid
sequences, the sequences are aligned for optimal comparison
purposes (e.g., gaps can be introduced in the sequence of a first
amino acid or nucleic acid sequence for optimal alignment with a
second amino acid or nucleic acid sequence). The nucleotides at
corresponding nucleotide positions are then compared. When a
position in the first sequence is occupied by the same nucleotide
as the corresponding position in the second sequence, then the
molecules are identical at that position. The percent identity
between the two sequences is a function of the number of identical
positions shared by the sequences (i.e., % identity=number of
identical overlapping positions/total number of positions
.times.100%). In some aspects, the two sequences are the same
length.
[0030] The determination of percent identity between two sequences
can also be accomplished using a mathematical algorithm. A
non-limiting example of a mathematical algorithm utilized for the
comparison of two sequences is the algorithm of Karlin and
Altschul, 1990, PNAS 87:2264 2268, modified as in Karlin and
Altschul, 1993, PNAS. 90:5873 5877. Such an algorithm is
incorporated into the NBLAST and XBLAST programs of Altschul et
al., 1990, J. Mol. Biol. 215:403. BLAST nucleotide searches are
performed with the NBLAST nucleotide program parameters set, e.g.,
for score=100, wordlength=12 to obtain nucleotide sequences
homologous to a nucleic acid molecules of the present invention.
BLAST protein searches are performed with the XBLAST program
parameters set, e.g., to score 50, wordlength=3 to obtain amino
acid sequences homologous to a protein molecule of the present
invention. To obtain gapped alignments for comparison purposes,
Gapped BLAST are utilized as described in Altschul et al., 1997,
Nucleic Acids Res. 25:3389 3402. Alternatively, PSI BLAST is used
to perform an iterated search which detects distant relationships
between molecules (Id.). When utilizing BLAST, Gapped BLAST, and
PSI Blast programs, the default parameters of the respective
programs (e.g., of XBLAST and NBLAST) are used (see, e.g., the NCBI
website). Another non-limiting example of a mathematical algorithm
utilized for the comparison of sequences is the algorithm of Myers
and Miller, 1988, CABIOS 4:11 17. Such an algorithm is incorporated
in the ALIGN program (version 2.0) which is part of the GCG
sequence alignment software package. When utilizing the ALIGN
program for comparing amino acid sequences, a PAM120 weight residue
table, a gap length penalty of 12, and a gap penalty of 4 is
used.
[0031] The percent identity between two sequences is determined
using techniques similar to those described herein or otherwise
known in the art, with or without allowing gaps. In calculating
percent identity, typically only exact matches are counted.
[0032] As used herein, the terms "subject" and "patient" are
synonymous and refer to a human or non-human animal, optionally a
mammal including a human, a non-primate such as cows, pigs, horses,
goats, sheep, cats, dogs, avian species and rodents; and a
non-human primate such as monkeys, chimpanzees, and apes; and a
human, also optionally denoted specifically as a "human
subject".
[0033] Processes are described that provide a rapid, specific, and
sensitive assay for detection of Nm in a sample by amplifying one
or more nucleotide sequences of the sodC gene by processes similar
to the polymerase chain reaction (PCR). Processes are similarly
provided for diagnosing the presence or absence of Nm infection in
a subject. The presence of Nm detected in a sample from the subject
diagnoses or confirms a prior diagnosis of infection of the subject
by Nm. The absence of Nm in a sample from a subject diagnoses the
absence of an infection of the subject by Nm.
[0034] An oligonucleotide forward primer with a nucleotide sequence
complementary to a unique sequence in a sodC nucleotide sequence
corresponding to the sodC sequence in one or more Nm is hybridized
to its complementary sequence and extended. A nucleotide sequence
is complementary if it hybridizes under stringent conditions.
Similarly, a reverse oligonucleotide primer complementary to a
second strand of Nm DNA in a separate sodC region is hybridized and
extended. This system allows for amplification of specific gene
sequences and is suitable for simultaneous or sequential detection
systems. It is appreciated that while the description is generally
directed to sequences of the sodC gene, or a Nm consensus sequence
thereof, that detection of mRNA encoding at least a portion of SodC
protein is equally detectable by the processes and compositions of
the inventions.
[0035] The present invention relates to the use of the sequence
information of Nm for diagnostic processes. In particular, the
present invention provides a process for detecting the presence or
absence of nucleic acid molecules of Nm, natural or artificial
variants, analogs, or derivatives thereof, in a sample. In some
aspects, processes involve obtaining a biological sample from one
or more of various sources and contacting the sample with a
compound or an agent capable of detecting a nucleic acid sequence
of sodC, natural or artificial variants, analogs, or derivatives
thereof, such that the presence of Nm, natural or artificial
variants, analogs, or derivatives thereof, is detected in the
sample. Optionally, infection by Neisseria meningitidis is
diagnosed by positively detecting one or more Nm in the sample. In
some aspects, the presence of Nm, natural or artificial variants,
analogs, or derivatives thereof, is detected in the sample using a
PCR reaction or real-time polymerase chain reaction (RT-PCR)
including primers that are constructed based on a partial
nucleotide sequence of the Nm organism. As sodC is present in both
Nm and H. influenzae, simple identification of primers such as by a
software program alone is insufficient for use in an inventive
assay. Primers must be designed to amplify sodC from the greatest
number of Nm strains while not amplifying sodC from H. influenzae
so as to prevent false positives. In a non-limiting aspect, a
forward primer designed to be successful for selective
amplification in a PCR based assay such as in a RT-PCR process is
illustratively 5'-GCACACTTAGGTGATTTACCTGCAT-3' (SEQ ID NO: 1). In
some aspects, a reverse primer designed to be successful for
selective amplification in a PCR based assay such as in a RT-PCR
process is illustratively 5'-CCACCCGTGTGGATCATAATAGA-3' (SEQ ID NO:
2). In some aspects, the primers used in a process are the nucleic
acid sequences of SEQ ID NOs: 1 and 2. As used herein, the term
"amplify" is defined as producing one or more copies of a target
molecule, or a complement thereof. A nucleic acid such as DNA or
RNA is amplified to produce one or more amplification products.
Illustratively, a forward primer and an optional reverse primer are
contacted with a target under conditions suitable for a polymerase
chain reaction to produce an amplification product.
[0036] An agent for detecting Neisseria meningitidis nucleic acid
sequences is a labeled nucleic acid probe capable of hybridizing to
a portion of the sodC gene, mRNA, or amplification products derived
therefrom. In some aspects, the nucleic acid probe is a nucleic
acid molecule of the nucleic acid sequence of
5'-CATGATGGCACAGCAACAAATCCTGTTT-3' (SEQ ID NO: 3), which
sufficiently specifically hybridizes under stringent conditions to
a Neisseria meningitidis nucleic acid sequence. A probe is
optionally labeled with a fluorescent molecule such as a
fluorescein (FAM) molecule and optionally a quencher such as the
black hole quencher BHQ1.
[0037] Primers are optionally used for the sequencing of the sodC
gene of an Nm. Illustratively, primers for PCR include a forward
primer 5'-CCTTATTAGCACTAGCGGTTAG-3' (SEQ ID NO: 4 and a reverse
primer 5'-CCGGTCATCTTTTATGCTCCAA-3' (SEQ ID NO: 5).
