CA2560521C - Compositions for use in identification of bacteria - Google Patents

Abstract

The present invention provides oligonucleotide primers and compositions and kits containing the same for rapid identification of bacteria by amplification of a segment of bacterial nucleic acid followed by molecular mass analysis.

Description

DEMANDES OU BREVETS VOLUMINEUX
LA PRESENTE PARTIE DE CETTE DEMANDE OU CE BREVETS
COMPREND PLUS D'UN TOME.
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COMPOSITIONS FOR USE IN IDENTIFICATION OF BACTERIA

STATEMENT OF GOVERNMENT SUPPORT
[00021 This invention was made with United States Government support under DARPA/SPO
contract BAA00-09. The United States Government may have certain rights in the invention.
FIELD OF THE INVENTION
[00031 The present invention relates generally to the field of genetic identification of bacteria and provides nucleic acid compositions and kits useful for this purpose when combined with molecular mass analysis.

BACKGROUND OF THE INVENTION
[0004] A problem in determining the cause of a natural infectious outbreak or a bioterrorist attack is the sheer variety of organisms that can cause human disease. There are over 1400 organisms infectious to humans; many of these have the potential to emerge suddenly in a natural epidemic or to be used in a malicious attack by bioterrorists (Taylor et al. Philos. Trans.
R. Soc. London B. Biol. Sci., 2001, 356, 983-989). This number does not include numerous strain variants, bioengineered versions, or pathogens that infect plants or animals.

[0005] Much of the new technology being developed for detection of biological weapons incorporates a polymerase chain reaction (PCR) step based upon the use of highly specific primers and probes designed to selectively detect certain pathogenic organisms. Although this approach is appropriate for the most obvious bioterrorist organisms, like smallpox and anthrax, experience has shown that it is very difficult to predict which of hundreds of possible pathogenic organisms might be employed in a terrorist attack. Likewise, naturally emerging human disease that has caused devastating consequence in public health has come from unexpected families of bacteria, viruses, fungi, or protozoa. Plants and animals also have their natural burden of infectious disease agents and there are equally important biosafety and security concerns for agriculture.

[0006] A major conundrum in public health protection, biodefense, and agricultural safety and security is that these disciplines need to be able to rapidly identify and characterize infectious agents, while there is no existing technology with the breadth of function to meet this need.
Currently used methods for identification of bacteria rely upon culturing the bacterium to effect isolation from other organisms and to obtain sufficient quantities of nucleic acid followed by sequencing of the nucleic acid, both processes which are time and labor intensive.

[0007] Mass spectrometry provides detailed information about the molecules being analyzed, including high mass accuracy. It is also a process that can be easily automated. DNA chips with specific probes can only determine the presence or absence of specifically anticipated organisms.
Because there are hundreds of thousands of species of benign bacteria, some very similar in sequence to threat organisms, even arrays with 10,000 probes lack the breadth needed to identify a particular organism.

[0008] There is a need for a method for identification of bioagents which is both specific and rapid, and in which no culture or nucleic acid sequencing is required.
Disclosed in I
U.S. published Patents: 2003-0027135; 2003-0228571; 2004-0209260; 2004-0219517;
2009-0280471; 2005-0266397 and in U.S. issued Patent Nos.: 7,217,510;
7,226,739;
7,255,992, each of which is commonly owned, are methods for identification of bioagents (any organism, cell, or virus, living or dead, or a nucleic acid derived from such an organism, cell or virus) in an unbiased manner by molecular mass and base composition analysis of "bioagent identifying amplicons" which are obtained by amplification of segments of essential and conserved genes which are involved in, for example, translation, replication, recombination and repair, transcription, nucleotide metabolism, amino acid metabolism, lipid metabolism, energy generation, uptake, secretion and the like. Examples of these proteins include, but are not limited to, ribosomal RNAs, ribosomal proteins, DNA and RNA polymerases, elongation factors, tRNA synthetases, protein chain initiation factors, heat shock protein groEL, phosphoglycerate kinase, NADH dehydrogenase, DNA ligases, DNA gyrases and DNA
topoisomerases, metabolic enzymes, and the like.

[0009] To obtain bioagent identifying amplicons, primers are selected to hybridize to conserved sequence regions which bracket variable sequence regions to yield a segment of nucleic acid which can be amplified and which is amenable to methods of molecular mass analysis. The variable sequence regions provide the variability of molecular mass which is used for bioagent identification. Upon amplification by PCR or other amplification methods with the specifically chosen primers, an amplification product that represents a bioagent identifying amplicon is obtained. The molecular mass of the amplification product, obtained by mass spectrometry for example, provides the means to uniquely identify the bioagent without a requirement for prior knowledge of the possible identity of the bioagent. The molecular mass of the amplification product or the corresponding base composition (which can be calculated from the molecular mass of the amplification product) is compared with a database of molecular masses or base compositions and a match indicates the identity of the bioagent. Furthermore, the method can be applied to rapid parallel analyses (for example, in a multi-well plate format) the results of which can be employed in a triangulation identification strategy which is amenable to rapid throughput and does not require nucleic acid sequencing of the amplified target sequence for bioagent identification.

[0010] The result of determination of a previously unknown base composition of a previously unknown bioagent (for example, a newly evolved and heretofore unobserved bacterium or virus) has downstream utility by providing new bioagent indexing information with which to populate base composition databases. The process of subsequent bioagent identification analyses is thus greatly improved as more base composition data for bioagent identifying amplicons becomes available.

[0011] The present invention provides oligonucleotide primers and compositions and kits containing the oligonucleotide primers, which define bacterial bioagent identifying amplicons and, upon amplification, produce corresponding amplification products whose molecular masses provide the means to identify bacteria, for example, at and below the species taxonomic level.
SUMMARY OF THE INVENTION
[0012] The present invention provides primers and compositions comprising pairs of primers, and kits containing the same for use in identification of bacteria. The primers are designed to produce bacterial bioagent identifying amplicons of DNA encoding genes essential to life such as, for example, 16S and 23S rRNA, DNA-directed RNA polymerase subunits (rpoB
and rpoC), valyl-tRNA synthetase (va1S), elongation factor EF-Tu (TufB), ribosomal protein L2 (rplB), protein chain initiation factor (infB), and spore protein (sspE). The invention further provides drill-down primers, compositions comprising pairs of primers and kits containing the same, which are designed to provide sub-species characterization of bacteria.

[0013] The present invention also provides an oligonucleotide primer 21 to 35 nucleobases in length comprising 70% to 100% sequence identity with SEQ ID NO: 97, or a composition comprising the same; an oligonucleotide primer 20 to 35 nucleobases in length comprising 70%
to 100% sequence identity with SEQ ID NO: 451, or a composition comprising the same; a composition comprising both primers; and a composition comprising a first oligonucleotide primer 21 to 35 nucleobases in length comprising between 70% to 100% sequence identity of SEQ ID NO: 97, and a second oligonucleotide primer 20 to 35 nucleobases in length comprising between 70% to 100% sequence identity of SEQ ID NO: 451.

[0014] The present invention also provides an oligonucleotide primer 19 to 35 nucleobases in length comprising 70% to 100% sequence identity with SEQ ID NO: 127, or a composition comprising the same; an oligonucleotide primer 14 to 35 nucleobases in length comprising 70%
to 100% sequence identity with SEQ ID NO: 482, or a composition comprising the same; a composition comprising both primers; and a composition comprising a first oligonucleotide primer 19 to 35 nucleobases in length comprising between 70% to 100% sequence identity of SEQ ID NO: 127, and a second oligonucleotide primer 14 to 35 nucleobases in length comprising between 70% to 100% sequence identity of SEQ ID NO: 482.

[0015] The present invention also provides an oligonucleotide primer 19 to 35 nucleobases in length comprising 70% to 100% sequence identity with SEQ ID NO: 174, or a composition comprising the same; an oligonucleotide primer 21 to 35 nucleobases in length comprising 70%
to 100% sequence identity with SEQ ID NO: 530, or a composition comprising the same; a composition comprising both primers; and a composition comprising a first oligonucleotide primer 19 to 35 nucleobases in length comprising between 70% to 100% sequence identity of SEQ ID NO: 174, and a second oligonucleotide primer 21 to 35 nucleobases in length comprising between 70% to 100% sequence identity of SEQ ID NO: 530.

[0016] The present invention also provides an oligonucleotide primer 21 to 35 nucleobases in length comprising 70% to 100% sequence identity with SEQ ID NO: 310, or a composition comprising the same; an oligonucleotide primer 19 to 35 nucleobases in length comprising 70%
to 100% sequence identity with SEQ ID NO: 668, or a composition comprising the same; a composition comprising both primers; and a composition comprising a first oligonucleotide primer 21 to 35 nucleobases in length comprising between 70% to 100% sequence identity of SEQ ID NO: 310, and a second oligonucleotide primer 19 to 35 nucleobases in length comprising between 70% to 100% sequence identity of SEQ ID NO: 668.

[0017] The present invention also provides an oligonucleotide primer 21 to 35 nucleobases in length comprising 70% to 100% sequence identity with SEQ ID NO: 313, or a composition comprising the same; an oligonucleotide primer 21 to 35 nucleobases in length comprising 70%
to 100% sequence identity with SEQ ID NO: 670, or a composition comprising the same; a composition comprising both primers; and a composition comprising a first oligonucleotide primer 21 to 35 nucleobases in length comprising between 70% to 100% sequence identity of SEQ ID NO: 313, and a second oligonucleotide primer 21 to 35 nucleobases in length comprising between 70% to 100% sequence identity of SEQ ID NO: 670.