[0038] Processes optionally involve a real-time PCR assay (RT-PCR),
optionally, a real-time quantitative PCR assay. In some aspects,
the PCR assay is a TaqMan assay (Holland et al., PNAS 88(16):7276
(1991)). It is appreciated that the processes are amenable to
performance on other RT-PCR systems and protocols that use
alternative reagents illustratively including, but not limited to
Molecular Beacons probes, Scorpion probes, multiple reporters for
multiplex PCR, combinations thereof, or other DNA detection
systems.
[0039] The assays are performed on an instrument designed to
perform such assays, for example those available from Applied
Biosystems (Foster City, Calif.). In some aspects, a real-time
quantitative PCR assay is used to detect the presence of Nm,
natural or artificial variants, analogs, or derivatives thereof, in
a sample by subjecting the Nm nucleic acid from the sample to PCR
reactions using specific primers, and detecting the amplified
product using a probe. In some aspects, the probe is a TaqMan probe
which consists of an oligonucleotide with a 5'-reporter dye and a
3'-quencher dye.
[0040] A fluorescent reporter dye, such as FAM dye (illustratively
6-carboxyfluorescein), is covalently linked, optionally to the 5'
end of the oligonucleotide probe. Other dyes illustratively include
TAMRA, AlexaFluor dyes such as AlexaFluor 495 or 590, Cascade Blue,
Marina Blue, Pacific Blue, Oregon Green, Rhodamine, Fluorescein,
TET, HEX, Cy5, Cy3, and Tetramethylrhodamine. A reporter is
optionally quenched by a dye at the 3' end or other non-fluorescent
quencher. Quenching molecules are optionally suitably matched to
the fluorescence maximum of the dye. Any suitable fluorescent probe
for use in RT-PCR detection systems is illustratively operable in
the instant invention. Similarly, any quenching molecule for use in
RT-PCR systems is illustratively operable. In some aspects, a
6-carboxyfluorescein reporter dye is present at the 5'-end and
matched to BLACK HOLE QUENCHER (BHQ1, Biosearch Technologies, Inc.,
Novato, Calif.) The fluorescence signals from these reactions are
captured at the end of extension steps as PCR product is generated
over a range of the thermal cycles, thereby allowing the
quantitative determination of the bacterial load in the sample
based on an amplification plot.
[0041] The Neisseria meningitidis nucleic acid sequences are
optionally amplified before or simultaneous with being detected.
The term "amplified" defines the process of making multiple copies
of the nucleic acid from a single or lower copy number of nucleic
acid sequence molecule. The amplification of nucleic acid sequences
is carried out in vitro by biochemical processes known to those of
skill in the art, illustratively by PCR techniques. The
amplification agent may be any compound or system that will
function to accomplish the synthesis of primer extension products,
including enzymes. Suitable enzymes for this purpose include, for
example, E. coli DNA polymerase I, Taq polymerase, Klenow fragment
of E. coli DNA polymerase I, T4 DNA polymerase, AmpliTaq Gold DNA
Polymerase from Applied Biosystems, other available DNA
polymerases, reverse transcriptase (preferably iScript RNase H+
reverse transcriptase), ligase, and other enzymes, including
heat-stable enzymes (i.e., those enzymes that perform primer
extension after being subjected to temperatures sufficiently
elevated to cause denaturation). In some aspects, the enzyme is
hot-start iTaq DNA polymerase from Bio-rad (Hercules, Calif.).
Suitable enzymes will facilitate combination of the nucleotides in
the proper manner to form the primer extension products that are
complementary to each mutant nucleotide strand. Generally, the
synthesis is initiated at the 3'-end of each primer and proceed in
the 5'-direction along the template strand, until synthesis
terminates, producing molecules of different lengths. There may be
amplification agents, however, that initiate synthesis at the
5'-end and proceed in the other direction, using the same or
similar processes as described herein. In any event, the processes
of the invention are not to be limited to the aspects of
amplification described herein.
[0042] One process of in vitro amplification, which optionally is
used according to this invention, is the polymerase chain reaction
(PCR) described in U.S. Pat. Nos. 4,683,202 and 4,683,195. The term
"polymerase chain reaction" refers to a process for amplifying a
DNA base sequence using a heat-stable DNA polymerase and two
oligonucleotide primers, one complementary to the (+)-strand at one
end of the sequence to be amplified and the other complementary to
the (-)-strand at the other end. Because the newly synthesized DNA
strands can subsequently serve as additional templates for the same
primer sequences, successive rounds of primer annealing, strand
elongation, and dissociation produce rapid and highly specific
amplification of the desired sequence. Many polymerase chain
processes are known to those of skill in the art and may be used in
the process of the invention. For example, DNA is subjected to 30
to 35 cycles of amplification in a thermocycler as follows: 2
minutes at 50.degree. C., 10 minutes at 95.degree. C., and then
50.times. (15 seconds at 95.degree. C. plus 1 minute at 60.degree.
C.).
[0043] The primers for use in amplifying the mRNA or genomic DNA of
Nm may be prepared using any suitable process, such as conventional
phosphotriester and phosphodiester processes or automated aspects
thereof so long as the primers are capable of hybridizing to the
nucleic acid sequences of interest. One process for synthesizing
oligonucleotides on a modified solid support is described in U.S.
Pat. No. 4,458,066. The exact length of primer will depend on many
factors, including temperature, buffer, and nucleotide composition.
The primer must prime the synthesis of extension products in the
presence of the inducing agent for amplification.
[0044] Primers used according to the process of the invention are
complementary to each strand of nucleotide sequence to be
amplified. The term "complementary" means that the primers
hybridize with their respective strands under conditions, which
allow the agent for polymerization to function, such as stringent
hybridization conditions. In other words, the primers that are
complementary to the flanking sequences hybridize with the flanking
sequences and permit amplification of the nucleotide sequence.
Optionally, the 3' terminus of the primer that is extended is
perfectly (100%) base paired with the complementary flanking
strand. Probes optionally possess nucleotide sequences
complementary to one or more strands of the sodC gene of Nm.
Optionally, primers contain the nucleotide sequences of SEQ ID NOs:
1 and 2. It is appreciated that the complements of SEQ ID NOs: 1
and 2 are similarly suitable for use in the instant inventions. It
is further appreciated that oligonucleotide sequences that
hybridize with SEQ ID NOs 1 or 2 are also similarly suitable.
Finally, multiple positions are available for hybridization on the
sodC gene and will be also suitable hybridization with a probe when
used with the proper forward and reverse primers.
[0045] Those of ordinary skill in the art will know of various
amplification processes that can also be utilized to increase the
copy number of target Nm nucleic acid sequence. The nucleic acid
sequences detected in the process of the invention are optionally
further evaluated, detected, cloned, sequenced, and the like,
either in solution or after binding to a solid support, by any
process usually applied to the detection of a specific nucleic acid
sequence such as another polymerase chain reaction, oligomer
restriction (Saiki et al., BioTechnology 3:1008 1012 (1985)),
allele-specific oligonucleotide (ASO) probe analysis (Conner et
al., PNAS 80: 278 (1983)), oligonucleotide ligation assays (OLAs)
(Landegren et al., Science 241:1077 (1988)), RNase Protection
Assay, among others. Molecular techniques for DNA analysis have
been reviewed (Landegren et al, Science 242:229 237 (1988)).
Following DNA amplification, the reaction product may be detected
by Southern blot analysis, with or without using radioactive
probes. In such a process, for example, a small sample of DNA
containing the nucleic acid sequence obtained from the tissue or
subject is amplified, and analyzed via a Southern blotting
technique. The use of non-radioactive probes or labels is
facilitated by the high level of the amplified signal. In some
aspects of the invention, one nucleoside triphosphate is
radioactively labeled, thereby allowing direct visualization of the
amplification product by autoradiography. In some aspects,
amplification primers are fluorescently labeled and run through an
electrophoresis system. Visualization of amplified products is by
light detection followed by computer assisted graphic display,
without a radioactive signal.
[0046] Other methods of detection amplified oligonucleotide
illustratively include gel electrophoresis, mass spectrometry,
liquid chromatography, fluorescence, luminescence, gel mobility
shift assay, fluorescence resonance energy transfer, nucleotide
sequencing, enzyme-linked immunoadsorbent assay, affinity
chromatography, other chromatography methods, immunoenzymatic
methods (Ortiz, A and Ritter, E, Nucleic Acids Res., 1996;
24:3280-3281), streptavidin-conjugated enzymes, DNA branch
migration (Lishanski, A, et al., Nucleic Acids Res., 2000;
28(9):e42), enzyme digestion (U.S. Pat. No. 5,580,730),
colorimetric methods (Lee, K., Biotechnology Letters, 2003;
25:1739-1742), or combinations thereof. A detection signal is
produced that is related to the detection method employed, be it
RT-PCR or other detection method. A test sample optionally produces
a first detection signal upon amplification of a target. A control
sample optionally produces a second detection signal upon
amplification of a control molecule.
[0047] The term "labeled" with regard to the probe is intended to
encompass direct labeling of the probe by coupling (i.e.,
physically linking) a detectable substance to the probe, as well as
indirect labeling of the probe by reactivity with another reagent
that is directly labeled. Examples of indirect labeling include
detection of a probe using a fluorescently labeled antibody and
end-labeling or centrally labeling of a DNA probe with biotin such
that it can be detected with fluorescently labeled streptavidin.
The detection methods can be used to detect RNA (particularly
mRNA), genomic nucleic acid, or amplification products thereof, in
a sample in vitro as well as in vivo. For example, in vitro
techniques for detection of nucleic acid include northern
hybridizations, in situ hybridizations, reverse transcription-PCR,
real-time-PCR, and DNase protection. In vivo techniques for
detection of Nm include introducing into a subject organism a
labeled antibody directed against a polypeptide component or
directed against a particular nucleic acid sequence of Nm. For
example, the antibody can be labeled with a radioactive marker
whose presence and location in the subject organism can be detected
by standard imaging techniques, including autoradiography.
[0048] The size of the primers used to amplify a portion of the
nucleic acid sequence of Nm is at least 5, and often 10, 15, 20,
25, 30 or more nucleotides in length, optionally any value or range
between 5 and 30 nucleotides in length. Optionally, the GC ratio is
above 30%, 35%, 40%, 45%, 50%, 55%, or 60% so as to prevent
hair-pin structure on the primer. The amplicon is optionally of
sufficient length to be detected by standard molecular biology
methodologies. The forward primer is optionally shorter than the
reverse primer or vice versa. Techniques for modifying the T.sub.m
of either primer are operable herein. An illustrative forward
primer contains LNA-dA and LNA-dT (Glen Research Corporation) so as
to match T.sub.m with a corresponding alternate primer.
[0049] An inventive process uses a polymerization reaction which
employs a nucleic acid polymerizing enzyme, illustratively a DNA
polymerase, RNA polymerase, reverse transcriptase, or mixtures
thereof. It is further appreciated that accessory proteins or
molecules are present to form the replication machinery. A
polymerizing enzyme is optionally a thermostable polymerase or
thermodegradable polymerase. Use of thermostable polymerases is
well known in the art such as Taq polymerase available from
Invitrogen Corporation, Carlsbad, Calif. Thermostable polymerases
allow a polymerization reaction to be initiated or shut down by
changing the temperature other condition in the reaction mixture
without destroying activity of the polymerase.
[0050] Accuracy of the base pairing of DNA sequence amplification
is provided by the specificity of the enzyme. Error rates for Taq
polymerase tend to be false base incorporation of 10.sup.-5 or
less. (Johnson, Annual Reviews of Biochemistry, 1993: 62:685-713;
Kunkel, Journal of Biological Chemistry, 1992; 267:18251-18254).
Specific examples of thermostable polymerases illustratively
include those isolated from Thermus aquaticus, Thermus
thermophilus, Pyrococcus woesei, Pyrococcus furiosus, Thermococcus
litoralis and Thermotoga maritima. Thermodegradable polymerases
illustratively include E. coli DNA polymerase, the Klenow fragment
of E. coli DNA polymerase, T4 DNA polymerase, T7 DNA polymerase and
other examples known in the art. It is recognized in the art that
other polymerizing enzymes are similarly suitable illustratively
including E. coli, T7, T3, SP6 RNA polymerases and AMV, M-MLV, and
HIV reverse transcriptases.
[0051] The polymerases are optionally bound to the primer. When the
Nm sodC gene sequence is a single-stranded DNA molecule due to heat
denaturing, the polymerase is bound at the primed end of the
single-stranded nucleic acid at an origin of replication. A binding
site for a suitable polymerase is optionally created by an
accessory protein or by any primed single-stranded nucleic
acid.
[0052] In some aspects, detection of PCR products of sodC or sodC
nucleic acid sequence is achieved by mass spectrometry. Mass
spectrometry has several advantages over real-time PCR systems in
that it can be used to simultaneously detect the presence of Nm and
decipher mutations in target nucleic acid sequences allowing
identification and monitoring of emerging strains. Further, mass
spectrometers are prevalent in the clinical laboratory. Similar to
fluorescence based detection systems, mass spectrometry is capable
of simultaneously detecting multiple amplification products for a
multiplexed and controlled approach to accurately quantifying
components of biological or environmental samples.
[0053] Multiple mass spectrometry platforms are suitable for use in
the invention illustratively including matrix assisted laser
desorption ionization time of flight mass spectrometry (MALDI),
electrospray mass spectrometry, electrospray ionization-Fourier
transform ion cyclotron resonance mass spectrometry (ESI-FTICR),
multi-stage mass spectrometry fragmentation analysis (MS/MS), mass
spectrometry coupled with liquid chromatography such as high
performance liquid chromatography mass spectrometry (HPLC) and
ultra performance liquid chromatography isotope dilution tandem
mass spectrometry (UPLC-ID/MS/MS), and variations thereof.