[0018] The present invention also provides an oligonucleotide primer 17 to 35 nucleobases in length comprising 70% to 100% sequence identity with SEQ ID NO: 277, or a composition comprising the same; an oligonucleotide primer 21 to 35 nucleobases in length comprising 70%
to 100% sequence identity with SEQ ID NO: 632, or a composition comprising the same; a composition comprising both primers; and a composition comprising a first oligonucleotide primer 17 to 35 nucleobases in length comprising between 70% to 100% sequence identity of SEQ ID NO: 277, and a second oligonucleotide primer 21 to 35 nucleobases in length comprising between 70% to 100% sequence identity of SEQ ID NO: 632.

[0019] The present invention also provides an oligonucleotide primer 21 to 35 nucleobases in length comprising 70% to 100% sequence identity with SEQ ID NO: 285, or a composition comprising the same; an oligonucleotide primer 19 to 35 nucleobases in length comprising 70%
to 100% sequence identity with SEQ ID NO: 640, or a composition comprising the same; a composition comprising both primers; and a composition comprising a first oligonucleotide primer 21 to 35 nucleobases in length comprising between 70% to 100% sequence identity of SEQ ID NO: 285, and a second oligonucleotide primer 19 to 35 nucleobases in length comprising between 70% to 100% sequence identity of SEQ ID NO: 640.

[0020] The present invention also provides an oligonucleotide primer 21 to 35 nucleobases in length comprising 70% to 100% sequence identity with SEQ ID NO: 301, or a composition comprising the same; an oligonucleotide primer 21 to 35 nucleobases in length comprising 70%
to 100% sequence identity with SEQ ID NO: 656, or a composition comprising the same; a composition comprising both primers; and a composition comprising a first oligonucleotide primer 21 to 35 nucleobases in length comprising between 70% to 100% sequence identity of SEQ ID NO: 301, and a second oligonucleotide primer 21 to 35 nucleobases in length comprising between 70% to 100% sequence identity of SEQ ID NO: 656.

[0021] The present invention also provides an oligonucleotide primer 18 to 35 nucleobases in length comprising 70% to 100% sequence identity with SEQ ID NO: 308, or a composition comprising the same; an oligonucleotide primer 18 to 35 nucleobases in length comprising 70%
to 100% sequence identity with SEQ ID NO: 663, or a composition comprising the same; a composition comprising both primers; and a composition comprising a first oligonucleotide primer 18 to 35 nucleobases in length comprising between 70% to 100% sequence identity of SEQ ID NO: 308, and a second oligonucleotide primer 18 to 35 nucleobases in length comprising between 70% to 100% sequence identity of SEQ ID NO: 663.

[0022] The present invention also provides compositions, such as those described herein, wherein either or both of the first and second oligonucleotide primers comprise at least one modified nucleobase, a non-templated T residue on the 5'-end, at least one non-template tag, or at least one molecular mass modifying tag, or any combination thereof.

[0023] The present invention also provides kits comprising any of the compositions described herein. The kits can comprise at least one calibration polynucleotide, or at least one ion exchange resin linked to magnetic beads, or both.

[0024] The present invention also provides methods for identification of an unknown bacterium.
Nucleic acid from the bacterium is amplified using any of the compositions described herein to obtain an amplification product. The molecular mass of the amplification product is determined.
Optionally, the base composition of the amplification product is determined from the molecular mass. The base composition or molecular mass is compared with a plurality of base compositions or molecular masses of known bacterial bioagent identifying amplicons, wherein a match between the base composition or molecular mass and a member of the plurality of base compositions or molecular masses identifies the unknown bacterium. The molecular mass can be measured by mass spectrometry. In addition, the prrsence or absence of a particular Glade, genus, species, or sub-species of a bioagent can be determined by the methods described herein.

[0025] The present invention also provides methods for determination of the quantity of an unknown bacterium in a sample. The sample is contacted with any of the compositions described herein and a known quantity of a calibration polynucleotide comprising a calibration sequence.
Concurrently, nucleic acid from the bacterium in the sample is amplified with any of the compositions described herein and nucleic acid from the calibration polynucleotide in the sample is amplified with any of the compositions described herein to obtain a first amplification product comprising a bacterial bioagent identifying amplicon and a second amplification product comprising a calibration amplicon. The molecular mass and abundance for the bacterial bioagent identifying amplicon and the calibration amplicon is determined. The bacterial bioagent identifying amplicon is distinguished from the calibration amplicon based on molecular mass, wherein comparison of bacterial bioagent identifying amplicon abundance and calibration amplicon abundance indicates the quantity of bacterium in the sample. The method can also comprise determining the base composition of the bacterial bioagent identifying amplicon.

BRIEF DESCRIPTION OF THE DRAWINGS
[0026] Figure 1 is a represenataive pseudo-four dimensional plot of base compositions of bioagent identifying amplicons of enterobacteria obtained with a primer pair targeting the rpoB
gene (primer pair no 14 (SEQ ID NOs: 37:362). The quantity each of the nucleobases A, G and C are represented on the three axes of the plot while the quantity of nucleobase T is represented by the diameter of the spheres. Base composition probability clouds surrounding the spheres are also shown.

[0027] Figure 2 is a represenataive diagram illustrating the primer selection process.

[0028] Figure 3 lists common pathogenic bacteria and primer pair coverage. The primer pair number in the upper right hand corner of each polygon indicates that the primer pair can produce a bioagent identifying amplicon for all species within that polygon.

[0029] Figure 4 is a represenataive 3D diagram of base composition (axes A, G
and C) of bioagent identifying amplicons obtained with primer pair number 14 (a precursor of primer pair number 348 which targets 16S rRNA). The diagram indicates that the experimentally determined base compositions of the clinical samples (labeled NHRC samples) closely match the base compositions expected for Streptococcus pyogenes and are distinct from the expected base compositions of other organisms.

[0030] Figure 5 is a represenataive mass spectrum of amplification products representing bioagent identifying amplicons of Streptococcus pyogenes, Neisseria meningitidis, and Haemophilus influenzae obtained from amplification of nucleic acid from a clinical sample with primer pair number 349 which targets 23S rRNA. Experimentally determined molecular masses and base compositions for the sense strand of each amplification product are `shown.

[0031] Figure 6 is a represenataive mass spectrum of amplification products representing a bioagent identifying amplicon of Streptococcus pyogenes, and a calibration amplicon obtained from amplification of nucleic acid from a clinical sample with primer pair number 356 which targets rplB. The experimentally determined molecular mass and base composition for the sense strand of the Streptococcus pyogenes amplification product is shown.

[0032] Figure 7 is a represenataive process diagram for identification and determination of the quantity of a bioagent in a sample.

[0033] Figure 8 is a represenataive mass spectrum of an amplified nucleic acid mixture which contained the Ames strain of Bacillus anthracis, a known quantity of combination calibration polynucleotide (SEQ ID NO: 741), and primer pair number 350 which targets the capC gene on the virulence plasmid pX02 of Bacillus anthracis. Calibration amplicons produced in the amplification reaction are visible in the mass spectrum as indicated and abundance data (peak height) are used to calculate the quantity of the Ames strain of Bacillus anthracis.
DESCRIPTION OF EMBODIMENTS
[0034] The present invention provides oligonucleotide primers which hybridize to conserved regions of nucleic acid of genes encoding, for example, proteins or RNAs necessary for life which include, but are not limited to: 16S and 23S rRNAs, RNA polymerase subunits, t-RNA
synthetases, elongation factors, ribosomal proteins, protein chain initiation factors, cell division proteins, chaperonin groEL, chaperonin dnaK, phosphoglycerate kinase, NADH
dehydrogenase, DNA ligases, metabolic enzymes and DNA topoisomerases. These primers provide the functionality of producing, for example, bacterial bioagent identifying amplicons for general identification of bacteria at the species level, for example, when contacted with bacterial nucleic acid under amplification conditions.

[00351 Referring to Figure 2, primers are designed as follows: for each group of organisms, candidate target sequences are identified (200) from which nucleotide alignments are created (210) and analyzed (220). Primers are designed by selecting appropriate priming regions (230) which allows the selection of candidate primer pairs (240). The primer pairs are subjected to in silico analysis by electronic PCR (ePCR) (300) wherein bioagent identifying amplicons are obtained from sequence databases such as, for example, GenBank or other sequence collections (310), and checked for specificity in silico (320). Bioagent identifying amplicons obtained from GenBank sequences (310) can also be analyzed by a probability model which predicts the capability of a particular amplicon to identify unknown bioagents such that the base compositions of amplicons with favorable probability scores are stored in a base composition database (325). Alternatively, base compositions of the bioagent identifying amplicons obtained from the primers and GenBank sequences can be directly entered into the base composition database (330). Candidate primer pairs (340) are validated by in vitro amplification by a method such as, for example, PCR analysis (400) of nucleic acid from a collection of organisms (410).
Amplification products that are obtained are optionally analyzed to confirm the sensitivity, specificity and reproducibility of the primers used to obtain the amplification products (420).
[00361 Synthesis of primers is well known and routine in the art. The primers may be conveniently and routinely made through the well-known technique of solid phase synthesis.
Equipment for such synthesis is sold by several vendors including, for example, Applied Biosystems (Foster City, CA). Any other means for such synthesis known in the art may additionally or alternatively be employed.