[0054] It is appreciated that numerous other detection processes
are similarly suitable for measuring an amplification product by
detecting a detection signal. Illustrative examples include, but
are not limited to, liquid chromatography, mass spectrometry,
liquid chromatography/mass spectrometry, static fluorescence,
dynamic fluorescence, high performance liquid chromatography,
ultra-high performance liquid chromatography, enzyme-linked
immunoadsorbent assay, real-time PCR (RT-PCR), gel electrophoresis,
or combinations thereof.
[0055] Optionally, PCR amplification products are generated using
complementary forward and reverse oligonucleotide primers. In a
non-limiting example, Nm genetic sequences or fragments thereof are
amplified by the primer pair SEQ ID NOs: 1 and 2 that amplify a
conserved sequence in the sodC gene encompassing nucleotides
351-478. The resulting amplification product is either directly
detected such as by a probe, or is subsequently processed and
prepared for detection by processes known in the art. It is
appreciated that the complements of SEQ ID NOs: 1 and 2 are
similarly suitable for use in the invention. It is further
appreciated that oligonucleotide sequences that hybridize with SEQ
ID NOs: 1 or 2 are also similarly suitable. Finally, multiple
positions are available for hybridization on the Nm genome and in
the sodC gene, gene product, or other and will be also suitable
hybridization with forward and reverse primers that may or may not
be used with a probe for real-time PCR.
[0056] Optionally, multiple amplification products are
simultaneously produced in a PCR reaction that are then available
for simultaneous detection and quantification. Thus, multiple
detection signals are inherently produced or emitted that are
separately and uniquely detected in one or more detection systems.
It is appreciated that multiple detection signals are optionally
produced in parallel. Optionally, a single biological sample is
subjected to analysis for the simultaneous or sequential detection
of Nm genetic sequences. It is appreciated that three or more
independent or overlapping sequences are simultaneously or
sequentially measured in the inventive processes. Oligonucleotide
matched primers (illustratively SEQ ID NOs: 1 and 2) are
simultaneously or sequentially added and the biological sample, or
a portion thereof, is subjected to proper thermocycling reaction
parameters. For detection by mass spectrometry, a single sample of
the amplification products from each gene are simultaneously
analyzed allowing for rapid and accurate determination of the
presence of Nm. Optionally, analysis by real-time PCR is employed
capitalizing on multiple probes with unique fluorescent signatures.
Thus, each gene is detected without interference by other
amplification products. This multi-target approach increases
confidence in quantification and provides for additional internal
control.
[0057] In some aspects, the processes further involve optionally
obtaining a control sample from a control subject, contacting a
control sample, optionally from said subject, with a compound or
agent capable of detecting the presence of Nm nucleic acid in the
sample, and comparing the presence or absence of mRNA or DNA in the
control sample with the presence of mRNA or DNA in the test sample.
A control sample is optionally a portion of a test sample processed
in parallel with the test sample. A control sample is optionally a
purified, isolated, or otherwise processed nucleic acid sequence of
known concentration optionally including at least a portion of the
sodC sequence, where the nucleic acid sequence or portion thereof
will hybridize under stringent conditions with a forward primer, a
reverse primer, and, optionally, a probe. A control sample is used
to produce a complementary amplification product produced either
simultaneously with, or sequentially to the first amplification
product produced from a target. The complementary amplification
product is optionally detected by detecting a second detection
signal by the same of a different method than that used to detect
the first amplification product. Illustratively, a second
amplification product is detected using a second probe of the same
or of a different sequence than that use to detect the first
amplification product. A second probe optionally has one or more
labels that are the same or different than that of a first probe,
when present. Illustratively, a control sample is subjected to the
identical amplification conditions in the same or other parallel
analysis, such as on the same instrument, as the test sample. If
the test sample and the control sample are processed in different
reaction chambers, the same probes with the same labels may be
used.
[0058] Some aspects include using a nucleic acid calibrator to
produce a signal from a known quantity of sample molecule. A
nucleic acid calibrator is optionally identical to or different
from a target molecule. Amplification of a nucleic acid calibrator
optionally produces a third detection signal, the presence of
intensity of, or size of is optionally compared to a first
detection signal to quantify the amount of target, or amplification
product in the test sample. Optionally, a plurality of nucleic acid
calibrators are used. A plurality of nucleic acid calibrators are
optionally of differing concentrations such as those suitable to
produce a standard curve. The detection signal from the test sample
is optionally compared to the standard curve to quantify the amount
of amplification product or target in the test sample. A nucleic
acid calibrator optionally includes a known amount of Neisseria
meningitidis sodC nucleic acid sequence, or a portion of a
Neisseria meningitidis sodC nucleic acid sequence.
[0059] The invention also encompasses kits for detecting the
presence of Nm nucleic acids in a test sample. The kit, for
example, includes a labeled compound or agent capable of detecting
a nucleic acid molecule in a test sample and, in certain aspects,
for determining the quantity of Nm in the sample.
[0060] For oligonucleotide-based kits, the kit includes, for
example: (1) an oligonucleotide, e.g., a detectably labeled
oligonucleotide, which hybridizes to a nucleic acid sequence of Nm
and/or (2) one or a pair of primers (one forward and one reverse)
useful for amplifying a nucleic acid molecule containing at least a
portion the Nm sequence such as the sodC sequence. The kit can also
include, e.g., a buffering agent, a preservative, or a protein
stabilizing agent. The kit can also include components necessary
for detecting the detectable agent (e.g., an enzyme or a
substrate). The kit can also contain a control sample or a series
of control samples which is assayed and compared to the test sample
contained. Each component of the kit is usually enclosed within an
individual container and all of the various containers are usually
enclosed within a single package along with instructions for
use.
[0061] The processes are amenable to use for diagnosis of Nm
infection or simple detection of the presence of Nm in a subject,
such as insects, and any other organism capable of infection or
transfection by or with Nm.
[0062] To increase confidence and to serve as an internal or
external control, a purified solution containing Nm is used as a
sample. Optionally, by amplification of a single sample with known
quantities of Nm or of a set of samples representing a titration of
Nm, the level of Nm in the unknown biological sample is determined,
optionally as a control. Optionally, the purified and quantified Nm
solution is analyzed in parallel with the unknown biological sample
to reduce inter assay error or to serve as a standard curve for
quantitation of unknown Nm in the test sample. Using purified and
quantified Nm solution provides for a similar complete genetic base
DNA strand for amplification.
[0063] In some aspects, a subgenomic fragment is cloned into a
plasmid for amplification, purification, and use as a quantitative
comparator or nucleic acid calibrator. In a non-limiting example, a
DNA subgenomic fragment of Nm is optionally amplified from a
positive nasal swab using primers bracketing the RT-PCR target
regions in sodC of Nm. It is appreciated that other sequences are
similarly suitable for use as a quantitative control. The known
concentration of the subgenomic fragment is used to create a
standard curve for quantitative determinations and to access
amplification efficiency.
[0064] Also provided is a kit for detecting or diagnosing Nm
infection that contains reagents for the amplification, or direct
detection of Nm or portions thereof in a sample. An exemplary kit
optionally includes a forward and reverse primer pair, and a probe.