[00371 The primers can be employed as compositions for use in, for example, methods for identification of bacterial bioagents as follows. In some embodiments, a primer pair composition is contacted with nucleic acid of an unknown bacterial bioagent. The nucleic acid is amplified by a nucleic acid amplification technique, such as PCR for example, to obtain an amplification product that represents a bioagent identifying amplicon. The molecular mass of one strand or each strand of the double-stranded amplification product is determined by a molecular mass measurement technique such as, for example, mass spectrometry wherein the two strands of the double-stranded amplification product are separated during the ionization process. In some embodiments, the mass spectrometry is electrospray Fourier transform ion cyclotron resonance mass spectrometry (ESI-FTICR-MS) or electrospray time of flight mass spectrometry (ESI-TOF-MS). A list of possible base compositions can be generated for the molecular mass value obtained for each strand and the choice of the correct base composition from the list is facilitated by matching the base composition of one strand with a complementary base composition of the other strand. The molecular mass or base composition thus determined is compared with a database of molecular masses or base compositions of analogous bioagent identifying amplicons for known bacterial bioagents. A match between the molecular mass or base composition of the amplification product from the unknown bacterial bioagent and the molecular mass or base composition of an analogous bioagent identifying amplicon for a known bacterial bioagent indicates the identity of the unknown bioagent.

[0038] In some embodiments, the primer pair used is one of the primer pairs of Table 1. In some embodiments, the method is repeated using a different primer pair to resolve possible ambiguities in the identification process or to improve the confidence level for the identification assignment.

[0039] In some embodiments, a bioagent identifying amplicon may be produced using only a single primer (either the forward or reverse primer of any given primer pair), provided an appropriate amplification method is chosen, such as, for example, low stringency single primer PCR (LSSP-PCR). Adaptation of this amplification method in order to produce bioagent identifying amplicons can be accomplished by one with ordinary skill in the art without undue experimentation.

[0040] In some embodiments, the oligonucleotide primers are "broad range survey primers"
which hybridize to conserved regions of nucleic acid encoding RNA, such as ribosomal RNA
(rRNA), of all, or at least 70%, at least 80%, at least 85%, at least 90%, or at least 95% of known bacteria and produce bacterial bioagent identifying amplicons. As used herein, the term "broad range survey primers" refers to primers that bind to nucleic acid encoding rRNAs of all, or at least 70%, at least 80%, at least 85%, at least 90%, or at least 95% known species of bacteria. In some embodiments, the rRNAs to which the primers hybridize are 16S and 23S
rRNAs. In some embodiments, the broad range survey primer pairs comprise oligonucleotides ranging in length from 13 to 35 nucleobases, each of which have from 70% to 100% sequence identity with primer pair numbers 3, 10, 11, 14, 16, and 17 which consecutively correspond to SEQ
ID NOs: 6:369, 26:388, 29:391, 37:362, 48:404, and 58:414.

[00411 In some cases, the molecular mass or base composition of a bacterial bioagent identifying amplicon defined by a broad range survey primer pair does not provide enough resolution to unambiguously identify a bacterial bioagent at the species level. These cases benefit from further analysis of one or more bacterial bioagent identifying amplicons generated from at least one additional broad range survey primer pair or from at least one additional "division-wide" primer pair (vide infra). The employment of more than one bioagent identifying amplicon for identification of a bioagent is herein referred to as "triangulation identification" (vide infra).
[00421 In other embodiments, the oligonucleotide primers are "division-wide"
primers which hybridize to nucleic acid encoding genes of broad divisions of bacteria such as, for example, members of the Bacillus/Clostridia group or members of the a-, 0-,'Y-, and c-proteobacteria. In some embodiments, a division of bacteria comprises any grouping of bacterial genera with more than one genus represented. For example, the 0-proteobacteria group comprises members of the following genera: Eikenella, Neisseria, Achromobacter, Bordetella, Burkholderia, and Raltsonia.
Species members of these genera can be identified using bacterial bioagent identifying amplicons generated with primer pair 293 (SEQ ID NOs: 344:700) which produces a bacterial bioagent identifying amplicon from the tufB gene of (3-proteobacteria. Examples of genes to which division-wide primers may hybridize to include, but are not limited to: RNA
polymerase subunits such as rpoB and rpoC, tRNA synthetases such as valyl-tRNA synthetase (valS) and aspartyl-tRNA synthetase (aspS), elongation factors such as elongation factor EF-Tu (tufB), ribosomal proteins such as ribosomal protein L2 (rplB), protein chain initiation factors such as protein chain initiation factor infB, chaperonins such as groL and dnaK, and cell division proteins such as peptidase ftsH (hflB). In some embodiments, the division-wide primer pairs comprise oligonucleotides ranging in length from 13 to 35 nucleobases, each of which have from 70% to 100% sequence identity with primer pair numbers 34, 52, 66, 67, 71, 72, 289, 290 and 293 which consecutively correspond to SEQ ID NOs: 160:515, 261:624, 231:591, 235:587, 349:711, 240:596, 246:602, 256:620, 344:700.

[00431 In other embodiments, the oligonucleotide primers are designed to enable the identification of bacteria at the Glade group level, which is a monophyletic taxon referring to a group of organisms which includes the most recent common ancestor of all of its members and all of the descendants of that most recent common ancestor. The Bacillus cereus Glade is an example of a bacterial Glade group. In some embodiments, the Glade group primer pairs comprise oligonucleotides ranging in length from 13 to 35 nucleobases, each of which have from 70% to 100% sequence identity with primer pair number 58 which corresponds to SEQ 1D
NOs:
322:686.

[0044] In other embodiments, the oligonucleotide primers are "drill-down"
primers which enable the identification of species or "sub-species characteristics." Sub-species characteristics are herein defined as genetic characteristics that provide the means to distinguish two members of the same bacterial species. For example, Escherichia coli 0157:H7 and Escherichia coli K12 are two well known members of the species Escherichia coli. Escherichia coli 0157:H7, however, is highly toxic due to the its Shiga toxin gene which is an example of a sub-species characteristic.
Examples of sub-species characteristics may also include, but are not limited to: variations in genes such as single nucleotide polymorphisms (SNPs), variable number tandem repeats (VNTR5). Examples of genes indicating sub-species characteristics include, but are not limited to, housekeeping genes, toxin genes, pathogenicity markers, antibiotic resistance genes and virulence factors. Drill-down primers provide the functionality of producing bacterial bioagent identifying amplicons for drill-down analyses such as strain typing when contacted with bacterial nucleic acid under amplification conditions. Identification of such sub-species characteristics is often critical for determining proper clinical treatment of bacterial infections. Examples of pairs of drill-down primers include, but are not limited to, a trio of primer pairs for identification of strains of Bacillus anthracis. Primer pair 24 (SEQ ID NOs: 97:451) targets the capC gene of virulence plasmid pX02, primer pair 30 (SEQ ID NOs: 127:482) targets the cyA
gene of virulence plasmid pX02, and primer pair 37 (SEQ ID NOs: 174:530) targets the lef gene of virulence plasmid pX02. Additional examples of drill-down primers include, but are not limited to, six primer pairs that are used for determining the strain type of group A
Streptococcus.
Primer pair 80 (SEQ ID NOs: 310:668) targets the gki gene, primer pair 81 (SEQ
ID NOs:
313:670) targets the gtr gene, primer pair 86 (SEQ ID NOs: 227:632) targets the murl gene, primer pair 90 (SEQ ID NOs: 285:640) targets the mutS gene, primer pair 96 (SEQ ID NOs:
301:656) targets the xpt gene, and primer pair 98 (SEQ ID NOs: 308:663) targets the yqiL gene.
[0045] In some embodiments, the primers used for amplification hybridize to and amplify genomic DNA, DNA of bacterial plasmids, or DNA of DNA viruses.

[0046] In some embodiments, the primers used for amplification hybridize directly to ribosomal RNA or messenger RNA (mRNA) and act as reverse transcription primers for obtaining DNA
from direct amplification of bacterial RNA or rRNA. Methods of amplifying RNA
using reverse transcriptase are well known to those with ordinary skill in the art and can be routinely established without undue experimentation.

[0047] One with ordinary skill in the art of design of amplification primers will recognize that a given primer need not hybridize with 100% complementarity in order to effectively prime the synthesis of a complementary nucleic acid strand in an amplification reaction.
Moreover, a primer may hybridize over one or more segments such that intervening or adjacent segments are not involved in the hybridization event (e.g., a loop structure or a hairpin structure). The primers of the present invention may comprise at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or at least 99% sequence identity with any of the primers listed in Table 1. Thus, in some embodiments of the present invention, an extent of variation of 70% to 100%, or any range therewithin, of the sequence identity is possible relative to the specific primer sequences disclosed herein. Determination of sequence identity is described in the following example: a primer 20 nucleobases in length which is otherwise identical to another 20 nucleobase primer but having two non-identical residues has 18 of 20 identical residues (18/20 =
0.9 or 90% sequence identity). In another example, a primer 15 nucleobases in length having all residues identical to a 15 nucleobase segment of primer 20 nucleobases in length would have 15/20 = 0.75 or 75% sequence identity with the 20 nucleobase primer.

[0048] Percent homology, sequence identity or complementarity, can be determined by, for example, the Gap program (Wisconsin Sequence Analysis Package, Version 8 for Unix, Genetics Computer Group, University Research Park, Madison WI), using default settings, which uses the algorithm of Smith and Waterman (Adv. Appl. Math., 1981, 2, 482-489). In some embodiments, homology, sequence identity, or complementarity of primers with respect to the conserved priming regions of bacterial nucleic acid, is at least 70%, at least 80%, at least 90%, at least 92%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or is 100%. ' [0049] In some embodiments, the primers described herein comprise at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 92%, at least 94%, at least 95%, at least 96%, at least 98%, or at least 99%, or 100% (or any range therewithin) sequence identity with the primer sequences specifically disclosed herein. Thus, for example, a primer may have between 70% and 100%, between 75% and 100%, between 80% and 100%, and between 95% and 100%
sequence identity with SEQ ID NO: 26. Likewise, a primer may have similar sequence identity with any other primer whose nucleotide sequence is disclosed herein.