In some aspects, the forward and reverse primers have the
oligonucleotide sequence SEQ ID NOs: 1 and 2 and a probe of the
sequence SEQ ID NO 3. It is appreciated that a diagnostic kit
optionally contains primers and probes that are the complements of
SEQ ID NOs 1-3 or that hybridize with oligonucleotides SEQ ID NOs
1-3. It is further appreciated that a diagnostic kit optionally
includes ancillary reagents such as buffers, solvents, thermostable
polymerases, nucleotides, and other reagents necessary and
recognized in the art for amplification and detection of Nm in a
sample.
[0065] A kit for detection of Nm infection in a subject optionally
contains reagents for PCR based detection of Nm genetic sequences,
either structural or non-structural, and optionally for detection
of antibodies directed to Nm proteins. The components of the kits
are any of the reagents described above or other necessary and
non-necessary reagents known in the art for solubilization,
detection, washing, storage, or other need for in a diagnostic
assay kit.
[0066] Various aspects of the present invention are illustrated by
the following non-limiting examples. The examples are for
illustrative purposes and are not a limitation on any practice of
the present invention. It will be understood that variations and
modifications can be made without departing from the spirit and
scope of the invention. While the examples are generally directed
to samples derived from a human, a person having ordinary skill in
the art recognizes that similar techniques and other techniques
known in the art readily translate the examples to other organisms.
Reagents illustrated herein are commonly cross reactive between
mammalian species or alternative reagents with similar properties
are commercially available, and a person of ordinary skill in the
art readily understands where such reagents may be obtained.
Example 1
RT-PCR Assay Design
[0067] Control isolates for assay design and optimization are
provided in Table 1:
TABLE-US-00001 TABLE 1 Used in This Re- Isolated Isolate Study for
ceived From From M10481 sodC sequencing 2003 North Carolina, USA
Eye M16204 sodC sequencing 2007 Connecticut, USA Blood M12221 sodC
sequencing 2004 Kenya Unknown M12746 sodC sequencing 2004 Mali CSF
M10697 sodC sequencing 2003 Oregon, USA Blood M13169 sodC
sequencing 2005 Kentucky, USA Blood M12075 sodC sequencing 2001
Canada Blood M11880 sodC sequencing 2004 Georgia, USA CSF M12881
sodC sequencing 2004 Hawaii, USA Blood M11701 sodC sequencing 2004
South Africa Unknown M11986 sodC sequencing 2004 California, USA
Blood M14563 sodC sequencing 2006 Ohio, USA Blood M15470 sodC
sequencing 2006 Colorado, USA Blood M10949 sodC sequencing 2003
Pennsylvania, USA Sputum M5178 Primer & probe 1998 Oregon, USA
Blood optimization, standard curves M3702 Standard curves 1992 CDC
Reference Lab Unknown Surveillance Collection M18631 Standard
curves 2009 Oregon, USA Blood M18634 Standard curves 2009 Oregon,
USA Blood .sup.1 There is no Z or 29E PCR serogrouping assay
currently in use in the CDC Meningitis Laboratory. However, these
isolates tested negative by rt-PCR for serogroup A, B, C, W135, X,
and Y genes. .sup.2 CTA sugar results
[0068] Genomic DNA from each isolate was prepared for use in the
various steps of assay design and optimization with the QIAamp DNA
Mini Kit (QIAGEN, Valencia, Calif.) using Protocol C then
quantified for use in standard curve experiments using a NanoDrop
ND-1000 or 8000 spectrophotometer (Nanodrop Technologies,
Wilmington, Del.). Preparation of DNA from bacterial isolates was
performed as previously described by Mothershed, E A, et al., J
Clin Microbiol, 2004; 42:320-328.
[0069] The sodC sequencing templates were prepared by conventional
PCR using Expand High Fidelity Enzyme Mix (Roche Diagnostics GmbH)
per the manufacturer's instructions and forward and reverse primers
were designed based on a consensus of sodC from meningococcal
strains Z2491 (nts 1521721-1522258), FAM18, and MC58 (respective
GenBank accession numbers AL157959.1, AM421808.1, and AE002098.2),
and provided as SEQ ID NOs: 4 and 5 respectively. DNA sequencing
was performed using primers of SEQ ID NO: 4 (forward) and SEQ ID
NO: 5 (reverse) at 400 nM and 100 nM respectively using the BigDye
Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems, Foster
City, Calif.) and an ABI PRISM.RTM. 3130xl Genetic Analyzer
(Applied Biosystems) and a consensus sequence was generated using
Lasergene DNAStar v. 7 Program SeqMan.
[0070] Over the 439-473 out of 560 sodC nucleotides sequenced in
these meningococcal isolates, these meningococcal sodC sequences
are 99-100% were identical to each other and are only 81% identical
to an H. influenzae (Hi) sodC consensus that was built using two
sequences from GenBank (accession numbers M84012 and AF549211).
[0071] Primers for sodC specific amplification were designed based
on the consensus sodC sequence to be located at positions not only
containing at least three nucleotide differences between Nm and Hi,
but also where sodC nucleotide sequence was conserved among the
meningococcal isolates that were sequenced. The sodC consensus
sequence was entered into Primer Express 3.0 (Applied Biosystems).
Given that sodC was likely acquired by Nm via horizontal transfer
from Hi, primers and a probe that differed by at least three
nucleotides per oligo between Nm and Hi sodC were chosen. Primers
and probes were analyzed for homology to other known sequences
using the Basic Local Alignment Search Tool (BLAST). Altschul S F,
et al., J Mol Biol, 1990; 215: 403-410. BLAST results showed that
the primers of SEQ ID NOs: 1 and 2 had no homology that was over
78% nucleotide identity with any genes but meningococcal sodC. The
only notable homology found for the probe of SEQ ID NO: 3 was a
two-nucleotide difference with H. parainfluenzae sodC; the primers,
however, showed no homology (less than two sequential nucleotides
identical) to this gene.
[0072] To determine if the primers were capable of amplifying sodC,
primers were tested for optimal concentration in triplicate or
quadruplicate by RT-PCR in combinations of final concentrations of
100, 300, 600, and 900 nM; the probe was tested in triplicate at
final concentrations of 50, 100, 200, and 300 nM. The amplified
product is located at nt 1427446 in MC58 (GenBank accession number
AE002098.2).
[0073] RT-PCR was performed as follows: A Stratagene Mx3005P
(Agilent, La Jolla, Calif.) and QuantiTect SYBR Green Master Mix
(QIAGEN) were used to optimize primer concentrations. Cycle
parameters were 2 minutes at 50.degree. C., 10 minutes at
95.degree. C., and then 50.times. (15 seconds at 95.degree. C. plus
1 minute at 60.degree. C.). Product dissociation curves were
generated using one round of the following cycle parameters at the
end of the primer optimization run: one minute at 95.degree. C., 30
seconds at 55.degree. C., and 30 seconds at 95.degree. C. For
following studies, master mixes contained 4.5 .mu.l sterile PCR
grade water (Roche Diagnostics), 12.5 .mu.l TaqMan.RTM.2.times.PCR
Master Mix (Applied Biosystems), 300 nM forward primer (SEQ ID NO:
1), 600 nM reverse primer (SEQ ID NO: 2), 100 nM labeled probe (SEQ
ID NO: 3; FAM-CATGATGGCACAGCAACAAATCCTGTTT-BHQ1), and 2 .mu.l
template DNA per total reaction volume of 25 .mu.l. With each
reaction plate that was run, cell lysates from known Nm served as
positive external amplification controls, while no-template
controls (NTCs) served as negative external amplification controls.