[0050] One with ordinary skill is able to calculate percent sequence identity or percent sequence homology and able to determine, without undue experimentation, the effects of variation of primer sequence identity on the function of the primer in its role in priming synthesis of a complementary strand of nucleic acid for production of an amplification product of a corresponding bioagent identifying amplicon.

[0051] In some embodiments of the present invention, the oligonucleotide primers are between 13 and 35 nucleobases in length (13 to 35 linked nucleotide residues). These embodiments comprise oligonucleotide primers 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34 or 35 nucleobases in length, or any range therewithin.

[0052] In some embodiments, any given primer comprises a modification comprising the addition of a non-templated T residue to the 5' end of the primer (i.e., the added T residue does not necessarily hybridize to the nucleic acid being amplified). The addition of a non-templated T
residue has an effect of minimizing the addition of non-templated A residues as a result of the non-specific enzyme activity of Taq polymerase (Magnuson et al. Biotechniques, 1996, 21, 700-709), an occurrence which may lead to ambiguous results arising from molecular mass analysis.
[0053] In some embodiments of the present invention, primers may contain one or more universal bases. Because any variation (due to codon wobble in the 3rd position) in the conserved regions among species is likely to occur in the third position of a DNA
triplet, oligonucleotide primers can be designed such that the nucleotide corresponding to this position is a base which can bind to more than one nucleotide, referred to herein as a "universal nucleobase." For example, under this "wobble" pairing, inosine (I) binds to U, C or A; guanine (G) binds to U or C, and uridine (U) binds to U or C. Other examples of universal nucleobases include nitroindoles such as 5-nitroindole or 3-nitropyrrole (Loakes et al., Nucleosides and Nucleotides, 1995, 14, 1001-1003), the degenerate nucleotides dP or dK (Hill et al.), an acyclic nucleoside analog containing 5-nitroindazole (Van Aerschot et al., Nucleosides and Nucleotides, 1995, 14, 1053-1056) or the purine analog 1-(2-deoxy-(3-D-ribofuranosyl)-imidazole-4-carboxamide (Sala et al., Nucl. Acids Res., 1996, 24, 3302-3306).

100541 In some embodiments, to compensate for the somewhat weaker binding by the "wobble"
base, the oligonucleotide primers are designed such that the first and second positions of each triplet are occupied by nucleotide analogs which bind with greater affinity than the unmodified nucleotide. Examples of these analogs include, but are not limited to, 2,6-diaminopurine which binds to thymine, 5-propynyluracil which binds to adenine and 5 propynylcytosine and phenoxazines, including G-clamp, which binds to G. Propynylated pyrimidines are described in U.S. Patent Nos. 5,645,985, 5,830,653 and 5,484,908, each of which is commonly owned.
Propynylated primers are described in U. S. issued Patent 6,875,593 which is also commonly owned.
Phenoxazines are described in U.S. Patent Nos. 5,502,177, 5,763,588, and 6,005,096, G-clamps are described in U.S. Patent Nos. 6,007,992 and 6,028,183.

[0055] In some embodiments, non template primer tags are used to increase the melting temperature (T.) of a primer-template duplex in order to improve amplification efficiency. A
non-template tag is at least three consecutive A or T nucleotide residues on a primer which are not complementary to the template. In any given non-template tag, A can be replaced by C or G
and T can also be replaced by C or G. Although Watson-Crick hybridization is not expected to occur for a non-template tag relative to the template, the extra hydrogen bond in a G-C pair relative to a A-T pair confers increased stability of the primer-template duplex and improves amplification efficiency for subsequent cycles of amplification when the primers hybridize to strands synthesized in previous cycles.

[0056] In other embodiments, propynylated tags may be used in a manner similar to that of the non-template tag,. wherein two or more 5-propynylcytidine or 5-propynyluridine residues replace template matching residues on a primer. In other embodiments, a primer contains a modified internucleoside linkage such as a phosphorothioate linkage, for example.

[0057] In some embodiments, the primers contain mass-modifying tags. Reducing the total number of possible base compositions of a nucleic acid of specific molecular weight provides a means of avoiding a persistent source of ambiguity in determination of base composition of amplification products. Addition of mass-modifying tags to certain nucleobases of a given primer will result in simplification of de novo determination of base composition of a given bioagent identifying amplicon (vide infra) from its molecular mass.

[0058] In some embodiments of the present invention, the mass modified nucleobase comprises one or more of the following: for example, 7-deaza-2'-deoxyadenosine-5-triphosphate, 5-iodo-2'-deoxyuridine-5'-triphosphate, 5-bromo-2'-deoxyuridine-5'-triphosphate, 5-bromo-2'-deoxycytidine-5'-triphosphate, 5-iodo-2'-deoxycytidine-5'-triphosphate, 5-hydroxy-2'-deoxyuridine-5'-triphosphate, 4-thiothymidine-5'-triphosphate, 5-aza-2'-deoxyuridine-5'-triphosphate, 5-fluoro-2'-deoxyuridine-5'-triphosphate, 06-methyl-2'-deoxyguanosine-5'-triphosphate, N2-methyl-2'-deoxyguanosine-5'-triphosphate, 8-oxo-2'-deoxyguanosine-5'-triphosphate or thiothymidine-5'-triphosphate. In some embodiments, the mass-modified nucleobase comprises 15N or 13C or both 15N and 13C.

[0059] In some embodiments of the present invention, at least one bacterial nucleic acid segment is amplified in the process of identifying the bioagent. Thus,,the nucleic acid segments that can be amplified by the primers disclosed herein and that provide enough variability to distinguish each individual bioagent and whose molecular masses are amenable to molecular mass determination are herein described as "bioagent identifying amplicons." The term "amplicon" as used herein, refers to a segment of a polynucleotide which is amplified in an amplification reaction. In some embodiments of the present invention, bioagent identifying amplicons comprise from about 45 to about 200 nucleobases (i.e. from about 45 to about 200 linked nucleosides), from about 60 to about 150 nucleobases, from about 75 to about 125 nucleobases.
One of ordinary skill in the art will appreciate that the invention embodies compounds of 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99,100,101,102,103,104,105,106,107,108,109,110,111,112,113,114,115,116,117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, and 200 nucleobases in length, or any range therewithin. It is the combination of the portions of the bioagent nucleic acid segment to which the primers hybridize (hybridization sites) and the variable region between the primer hybridization sites that comprises the bioagent identifying amplicon. Since genetic data provide the underlying basis for identification of bioagents by the methods of the present invention, it is prudent to select segments of nucleic acids which ideally provide enough variability to distinguish each individual bioagent and whose molecular mass is amenable to molecular mass determination.

.10060] In some embodiments, bioagent identifying amplicons amenable to molecular mass determination which are produced by the primers described herein are either of a length, size or mass compatible with the particular mode of molecular mass determination or compatible with a means of providing a predictable fragmentation pattern in order to obtain predictable fragments of a length compatible with the particular mode of molecular mass determination. Such means of providing a predictable fragmentation pattern of an amplification product include, but are not limited to, cleavage with restriction enzymes or cleavage primers, for example. Methods of using restriction enzymes and cleavage primers are well known to those with ordinary skill in the art.
[00611 In some embodiments, amplification products corresponding to bacterial bioagent identifying amplicons are obtained using the polymerase chain reaction (PCR) which is a routine method to those with ordinary skill in the molecular biology arts. Other amplification methods may be used such as ligase chain reaction (LCR), low-stringency single primer PCR, and multiple strand displacement amplification (MDA) which are also well known to those with ordinary skill.

[00621 In the context of this invention, a "bioagent" is any organism, cell, or virus, living or dead, or a nucleic acid derived from such an organism, cell or virus. Examples of bioagents include, but are not limited, to cells, (including but not limited to human clinical samples, bacterial cells and other pathogens), viruses, fungi, protists, parasites, and pathogenicity markers (including but not limited to: pathogenicity islands, antibiotic resistance genes, virulence factors, toxin genes and other bioregulating compounds). Samples may be alive or dead or in a vegetative state (for example, vegetative bacteria or spores) and may be encapsulated or bioengineered. In the context of this invention, a "pathogen" is a bioagent which causes a disease or disorder.

[0063] In the context of this invention, the term "unknown bioagent" may mean either: (i) a bioagent whose existence is known (such as the well known bacterial species Staphylococcus aureus for example) but which is not known to be in a sample to be analyzed, or (ii) a bioagent whose existence is not known (for example, the SARS coronavirus was unknown prior to April 2003). For example, if the method for identification of coronaviruses disclosed in commonly owned 'U.S. published Patent 2005-02663 97 - was to be employed prior to April 2003 to identify the SARS coronavirus in a clinical sample, both meanings of "unknown" bioagent are applicable since the SARS coronavirus was unknown to science prior to April, 2003 and since it was not known what bioagent (in this case a coronavirus) was present in the sample. On the other hand, if the method of U.S. published Patent 2005-0266397' was to be employed subsequent to April 2003 to identify the SARS
coronavirus in a clinical sample, only the first meaning (i) of "unknown" bioagent would apply since the SARS coronavirus became known to science subsequent to April 2003 and since it was not known what bioagent was present in the sample.

[0064] The employment of more than one bioagent identifying amplicon for identification of a bioagent is herein referred to as "triangulation identification."
Triangulation identification is pursued by analyzing a plurality of bioagent identifying amplicons selected within multiple core genes. This process is used to reduce false negative and false positive signals, and enable reconstruction of the origin of hybrid or otherwise engineered bioagents. For example, identification of the three part toxin genes typical of B. anthracis (Bowen et al., J. Appl.
Microbiol., 1999, 87, 270-278) in the absence of the expected signatures from the B. anthracis genome would suggest a genetic engineering event.

[0065] In some embodiments, the triangulation identification process can be pursued by characterization of bioagent identifying amplicons in a massively parallel fashion using the polymerase chain reaction (PCR), such as multiplex PCR where multiple primers are employed in the same amplification reaction mixture, or PCR in multi-well plate format wherein a different and unique pair of primers is used in multiple wells containing otherwise identical reaction mixtures. Such multiplex and multi-well PCR methods are well known to those with ordinary skill in the arts of rapid throughput amplification of nucleic acids.

[0066] In some embodiments, the molecular mass of a particular bioagent identifying amplicon is determined by mass spectrometry. Mass spectrometry has several advantages, not the least of which is high bandwidth characterized by the ability to separate (and isolate) many molecular peaks across a broad range of mass to charge ratio (mlz). Thus, mass spectrometry is intrinsically a parallel detection scheme without the need for radioactive or fluorescent labels, since every amplification product is identified by its molecular mass. The current state of the art in mass spectrometry is such that less than femtomole quantities of material can be readily analyzed to afford information about the molecular contents of the sample. An accurate assessment of the molecular mass of the material can be quickly obtained, irrespective of whether the molecular weight of the sample is several hundred, or in excess of one hundred thousand atomic mass units (amu) or Daltons.

[0067] In some embodiments, intact molecular ions are generated from amplification products using one of a variety of ionization techniques to convert the sample to gas phase. These ionization methods include, but are not limited to, electrospray ionization (ES), matrix-assisted laser desorption ionization (MALDI) and fast atom bombardment (FAB). Upon ionization, several peaks are observed from one sample due to the formation of ions with different charges.
Averaging the multiple readings of molecular mass obtained from a single mass spectrum affords an estimate of molecular mass of the bioagent identifying amplicon.
Electrospray ionization mass spectrometry (ESI-MS) is particularly useful for very high molecular weight polymers such as proteins and nucleic acids having molecular weights greater than 10 kDa, since it yields a distribution of multiply-charged molecules of the sample without causing a significant amount of fragmentation.

[0068] The mass detectors used in the methods of the present invention include, but are not limited to, Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR-MS), time of flight (TOF), ion trap, quadrupole, magnetic sector, Q-TOF, and triple quadrupole.

[0069] In some embodiments, conversion of molecular mass data to a base composition is useful for certain analyses. As used herein, a "base composition" is the exact number of each nucleobase (A, T, C and G). For example, amplification of nucleic acid of Neisseria meningitidis with a primer pair that produces an amplification product from nucleic acid of 23S rRNA that has a molecular mass (sense strand) of 28480.75124, from which a base composition of A25 G27 C22 T18 is assigned from a list of possible base compositions calculated from the molecular mass using standard known molecular masses of each of the four nucleobases.

[0070] In some embodiments, assignment of base compositions to experimentally determined molecular masses is accomplished using "base composition probability clouds."
Base compositions, like sequences, vary slightly from isolate to isolate within species. It is possible to manage this diversity by building "base composition probability clouds" around the composition constraints for each species. This permits identification of organisms in a fashion similar to sequence analysis. A "pseudo four-dimensional plot" (Figure 1) can be used to visualize the concept of base composition probability clouds. Optimal primer design requires optimal choice of bioagent identifying amplicons and maximizes the separation between the base composition signatures of individual bioagents. Areas where clouds overlap indicate regions that may result in a misclassification, a problem which is overcome by a triangulation identification process using bioagent identifying amplicons not affected by overlap of base composition probability clouds.

[0071] In some embodiments, base composition probability clouds provide the means for screening potential primer, pairs in order to avoid potential misclassifications of base compositions. In other embodiments, base composition probability clouds provide the means for predicting the identity of a bioagent whose assigned base composition was not previously observed and/or indexed in a bioagent identifying amplicon base composition database due to evolutionary transitions in its nucleic acid sequence. Thus, in contrast to probe-based techniques, mass spectrometry determination of base composition does not require prior knowledge of the composition or sequence in order to make the measurement.

[0072] The present invention provides bioagent classifying information similar to DNA
sequencing and phylogenetic analysis at a level sufficient to identify a given bioagent.
Furthermore, the process of determination of a previously unknown base composition for a given bioagent (for example, in a case where sequence information is unavailable) has downstream utility by providing additional bioagent indexing information with which to populate base composition databases. The process of future bioagent identification is thus greatly improved as more BCS indexes become available in base composition databases.

[0073] In one embodiment, a sample comprising an unknown bioagent is contacted with a pair of primers which provide the means for amplification of nucleic acid from the bioagent, and a known quantity of a polynucleotide that comprises a calibration sequence. The nucleic acids of the bioagent and of the calibration sequence are amplified and the rate of amplification is reasonably assumed to be similar for the nucleic acid of the bioagent and of the calibration sequence. The amplification reaction then produces two amplification products:
a bioagent identifying amplicon and a calibration amplicon. The bioagent identifying amplicon and the calibration amplicon should be distinguishable by molecular mass while being amplified at essentially the same rate. Effecting differential molecular masses can be accomplished by choosing as a calibration sequence, a representative bioagent identifying amplicon (from a specific species of bioagent) and performing, for example, a 2 to 8 nucleobase deletion or insertion within the variable region between the two priming sites. The amplified sample containing the bioagent identifying amplicon and the calibration amplicon is then subjected to molecular mass analysis by mass spectrometry, for example. The resulting molecular mass analysis of the nucleic acid of the bioagent and of the calibration sequence provides molecular mass data and abundance data for the nucleic acid of the bioagent and of the calibration sequence. The molecular mass data obtained for the nucleic acid of the bioagent enables identification of the unknown bioagent and the abundance data enables calculation of the quantity of the bioagent, based on the knowledge of the quantity of calibration polynucleotide contacted with the sample.

[0074] In some embodiments, the identity and quantity of a particular bioagent is determined using the process illustrated in Figure 7. For instance, to a sample containing nucleic acid of an unknown bioagent are added primers (500) and a known quantity of a calibration polynucleotide (505). The total nucleic acid in the sample is subjected to an amplification reaction (510) to obtain amplification products. The molecular masses of amplification products are determined (515) from which are obtained molecular mass and abundance data. The molecular mass of the bioagent identifying amplicon (520) provides the means for its identification (525) and the molecular mass of the calibration amplicon obtained from the calibration polynucleotide (530) provides the means for its identification (535). The abundance data of the bioagent identifying amplicon is recorded (540) and the abundance data for the calibration data is recorded (545), both of which are used in a calculation (550) which determines the quantity of unknown bioagent in the sample.

[0075] In some embodiments, construction of a standard curve where the amount of calibration polynucleotide spiked into the sample is varied, provides additional resolution and improved confidence for the determination of the quantity of bioagent in the sample.
The use of standard curves for analytical determination of molecular quantities is well known to one with ordinary skill and can be performed without undue experimentation.

[0076] In some embodiments, multiplex amplification is performed where multiple bioagent identifying amplicons are amplified with multiple primer pairs which also amplify the corresponding standard calibration sequences. In this or other embodiments, the standard calibration sequences are optionally included within a single vector which functions as the calibration polynucleotide. Multiplex amplification methods are well known to those with ordinary skill and can be performed without undue experimentation.

[0077] In some embodiments, the calibrant polynucleotide is used as an internal positive control to confirm that amplification conditions and subsequent analysis steps are successful in producing a measurable amplicon. Even in the absence of copies of the genome of a bioagent, the calibration polynucleotide should give rise to a calibration amplicon.
Failure to produce a measurable calibration amplicon indicates a failure of amplification or subsequent analysis step such as amplicon purification or molecular mass determination. Reaching a conclusion that such failures have occurred is in itself, a useful, event.

[0078] In some embodiments, the calibration sequence is inserted into a vector which then itself functions as the calibration polynucleotide. In some embodiments, more than one calibration sequence is inserted into the vector that functions as the calibration polynucleotide. Such a calibration polynucleotide is herein termed a "combination calibration polynucleotide." The process of inserting polynucleotides into vectors is routine to those skilled in the art and can be accomplished without undue experimentation. Thus, it should be recognized that the calibration method should not be limited to the embodiments described herein. The calibration method can be applied for determination of the quantity of any bioagent identifying amplicon when an appropriate standard calibrant polynucleotide sequence is designed and used.
The process of choosing an appropriate vector for insertion of a calibrant is also a routine operation that can be accomplished by one with ordinary skill without undue experimentation.

[0079] The present invention also provides kits for carrying out, for example, the methods described herein. In some embodiments, the kit may comprise a sufficient quantity of one or more primer pairs to perform an amplification reaction on a target polynucleotide from a bioagent to form a bioagent identifying amplicon. In some embodiments, the kit may comprise from one to fifty primer pairs, from one to twenty primer pairs, from one to ten primer pairs, or from two to five primer pairs. In some embodiments, the kit may comprise one or more primer pairs recited in Table 1.

[0080] In some embodiments, the kit may comprise one or more broad range survey primer(s), division wide primer(s), Glade group primer(s) or drill-down primer(s), or any combination thereof. A kit may be designed so as to comprise particular primer pairs for identification of a particular bioagent. For example, a broad range survey primer kit may be used initially to identify an unknown bioagent as a member of the Bacillus/Clostridia group.
Another example of a division-wide kit may be used to distinguish Bacillus anthracis, Bacillus cereus and Bacillus thuringiensis from each other. A Glade group primer kit may be used, for example, to identify an unknown bacterium as a member of the Bacillus cereus Glade group. A drill-down kit may be used, for example, to identify genetically engineered Bacillus anthracis. In some embodiments, any of these kits may be combined to comprise a combination of broad range survey primers and division-wide primers, Glade group primers or drill-down primers, or any combination thereof, for identification of an unknown bacterial bioagent.

[0081] In some embodiments, the kit may contain standardized calibration polynucleotides for use as internal amplification calibrants.