The primers of SEQ ID NOs: 1 and 2, along with the labeled probe of
SEQ ID NO: 3 successfully amplified and detected sodC.
Example 2
Specificity and Characteristics of an Exemplary sodC RT-PCR
Assay
[0074] The specificity of an RT-PCR based sodC assay for detecting
only meningococci was determined using cell lysates from a total of
244 non-Nm isolates by the RT-PCR conditions of Example 1 using a
forward primer of SEQ ID NO: 1, a reverse primer of SEQ ID NO: 2,
and a probe of SEQ ID NO: 3. Each of the 244 non Nm isolates were
negative by the assay RT-PCR method. None of 35 non-Nm from various
sources and none of 209 non-meningococcal carriage study isolates
were detected (100% specificity) as seen in Table 2.
TABLE-US-00002 TABLE 2 Organism n sodC.sup.+ M. catarrhalis 22 0 H.
aphrophilus/paraphrophilus 2 0 H. aphrophilus 1 0 H. influenzae
biogroup aegyptius 1 0 H. influenzae serotype a 1 0 H. influenzae
serotype b 1 0 H. influenzae serotype c 1 0 H. influenzae serotype
d 1 0 H. influenzae serotype e 2 0 H. influenzae serotype f 1 0 H.
influenzae nontypeable (NTHi) 81 0 H. parainfluenzae 10 0 H.
haemolyticus 1 0 N. lactamica 93 0 N. spp. 3 0 N. polysaccharea 1 0
N. cinerea 2 0 N. subflava 1 0 N. sicca 2 0 N. gonorrhoeae 5 0 E.
coli K1 2 0 C. neoformans 1 0 S. aureus 1 0 S. pneumoniae 1 0 L.
monocytogenes 1 0 A. pleuropneumoniae 1 0 S. choleraesuis 1 0 S.
agalactiae 1 0 P. aeruginosa 1 0 C. diptheriae 1 0 B. pertussis 1 0
Total 244 0 .sup.1 92 N. lactamica, 4 N. gonorrhoeae, 19 M.
catarrhalis, and 2 H. spp. from this panel were collected in a
Georgia Nm carriage study (8). 3 N. spp., 1 N. polysaccharea, 2 M.
catarrhalis, 9 H. parainfluenzae, and 76 NTHi and 1 Hie were
collected in a Georgia Hib carriage study (Sharma, et al., in
preparation).
[0075] Interestingly, the sodC assay identified one isolate,
M16160, as Nm when other standard carriage study tests could not
correctly resolve its species. It was originally identified as N.
polysaccharea/N. spp. by NH strip, but upon re-investigation, did
not grow at room temperature on chocolate agar, indicating that it
is not N. polysaccharea. All 7 meningococcal housekeeping genes
were readily amplified during MLST, suggesting that M16160 is
indeed Nm (ST-7456, cc60), and again showing sodC to be a useful
tool for definitive identification of carriage isolates.
[0076] The lower limit of detection was determined using standard
curves generated by testing genomic DNA from four invasive
meningococcal isolates with the sodC assay. LLDs at a C.sub.t value
of 35 were found to be 39, 70, 101, and 82 genomes per reaction,
yielding an average of 73 genomes detected per reaction. The
average reaction efficiency was 100% and the average R.sup.2 value
was 0.9925.
Example 3
Sensitivity of the RT-PCR sodC Assay
[0077] The sensitivity of the RT-PCR assay of Example 1 using the
primers of SEQ ID NOs: 1 and 2 and the probe of SEQ ID NO: 3 was
determined using 626 cell lysates (listed in Dolan Thomas, J, et
al., 2011, PLoS ONE 6(5): e19361, doi:10.1371/journal.pone.0019361)
including lysates prepared from a temporally and geographically
dispersed convenience sample of isolates from the CDC Meningitis
Laboratory strain collection (received 1993-2008, n=106) and all
isolates from a US carriage study (n=520) known to be Nm by SASG,
RT-PCR serogrouping (Mothershed, et al.), NH strips
(bioMerieux.RTM. sa), and Cystine Trypticase Agar (CTA) sugars
(Remel). To further confirm identification, multilocus sequence
typing (MLST) was performed on all U.S. carriage study and
ctrA-negative NG isolates.
[0078] All isolates were positive for sodC using these primers and
probe, including 26 ctrA-negative NG isolates, with a median
C.sub.t value of 19.7, mean of 19.9.+-.1.9, and range of 16.4 to
26.0.
[0079] Additional samples from two U.S. carriage studies were also
tested. 518/520 (99.6%) of meningococcal carriage isolates from the
studies were positive for sodC (median C.sub.t 16.9, mean
17.0.+-.1.5, and range 13.5 to 29.3), while ctrA detected only
368/520 (70.8%) of these isolates (median C.sub.t 19.0, mean
19.2.+-.2.7, and range 13.5 to 34.0). The two sodC-negative
isolates carrying Nm were SASG NG, ctrA-negative but were confirmed
to be Nm ST-1117 and ST-4788, both cc1117; both were isolated from
the same study participant at different time points. Therefore,
176/178 (98.9%) SASG NG, ctrA-negative invasive and non-invasive Nm
isolates were sodC positive by this assay. Four sodC-negative
carriage study isolates that were identified as Nm by conventional
methods were re-investigated and shown to actually be non-Nm.
Example 4
Assay for Presence of Nm in Biological Samples from Clinical
Sources
[0080] The ability of the sodC assay of Example 2 to detect Nm was
assessed and was compared to ctrA as the target gene using
extracted DNA from 120 Turkish cerebrospinal fluid (CSF) specimens
and 37 U.S. clinical specimens.
[0081] For the clinical samples, CSF specimens from pediatric
meningitis patients were cultured as soon as possible after
collection. Specimens that were culture-negative were sent to CDC
on ice for detection of meningitis etiology by the Marmara
University School of Medicine in Istanbul, Turkey, and came from
patients who met the case definition for purulent meningitis
[leukocytosis (>100 cells/mm.sup.3) and either elevated protein
(>100 mg/dl) or decreased glucose (<40 mg/dl)]. DNA
extraction was performed as previously described for clinical
specimens with the QIAamp DNA Mini Kit (QIAGEN, Valencia, Calif.)
using Protocol C. After DNA extraction and real-time PCR testing of
all specimens for ctrA of Nm as described by Mothershed E A, et
al., J Clin Microbiol, 2004; 42:320-328, lytA of S. pneumoniae as
described by Carvalho M da G, et al., J Clin Microbiol, 2007;
45:2460-2466, and bexA and/or bcs2 of Hi, the subset of specimens
chosen to test the sodC assay (n=120) were either (1) positive for
ctrA (n=12) or (2) ctrA.sup.- lytA.sup.- bexA/bcs2.sup.-
(n=108).