[0082] In some embodiments, the kit may also comprise a sufficient quantity of reverse transcriptase (if an RNA virus is to be identified for example), a DNA
polymerase, suitable nucleoside triphosphates (including any of those described above), a DNA
ligase, and/or reaction buffer, or any combination thereof, for the amplification processes described above. A kit may further include instructions pertinent for the particular embodiment of the kit, such instructions describing the primer pairs and amplification conditions for operation of the method. A kit may also comprise amplification reaction containers such as microcentrifuge tubes and the like. A kit may also comprise reagents or other materials for isolating bioagent nucleic acid or bioagent identifying amplicons from amplification, including, for example, detergents, solvents, or ion exchange resins which may be linked to magnetic beads. A kit may also comprise a table of measured or calculated molecular masses and/or base compositions of bioagents using the primer pairs of the kit.

[0083] In order that the invention disclosed herein may be more efficiently understood, examples are provided below. It should be understood that these examples are for illustrative purposes only and are not to be construed as limiting the invention in any manner.
Throughout these examples, molecular cloning reactions, and other standard recombinant DNA techniques, were carried out according to methods described in Maniatis et al., Molecular Cloning - A
Laboratory Manual, 2nd ed., Cold Spring Harbor Press (1989), using commercially available reagents, except where otherwise noted.

EXAMPLES
[0084] Example 1: Selection of Primers That Define Bioagent Identifying Amplicons [0085] For design of primers that define bacterial bioagent identifying amplicons, relevant sequences from, for example, GenBank are obtained, aligned and scanned for regions where pairs of PCR primers would amplify products of about 45 to about 200 nucleotides in length and distinguish species from each other by their molecular masses or base compositions. A typical process shown in Figure 2 is employed.

[0086] A database of expected base compositions for each primer region is generated using an in silico PCR search algorithm, such as (ePCR). An existing RNA structure search algorithm (Macke et al., Nuc. Acids Res., 2001, 29,4724-4735) has been modified to include PCR parameters such as hybridization conditions, mismatches, and thermodynamic calculations (SantaLucia, Proc. Natl. Acad. Sci.
U.S.A., 1998, 95,1460-1465 ). This also provides information on primer specificity of the selected primer pairs.

[0087] Table 1 represents a collection of primers (sorted by forward primer name) designed to identify bacteria using the methods herein described. The forward or reverse primer name indicates the gene region of bacterial genome to which the primer hybridizes relative to a reference sequence eg: the forward primer name 168 EC_1077 1106 indicates that the primer hybridizes to residues 1077-1106 of the gene encoding 16S ribosomal RNA in an E. coil reference sequence represented by a sequence extraction of coordinates 4033120..4034661 from GenBank gi number 16127994 (as indicated in Table 2). As an additional example: the forward primer name BONTA X52066 450 473 indicates that the primer hybridizes to residues 450-437 of the gene encoding Clostridium botulinum neurotoxin type A (BoNT/A) represented by GenBank Accession No. X52066 (primer pair name codes appearing in Table 1 are defined in Table 2). In Table 1, Lr = 5-propynyluracil; Ca = 5-propynylcytosine; * =
phosphorothioate linkage. The primer pair number is an in-house database index number.
Table 1: Primer Pairs for Identification of Bacterial Bioagents Primer For. For. Rev.
pair primer SEQ ID Rev. primer SEQ ID
number name Forward sequence NO: name Reverse sequence NO:

16SEC_1177 16S EC 108 ATGTTGGGTTAAGTCCC 1196_lOG_1 TGACGTCATGGCCACCTTC

265 2 1100-F GC 2 1196 lOG R C 373 16S EC 109 16S_EC_1175 011112T TTTAAGTCCCGCAACGA _1196_TMOD_ TTGACGTCATCCCCACCTT

16S_EC_109 TAGTCCCGCAACGAGCG 16S EC 1174 GACGTCATCCCCACCTTCC

16s-EC-119 CAAGTCATCATGGCCCT 16S EC 1525 16s-EC-122 GCTACACACGTGCTACA 16SEC_1303 CGAGTTGCAGACTGCGATC

16s-EC-130 CGGATTGGAGTCTGCAA 1 6S EC 1389 16S_EC_133 AAGTCGGAATCGCTAGT 16S_EC_1389 16S_EC_136 TACGGTGAATACGTTCC 16S EC 1485 ACCTTGTTACGACTTCACC

16S_EC_138 GCCTTGTACACACCTCC 16S_EC_1494 CACGGCTACCTTGTTACGA

16S_EC_138 CTTGTACACACCGCCCG 16S EC 1525 16S-EC-139 TTGTACACACCGCCCGT 16S_EC_1486 CCTTGTTACGACTTCACCC

16S_EC_30_ TGAACGCTGGTGGCATG 16S_EC_105_ TACGCATTACTCACCCGTC

16S EC 314 CACTGGAACTGAGACAC 16SEC_556_ CTTTACGCCCAGTAATTCC

16S_EC_38_ GTGGCATGCCTAATACA 16S_EC_101_ TTACTCACCCGTCCGCCGC

16S_EC_405 TGAGTGATGAAGGCCTT 16S EC 507 CGGCTGCTGGCACGAAGTT

16S_EC_49_ TAACACATGCAAGTCGA 16SEC104_ 16S-EC-49- TAACACATGCAAGTCGA 16SEC_1061 16S_EC_49_ TAACACATGCAAGTCGA 16S EC 880 16S EC 556 CGGAATTACTGGGCGTA 16S_EC_683_ 16S_EC_556 CGGAATTACTGGGCGTA 16S-EC-774_ GTATCTAATCCTGTTTGCT

16S_EC683 GTGTAGCGGTGAAATGC 16SEC_1303 CGAGTTGCAGACTGCGATC

16S_EC_683 GTGTAGCGGTGAAATGC 16S_EC_774_ GTATCTAATCCTGTTTGCT
9 700 F G 24 795.-R CCC 387 16S EC 683 GTGTAGCGGTGAAATGC 16S_EC_967_ 16S EC 713 AGAACACCGATGGCGAA 16S_EC_789_ CGTGGACTACCAGGGTATC

16S_EC_713 _732_TMOD_ TAGAACACCGATGGCGA 16S_EC_789 TCGTGGACTACCAGGGTAT

16S EC 785 GGATTAGAGACCCTGGT 16S_EC_880_ 16SEC_785 _806_TMOD_ TGGATTAGAGACCCTGG 16S_EC_880_ 16S-EC-785 GGATTAGATACCCTGGT 16S_EC_880_ 12 810 F , AGTCCACGC 31 897 2 R GGCCGTACTCCCCAGGCG 391 16s-EC-789 TAGATACCCTGGTAGTC 16S_EC_880_ 16S_EC_789 TAGATACCCTGGTAGTC 168 EC_882_ 16S EC 791 GATACCCTGGTAGTCCA 16S_EC_886_ 16S_EC_8_2 AGAGTTTGATCATGGCT 16S EC_342_ 16S-EC-804 ACCACGCCGTAAACGAT 165_EC_909_ CCCCCGTCAATTCCTTTGA

16S_EC_937 AAGCGGTGGAGCATGTG 1 6S EC 1220 ATTGTAGCACGTGTGTAGC

16S_EC_960 TTCGATGCAACGCGAAG 16SEC_1054 ACGAGCTGACGACAGCCAT

16s-EC-960 16SEC_1054 _981_TMOD_ TTTCGATGCAACGCGAA 1073_TMOD_ TACGAGCTGACGACAGCCA
348 F GAACCT'' 38 R TG 363 16S_EC_969 ACGCGAAGAACCTTA 1 6S EC 1061 119 985 1P F UaC 39 1078 2P R ACGACACGAGUaCaGACGAC 364 16S_EC_969 168 EC 1389 16S_EC_971 GCGAAGAACCTTACCAG 1 6S EC 1043 ACAACCATGCACCACCTGT

16S_EC972 16S EC 1064 120 985 2P F CGAAGAAUaUaTTACC 42 1075 2P R ACACGAGUaCaGAC 365 16s-EC-972 16S EC 1064 23S_BRM_11 TGCGCGGAAGATGTAAC 23 8 BRM 117 TCGCAGGCTTACAGAACGC

23S_BS_- AAACTAGATAACAGTAG 23S BS 5 21 23s-EC-160 TACCCCAAACCGACACA 23S EC 1686 16 6 1'843-F C 48 1924 R GACCGTTATAGTTACGGCC 404 23SEC_182 238 EC 1906 618 43 TM0 TCTGACACCTGCCCGGT _1924_TMOD_ TGACCGTTATAGTTACGGC

23S_EC_183 ACCTGCCCAGTGCTGGA 23S EC 1919 23S_EC_187 GGGAACTGAAACATCTA 238 EC_242_ 23S_EC_23_ 23S-EC-115-23S_EC_243 AAGGTACTCCGGGGATA 23S_EC_2490 AGCCGACATCGAGGTGCCA

23S_EC_258 TAGAACGTCGCGAGACA 23S_EC_2658 AGTCCATCCCGGTCCTCTC

23S_EC_259 GACAGTTCGGTCCCTAT 23S_EC_2653 23S EC 264 CTGTCCCTAGTACGAGA 23SEC_2751 GTTTCATGCTTAGATGCTT

23S_EC_264 TCTGTCCCTAGTACGAG 23S EC 2744 23S_EC_264 CTGTTCTTAGTACGAGA 23S EC 2745 TTCGTGCTTAGATGCTTTC

6_2667_TMO AGGACC _2765_TMOD_ CAG
D F R

240 3 2-j69 -F TAGTACGAGAGGACCGG 62 2758 R ATC 412 23S_EC_493 GGGGAGTGAAAGAGATC 23S_EC_551_ ACAAAAGGCACGCCATCAC

23S-EC-493 GGGGAGTGAAAGAGATC 23S_EC_551_ ACAAAAGGTACGCCGTCAC

ABMLST-OIF007_120 TCGTGCCCGCAATTTGC 01F007 1266 TAATGCCGGGTAGTGCAAT

ABMLST-0IF007 120 TCGTGCCCGCAATTTGC OIF007_1299 ABMLST-OIF007_123 TTGTAGCACAGCAAGGC 01F007_1335 TGCCATCCATAATCACGCC

AB_MLST-OIF007_132 TAGGTTTACGTCAGTAT 0IF007_1422 TGCCAGTTTCCACATTTCA

ABMLST-OIF007_134 TCGTGATTATGGATGGC OIF007_1470 TCGCTTGAGTGTAGTCATG

ABMLST-11- AB_MLST-11-OIF007135 TTATGGATGGCAACGTG 01F007_1470 TCGCTTGAGTGTAGTCATG

ABMLST-OIF007_138 TCTTTGCCATTGAAGAT OIF007_1470 TCGCTTGAGTGTAGTCATG

ABMLST-ABMLST-OIF007_156 TTGCCAATGATATTCGT OIF007_1656 TGAGTCGGGTTCACTTTAC

ABMLST-OIF007_161 TCGGCGAAATCCGTATT 0IF007 1731 TACCGGAAGCACCAGCGAC

ABMLST-ABMLST-OIF007179 CTATTCAGTTGCTTGGT OIF007_1876 TGAATTATGCAAGAAGTGA

ABMLST-ABMLST-OIF007_185 TATTGTTTCAAATGTAC 01F007 291 TCACAGGTTCTACTTCATC

AB_MLST-OIF007_197 TGGTTATGTACCAAATA OIF007_2097 TGACGGCATCGATACCACC

ABMLST-1154 239F TCGATGCACTTGATGTA 78 344_R TTCACAGG 433 AB MLST-11- AB_MLST-11-ABMLST-M

-AB_MLST-11-07547 TCAACCTGACTGCGTGA OIF007_656_ TACGTTCTACGATTTCTTC

1156 F ATGGTTGT 81 686 P. ATCAGGTACATC 436 ABMLST-ABMLST-11- AB_MLST-11-OIF007_62_ TGAGATTGCTGAACATT 0IF007_169_ TTGTACATTTGAAACAATA

ASD_FRT_1_ TTGCTTAAAGTTGGTTT ASD_FRT_86_ TGAGATGTCGAAAAAAACG

ASD_FRT_43 TCAGTTTTAATGTCTCG ASD_FRT_129 TCCATATTGTTGCATAAAA

BONTA_X520 66450473 TCTAGTAATAATAGGAC BONTA_X5206 TAACCATTTCGCGTAAGAT

BONTAX520 T*Ua*CaAGTAATAATAG BONTA X5206 66_450_473 GA*Ua*Ua*Ua*Ca*UaAG 6 517 539P TAACCA*Ca*Ca*Ca*UaGC
486 P F C 87 R GTAAGA*Ca*Ca*UAA 441 BONTA_X520 66_538_552 BONTA_X5206 66538_552 TA*CaGGC*Ca*Ua*CaA 6 647 660P TG*Ca*CaA*Ua*CaG*Ua*C
482 P F *Ua*Ca*UaAA 88 R aGGAT 443 66_591620 TGAGTCACTTGAAGTTG BONTA_X5206 TCATGTGCTAATGTTACTG

BONTA_X520 66701720 GAATAGCAATTAATCCA BONTA_X5206 66_701_720 GAA*CaAG*UaAA*Ca*C 6 759 775P TTA*Ua*Ca*Ca*Ua*CaAA*
484 P F aAA*Ca*Ua*UaAAAT 90 R Ua*Ua*UaA*Ua*CaC 444 947_33407_ TCAGTTCCGTTATCGCC 4733494_33 TGCGGGCTGGTTCAACAAG

CAF1_AF053 CAF1_AF0539 94733435_ TGGAACTATTGCAACTG 47_3349933 CAF1_AF053 CAF1AF0539 947_33515_ TCACTCTTACATATAAG 473359533 TCCTGTTTTATAGCCGCCA

CAF1_AF053 CAF1AF0539 947_33687_ TCAGGATGGAAATAACC 473375533 TCAAGGTTCTCACCGTTTA

CAPC_BA_10 GTTATTTAGCACTCGTT CAPC_BA_180 TGAATCTTGAAACACCATA

CAPC_BA_11 ACTCGTTTTTAATCAGC CAPC_BA_185 TGAATCTTGAAACACCATA

CAPC_BA_27 GATTATTGTTATCCTGT CAPC_BA_349 GTAACCCTTGTCTTTGAAT

CAPC_BA27 4 303 TMOD TGATTATTGTTATCCTG CAPC_BA_349 TGTAACCCTTGTCTTTGAA

CAPC_BA27 TTATTGTTATCCTGTTA CAPC_BA_358 GGTAACCCTTGTCTTTGAA

CAPC_BA_28 GTTATCCTGTTATGCCA CAPC_BA_361 CAPC_BA31 CCGTGGTATTGGAGTTA CAPC_BA_361 27 5 334 F_ TTG 101 378-R TGGTAACCCTTGTCTTTG 454 CJSTCJ_12 AGTTATAAACACGGCTT CJSTCJ134 TCGGTTTAAGCTCTACATG

CJST_CJ_12 TGGCTTATCCAAATTTA CJST_CJ_140 TTTGCTCATGATCTGCATG

CJSTCJ_16 TTATCGTTTGTGGAGCT CJST_CJ_172 TGCAATGTGTGCTATGTCA

1045 68 1700 F TTGCTAAATTTAGAGA 106 4 1799 P. TTGGATG 460 CJSTCJ_16 TGATTTTGCTAAATTTA CJSTCJ_179 TATGTGTAGTTGAGCTTAC

CJST_CJ_18 TCCCAATTAATTCTGCC CJST_CJ_198 TGGTTCTTACTTGCTTTGC

CJSTCJ20 TCCCGGACTTAATATCA CJST_CJ 214 TCGATCCGCATCACCATCA

CJST_CJ21 TGCGGATCGTTTGGTGG CJSTCJ224 TCCACACTGGATTGTAATT
1059 65 2194 F TTGTAGATGAAAA 110 7 2278 P. TACCTTGTTCTTT 464 CJST_CJ_21 TCGTTTGGTGGTGGTAG CJST_CJ_228 TCTCTTTCAAAGCACCATT

CJST_CJ 21 TAGATGAAAAGGGCGAA CJSTCJ_228 TGAATTCTTTCAAAGCACC
1057 85 2212 F GTGGCTAATGG 112 3,2316 P. ATTGCTCATTATAGT 466 CJST_CJ 26 TGCCTAGAAGATCTTAA CJSTCJ_275 TTGCTGCCATAGCAAAGCC

CJST_CJ26 TCCCCAGGACACCCTGA CJST_CJ_276 TGTGCTTTTTTTGCTGCCA

CJST_CJ_28 TGGCATTTCTTATGAAG CJST_CJ_296 TGCTTCAAAACGCATTTTT

CJST_CJ_28 TGAAGCTTGTTCTTTAG CJST_CJ_297 TCCTCCTTGTGCCTCAAAA

CJST_CJ_32 TTTGATTTTACGCCGTC CJST_CJ_335 TCAAAGAACCCGCACCTAA

CJST_CJ_36 TCCTGTTATCCCTGAAG CJST_CJ_443 TACAACTGGTTCAAAAACA

TCCTGTTATCCCTGAAG
CJST_CJ_36 TAGTTAATCAAGTTTGT CJST_CJ_442 TCAACTGGTTCAAAAACAT

TAGGCGAAGATATACAA
CJST_CJ_5_ AGAGTATTAGAAGCTAG CJST_CJ_104 TCCCTTATTTTTCTTTCTA

CJST_CJ58 TCCAGGACAAATGTATG CJST_CJ_663 TTCATTTTCTGGTCCAAAG

CJSTCJ59 TGAAAAATGTCCAAGAA CJST_CJ711 TCCCGAACAATGAGTTGTA

CTXA_VBC_1 TCTTATGCCAAGAGGAC CTXA_VBC_19 TGCCTAACAAATCCCGTCT

CYA_BA_105 GAAAGAGTTCGGATTGG CYA_BA_1112 CYA_BA_134 ACAACGAAGTACAATAC CYA_BA_1426 CTTCTACATTTTTAGCCAT

CYA_BA_135 CGAAGTACAATACAAGA CYA_BA_1448 TGTTAACGGCTTCAAGACC

CYA_BA_135 CYA BA 1448 3137 9 TMO TCGAAGTACAATACAAG _1467_TMOD_ TTGTTAACGGCTTCAAGAC

CYA_BA_135 ACAATACAAGACAAAAG CYA_BA_1447 CYABA914 CAGGTTTAGTACCAGAA CYA_BA_999_ ACCACTTTTAATAAGGTTT

CYA_BA_916 GGTTTAGTACCAGAACA CYA_BA_1003 CCACTTTTAATAAGGTTTG

DNAK_EC_42 CGGCGTACTTCAACGAC DNAK_EC_503 CGCGGTCGGCTCGTTGATG

GALE FRT1 TTATCAGCTAGACCTTT GALE FRT~24 TCACCTACAGCTTTAAAGC
1102 68 199 F_ TAGGTAAAGCTAAGC 133 1 269 P. CAGCAAAATG 486 GALE FRT3 TCCAAGGTACACTAAAC GALE_FRT_39 TCTTCTGTAAAGGGTGGTT