[0082] 37 U.S. clinical specimens were referred to CDC for
detection or confirmation of bacterial meningitis etiology from
January to June 2009, including CSF (n=21), whole blood (n=6),
serum (n=6), and tissues (n=4).
[0083] The RT-PCR assay of Example 1 was used to examine each of
the samples for the presence or absence of Nm. Results are
illustrated in Table 3.
TABLE-US-00003 TABLE 3 n Nm Culture % Culture Specimen Total n n
sodC.sup.+ % sodC.sup.+ 95% CI n ctrA.sup.+ % ctrA.sup.+ 95%
CI.sup.1 Positive.sup.2 Positive 95% CI CSF 141 24 17.0 11% to 24%
20 14.2 9% to 21% 1 0.7 0% to 4% Blood 6 1 16.7 0% to 64% 1 16.7 0%
to 64% 0 0.0 0% to 46% Serum 6 0 0.0 0% to 46% 0 0.0 0% to 46% 0
0.0 0% to 46% Body Tissue 4 0 0.0 0% to 60% 0 0.0 0% to 60% 0 0.0
0% to 60% Total 157 25 15.9 n/a 21 13.4 n/a.sup.3 1 0.6 n/a
.sup.1Exact binomial 95% confidence interval .sup.2Culture was
attempted for all 120 Turkey CSF specimens. With the exception of
the one ctrA -negative, sodC-positive U.S. CSF specimen that was
culture -positive, culture was either not attempted, not reported,
or in one case, the isolate was nonviable two times for the other
36 U.S. clinical specimens. .sup.3n/a, not applicable
[0084] Briefly, screening for ctrA detected Nm in 21/157 (13.4%)
specimens (20 CSF and 1 blood), while the assay of Example 2 for
sodC was positive for those 21 plus four additional CSF specimens
(25/157, 15.9%). Therefore, the RT-PCR assay for sodC was 100% (95%
confidence interval [CI]: 84-100%) sensitive compared to ctrA at
detection of Nm from these clinical specimens. The C.sub.t values
for the four ctrA-negative, sodC-positive CSF extractions averaged
40.4 for ctrA while their sodC C.sub.t values averaged 32.0.
[0085] One ctrA-negative, sodC-positive CSF specimen was Nm
culture-positive (1/157, 0.6%); the remaining specimens were either
culture-negative (139/157) or culture was not attempted or not
reported (17/157). Therefore, sodC was 88% (95% CI: 82-93%)
specific compared to culture at detection of Nm from the clinical
specimens for which culture was attempted.
Example 5
Detection of Nm in Carriage Specimens
[0086] The assay of Example 1 was also used to screen for the
presence or absence of Nm in carriage specimens. The carriage
specimens were obtained from three carriage studies. In a first
study from Goiania, Brazil, nasopharyngeal (NP) swabs (n=223) were
obtained from 154 children (ranging from 2-163 months of age)
attending two daycare centers, 59 adult contacts of the attendees,
and 10 daycare workers. The specimens were placed into skim
milk-tryptone-glucose-glycerine (STGG) transport medium and sent
immediately to the Applied Microbiology Laboratory of Federal
University of Goias in Brazil for processing. The vials were then
kept frozen during transport to CDC, where DNA extractions and
RT-PCR were performed.
[0087] Second, 291 posterior NP swab specimens were collected from
a random sample of children 6 to 59 months of age who presented to
the Emergency Department at CHOA at Egleston from March to August,
2009. Each swab specimen was immediately placed into 1 ml STGG.
Specimens were transported at room temperature to the clinical
microbiology laboratory within 12 hours of collection for storage
at -80.degree. C. until processing. An aliquot of the STGG from
each specimen was transported on dry ice to the CDC Meningitis
Laboratory, where DNA extractions and RT-PCR were performed.
[0088] Third, a total of 33 NP swabs and 35 nasal washes (NWs) were
taken from 24 participants ages 21-57 years during .ltoreq.7 visits
in a Spring 2009 study conducted in the NIHR Biomedical Research
Centre in Microbial Diseases at the Liverpool School of Tropical
Medicine, Liverpool, United Kingdom (UK). Swab specimens were
collected as previously described (O'Brien K L, and Nohynek H,
Pediatr Infect Dis J, 2003; 22:e1-11) with some modifications and
placed directly into 1 ml STGG, then transported to the laboratory
on wet ice for culture and processing. 900 .mu.l of each specimen
was frozen at -80.degree. C. for subsequent DNA extraction and
rt-PCR in Liverpool.
[0089] For CSF specimens and the Brazilian NP swab eluates, DNA
extraction was conducted as previously described for clinical
specimens. O'Brien K L, et al., J Clin Microbiol, 2001;
39:1021-1024. DNA extractions were performed on the CHOA carriage
study NP swab eluates using the MagNA Pure LC instrument and the
DNA Isolation Kit III (Bacteria, Fungi) per the manufacturer's
instructions (Roche Diagnostics GmbH, Mannheim, Germany). DNA was
extracted from the UK NP and NW specimens using the QIAsymphony SP
System and the QIAsymphony Virus/Bacteria Midi Kit (QIAGEN Inc.,
UK) according to the manufacturer's instructions. All extracted DNA
was stored at -20.degree. C.
[0090] Results from all three carriage studies are summarized in
Table 4.
TABLE-US-00004 TABLE 4 n Nm Culture % Culture Specimen Total n n
sodC.sup.+ % sodC.sup.+ 95% CI.sup.1 n ctrA.sup.+ % ctrA.sup.+ 95%
CI Positive Positive 95% CI NP swab eluate 547 3 0.5 0% to 2% .sup.
1.sup.2 0.2 0% to 1% 0 0.0 0% to 1% Nasal wash 35 .sup. 2.sup.3 5.7
1% to 19% 3 8.6 2% to 23% .sup. 1.sup.4 2.9 0% to 15% Total 582 5
0.9 n/a.sup.5 4 0.7 n/a 1 0.2 n/a .sup.1Exact binomial 95%
confidence interval .sup.2This ctrA-positive NP swab eluate was
sodC -negative. .sup.3Both of these sodC-positive NP swab eluates
were ctrA -positive. .sup.4This Nm culture-positive NW was ctrA
-positive, sodC -positive. .sup.5 n/a, not applicable
[0091] None (0/223, 0%) of the Brazilian NP swab eluate extractions
tested at CDC were ctrA-positive, while 3 (3/223, 1.3%) were
sodC-positive, yielding C.sub.t values of 29.1, 26.2, and 25.5. The
ctrA-negative, sodC-positive specimens were negative for serogroups
A, B, C, W135, X, and Y by rt-PCR. In the 1/3 ctrA-negative,
sodC-positive specimen that had a sufficient amount of extraction
volume remaining, MLST confirmed the presence of meningococcal DNA
(ST-823, cc198). All 23 Hi culture-positive Brazilian specimens
were ctrA- and sodC-negative.
[0092] All (291/291, 100%) NP swab eluate extractions from the
Georgia CHOA carriage study were ctrA-negative and sodC-negative,
as expected, since no Nm was cultured. These specimens were,
however, culture-positive for N. spp. (n=3), N. polysaccharea
(n=1), M. catarrhalis (n=8), H. parainfluenzae (n=9), H.
haemolyticus (n=1), and Hi (n=76), further demonstrating the
specificity of sodC as a target for the presence of Nm in
samples.
[0093] S. aureus (11=17), alpha-hemolytic streptococci (n=18), M.
catarrhalis (n=5), diptheroids (n=10), N. polysaccharea (n=1), N.
cinerea (n=2), and N. meningitidis (n=1) were cultured from the 68
UK carriage study specimens. The Nm culture-positive NW was
ctrA-positive and sodC-positive. sodC and ctrA were negative for
all of the non-Nm culture-positive specimens except one
ctrA-positive, sodC-positive NW that grew N. cinerea and
alpha-hemolytic streptococci. 1/33 (3%) NP swab eluate extractions
from the UK carriage study were ctrA-positive, 0/33 were
sodC-positive, and 0/33 were Nm culture-positive. 3/35 (8.6%) NW
extractions were ctrA-positive, 2/35 (5.7%) were also
sodC-positive; of these, one (1/35, 2.9%) ctrA-positive,
sodC-positive NW was Nm culture-positive. The ctrA-positive,
sodC-negative NP specimen (average ctrA C.sub.t 35.0.+-.0.3,
average sodC C.sub.t 37.8.+-.0.8) and the ctrA-positive,
sodC-negative NW (average ctrA C.sub.t value of 34.3.+-.0.2 and an
average sodC C.sub.t value of 35.1.+-.0.2) were both from patient
8, visit 3; this patient had the Nm-positive culture at visit 1 and
the N. cinerea-positive culture at visit 2.
Example 6
Detection of Nm by PCR/LC/MS
[0094] The isolates of Table 1 are each rescreened using PCR
amplification with parameters similar to the RT-PCR assay of
Example 1. Genomic DNA is subjected to cycle parameters of 2
minutes at 50.degree. C., 10 minutes at 95.degree. C., and then
50.times. (15 seconds at 95.degree. C. plus 1 minute at 60.degree.
C.). For each amplification reaction, master mixes contain 4.5
.mu.l sterile PCR grade water (Roche Diagnostics), 12.5 .mu.l
TaqMan.RTM.2.times.PCR Master Mix (Applied Biosystems), 300 nM
forward primer (SEQ ID NO: 1), 600 nM reverse primer (SEQ ID NO:
2), and 2 .mu.l template DNA per total reaction volume of 25
.mu.l.
[0095] The reaction products are subjected to analyses by
electrospray ionization mass spectrometry substantially as
described by Naito, Y, et al., Rapid Communications in Mass
Spectrometry, 1995; 9:1484-1486; or Wunschel D S, et al., Rapid
Commun Mass Spectrom. 1996; 10(1):29-35. Each of the reaction
products from the PCR reactions are successfully and rapidly
detected.
Example 7
Detection of Nm by PCR/gel Electrophoresis
[0096] The isolates of Table 1 are each rescreened using PCR
amplification with parameters similar to the RT-PCR assay of
Example 1. Genomic DNA is subjected to cycle parameters of 2
minutes at 50.degree. C., 10 minutes at 95.degree. C., and then
50.times. (15 seconds at 95.degree. C. plus 1 minute at 60.degree.
C.). For each amplification reaction, master mixes contain 4.5
.mu.l sterile PCR grade water (Roche Diagnostics), 12.5 .mu.l
TaqMan.RTM.2.times.PCR Master Mix (Applied Biosystems), 300 nM
forward primer (SEQ ID NO: 1), 600 nM reverse primer (SEQ ID NO:
2), 100 nM labeled probe (SEQ ID NO: 3; Alexa
488-5'-CATGATGGCACAGCAACAAATCCTGTTT-3'), and 2 .mu.l template DNA
per total reaction volume of 25 .mu.l.
[0097] The amplified reaction products are separated by gel
electrophoresis and detected by fluorescent imaging. Each of the
isolates show detectable amplified sodC DNA.
[0098] Methods involving conventional biological techniques are
described herein. Such techniques are generally known in the art
and are described in detail in methodology treatises such as
Molecular Cloning: A Laboratory Manual, 3rd ed., vol. 1-3, ed.
Sambrook et al., Cold Spring Harbor Laboratory Press, Cold Spring
Harbor, N.Y., 2001; Current Protocols in Molecular Biology, ed.
Ausubel et al., Greene Publishing and Wiley-Interscience, New York,
1992 (with periodic updates); and Short Protocols in Molecular
Biology, ed. Ausubel et al., 52 ed., Wiley-Interscience, New York,
2002. Immunological methods (e.g., preparation of antigen-specific
antibodies, immunoprecipitation, and immunoblotting) are described,
e.g., in Current Protocols in Immunology, ed. Coligan et al., John
Wiley & Sons, New York, 1991; and Methods of Immunological
Analysis, ed. Masseyeff et al., John Wiley & Sons, New York,
1992.
[0099] Additional protocols such as PCR Protocols can be found in A
Guide to Methods and Applications Academic Press, NY. Methods for
protein purification include such methods as ammonium sulfate
precipitation, column chromatography, electrophoresis,
centrifugation, crystallization, and others. See, e.g., Ausubel, et
al. (1987 and periodic supplements); Deutscher (1990) "Guide to
Protein Purification," Methods in Enzymology vol. 182, and other
volumes in this series; Current Protocols in Protein Science, John
Wiley and Sons, New York, N.Y.; and manufacturer's literature on
use of protein purification products known to those of skill in the
art.
[0100] Various modifications of the present invention, in addition
to those shown and described herein, will be apparent to those
skilled in the art of the above description. Such modifications are
also intended to fall within the scope of the appended claims.
[0101] It is appreciated that all reagents are obtainable by
sources known in the art unless otherwise specified. Methods of
nucleotide amplification, cell transfection, and protein expression
and purification are similarly within the level of skill in the
art.
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[0147] Patents and publications mentioned in the specification are
indicative of the levels of those skilled in the art to which the
invention pertains. These patents and publications are incorporated
herein by reference to the same extent as if each individual
application or publication was specifically and individually
incorporated herein by reference.
[0148] The foregoing description is illustrative of particular
aspects of the invention, but is not meant to be a limitation upon
the practice thereof. The following claims, including all
equivalents thereof, are intended to define the scope of the
invention.
Sequence CWU 1
1
5125DNANeisseria meningitidismisc_featureSodC rt-pcr fwd primer
1gcacacttag gtgatttacc tgcat 25223DNANeisseria
meningitidismisc_featureSodC rt-pcr rev primer 2ccacccgtgt
ggatcataat aga 23328DNANeisseria meningitidismisc_featurert-pcr
probe 3catgatggca cagcaacaaa tcctgttt 28422DNANeisseria
meningitidismisc_featuresequencing fwd primer 4ccttattagc
actagcggtt ag 22522DNANeisseria meningitidismisc_featuresequencing
rev primer 5ccggtcatct tttatgctcc aa 22
* * * * *