GALE_FRT_8 TCAAAAAGCCCTAGGTA GALE_FRT_90 TAGCCTTGGCAACATCAGC

GLTA_RKP_1 TCCGTTCTTACAAATAG GLTA_RKP_11 TTGGCGACGGTATACCCAT
1092 023 1055 F CAATAGAACTTGAAGC 136 29 1156 P. AGCTTTATA 489 GLTA_RKP_1 0431072_2 TGGAGCTTGAAGCTATC GLTA_RKP11 TGAACATTTGCGACGGTAT
1093 F GCTCTTAAAGATG 137 38 1162 _R ACCCAT 490 GLTA_RKP_1 04310723 TGGAACTTGAAGCTCTC GLTA_RKP_11 TGTGAACATTTGCGACGGT

GLTARKP_1 TGGGACTTGAAGCTATC GLTARKP_11 TGAACATTTGCGACGGTAT

GLTARKP4 TCTTCTCATCCTATGGC GLTA_RKP_49 TGGTGGGTATCTTAGCAAT

GLTA_RKP_4 TCTTCTCATCCTATGGC GLTA_RKP_50 TGCGATGGTAGGTATCTTA

GROL_EC21 GGTGAAAGAAGTTGCCT GROL_EC_328 TTCAGGTCCATCGGGTTCA

GROLEC49 ATGGACAAGGTTGGCAA GROL_EC_577 TAGCCGCGGTCGAATTGCA

GROL EC 51 AAGGAAGGCGTGATCAC GROL_EC_571 CCGCGGTCGAATTGCATGC

GROL_EC_94 TGGAAGATCTGGGTCAG GROL_EC_103 CAATCTGCTGACGGATCTG

GYRAAF100 TCTGCCCGTGTCGTTGG 57_119142 TCGAACCGAAGTTACCCTG

GYRAAF100 GYRA_AF1005 55770_94 TCCATTGTTCGTATGGC 57178201 TGCCAGCTTAGTCATACGG

GYRB_AB008 GYRB_AB0087 700_19_40_ TCAGGTGGCTTACACGG 00_111_140 TATTGCGGATCACCATGAT

GYRB_AB008 GYRB_AB0087 700_265_29 TCTTTCTTGAATGCTGG 00369_395 TCGTTGAGATGGTTTTTAC

GYRB_ABO08 GYRE ABO087 700_368_39 TCAACGAAGGTAAAAAC 00_466_494_ TTTGTGAAACAGCGAACAT

GYRB_AB008 GYRBABOO87 700_477_50 TGTTCGCTGTTTCACAA 00_611_632_ TCACGCGCATCATCACCAG

GYRB_AB008 GYRB_AB0087 700_760_78 TACTTACTTGAGAATCC 00_862_888_ TCCTGCAATATCTAATGCA

GYRB_AB008 GYRB_AB0087 700_760_78 TACTTACTTGAGAATCC 00_862_888_ ACCTGCAATATCTAATGCA

HFLBEC_10 TGGCGAACCTGGTGAAC HFLB_EC_114 CTTTCGCTTTCTCGAACTC

HUPB_CJ_11 TAGTTGCTCAAACAGCT HUPB_CJ_157 TCCCTAATAGTAGAAATAA

HUPB_CJ_76 TCCCGGAGCTTTTATGA HUPB_CJ_114 TAGCCCAGCTGTTTGAGCA

ICDCXB17 TCGCCGTGGAAAAATCC ICD_CXB_224 TAGCCTTTTCTCCGGCGTA

ICD_CXB_93 TCCTGACCGACCCATTA ICD_CXB_172 TAGGATTTTTCCACGGCGG

INFB_EC_11 GTCGTGAAAACGAGCTG INFB_EC_117 INFB EC 13 TGCGTTTACCGCAATGC INFB_EC_141 INFBEC_13 TGCTCGTGGTGCACAAG INFB EC 143 TGCTGCTTTCGCATGGTTA

INFBEC_13 INFB_EC_143 65_1393_TM TTGCTCGTGGTGCACAA 914 67 TMOD TTGCTGCTTTCGCATGGTT

INFBEC_19 CGTCAGGGTAAATTCCG INFB EC 203 AACTTCGCCTTCGGTCATG

INV_U22457 1558_1581 TGGTAACAGAGCCTTAT INV_U22457_ TTGCGTTGCAGATTATCTT

INV_U22457 TGGCTCCTTGGTATGAC INV_U22457_ TGTTAAGTGTGTTGCGGCT

INV_U22457 TGCTGAGGCCTGGACCG INV_U22457_ TCACGCGACGAGTGCCATC

INV_U22457 TTATTTACCTGCACTCC INV U22457 TGACCCAAAGCTGAAAGCT
780 834_858F CACAACTG 166 942-966 R TTACTG 521 IPAH SGF 2 TGAGGACCGTGTCGCGC IPAHSGF_30 TCCTTCTGATGCCTGATGG

IPAH SGF 4 TCAGACCATGCTCGCAG IPAH_SGF_52 IS1111A_NC IS1111A NCO
002971_686 TCAGTATGTATCCACCG 029716928 TAAACGTCCGATACCAATG

IS1111A NC IS1111A_NCO
002971_745 TGGGTGACATTCATCAA 02971_7529_ TCAACAACACCTCCTTATT

LEF_BA_103 LEF BA 1119 LEF_BA_103 CAAGAAGAAAAAGAGCT LEF_BA_1119 AGATAAAGAATCACGAATA

LEF_BA_756 AGCTTTTGCATATTATA LEF_BA_843_ TCTTCCAAGGATAGATTTA

LEF_BA_756 _781_TMOD_ TAGCTTTTGCATATTAT LEF_BA_843_ TTCTTCCAAGGATAGATTT

LEF_BA_758 CTTTTGCATATTATATC LEF_BA_843_ AGGATAGATTTATTTCTTG

LEF_BA795 TTTACAGCTTTATGCAC LEF_BA883 40 899 F_ CAACGGATGCTGGCAAG 178 958 R G 533 3_2366996_ TGTAGCCGCTAAGCACT 2367073_23 TCTCATCCCGATATTACCG

3_2367172 TGGACGGCATCACGATT 236724923 TGGCAACAGCTCAACACCT

MECAY1405 MECA_Y14051 1_3645367 TGAAGTAGAAATGACTG 36903719_ TGATCCTGAATGTTTATAT

MECA_Y1405 MECA_Y14051 13774_380 TAAAACAAACTACGGTA 3828_3854 TCCCAATCTAACTTCCACA

879 0_F

MECA_Y1405 MECAY14051 1 4510_453 TGTACTGCTATCCACCC 45864610 TATTCTTCGTTACTCATGC
880 0^F TCAA 184 R CATACA 539 MECA_Y1405 MECA_Y14051 882 OP F TUaUAUaU'UaCaUAA 185 R CaAUaCaUaACaGU'UA 540 MECA_Y1405 MECAY14051 1_4520_453 4600 4610P
883 OP F TUaUaAUaUaUaCaUsAA 185 R CaACaCaUaCaCaUfGCaT 541 MECA_Y1405 MECA_Y14051 1_4669469 TCACCAGGTTCAACTCA _4765_4793_ TAACCACCCCAAGATTTAT

MECIA_Y140 MECIAY1405 51331533 TTACACATATCGTGAGC 1_33673393 TGTGATATGGAGGTGTAGA

OMPA_AY485 OMPAAY4852 22727230 TTACTCCATTATTGCTT 27_364388 GAGCTGCGCCAACGAATAA

OMPAAY485 OMPA_AY4852 OMPA_AY485 OMPA_AY4852 OMPA AY485 OMPA_AY4852 227_415_44 TGCCTCGAAGCTGAATA 27_514_546_ TCGGGCGTAGTTTTTAGTA

227_494_52 TCAACGGTAACTTCTAT 27_569_596_ TCGTCGTATTTATAGTGAC

OMPA_AY415 OMPAAY4852 OMPA_AY485 OMPA_AY4852 227_555_58 TCCGTACGTATTATTAG 27_635_662 TCAACACCAGCGTTACCTA

OMPA_AY485 OMPA_AY4852 227_556_58 TCGTACGTATTATTAGG 27_659_683 TCGTTTAAGCGCCAGAAAG

227_657_67 TGTTGGTGCTTTCTGGC 27_739765 TAAGCCAGCAAGAGCTGTA

OMPA_AY485 OMPA_AY4852 227_660_68 TGGTGCTTTCTGGCGCT 27_786807_ TACAGGAGCAGCAGGCTTC

OMPB_RKP_1 TCTACTGATTTTGGTAA OMPB_RKP_12 TAGCAGCAAAAGTTATCAC

OMPB_RKP_3 TGCAAGTGGTACTTCAA OMPB_RKP_35 TGGTTGTAGTTCCTGTAGT

OMPB_RKP8 TTACAGGAAGTTTAGGT OMPB_RKP97 TCCTGCAGCTCTACCTGCT

PAG BA 122 CAGAATCAAGTTCCCAG PAG_BA190_ CCTGTAGTAGAAGAGGTAA

PAG_BA_123 AGAATCAAGTTCCCAGG PAG BA 187_ CCCTGTAGTAGAAGAGGTA

PAG_BA_269 AATCTGCTATTTGGTCA PAG BA 326_ PAG_BA_655 GAAGGATATACGGTTGA PAG BA 755_ PAG_BA_753 TCCTGAAAAATGGAGCA PAG BA 849_ TCGGATAAGCTGCCACAAG

PAG_BA_763 TGGAGCACGGCTTCTGA PAG_BA_849 TCGGATAAGCTGCCACAAG

PARC_X9581 9123_147 GGCTCAGCCATTTAGTT PARCX95819 TCGCTCAGCAATAATTCAC

PARC_X9581 TCAGCGCGTACAGTGGG PARC_X95819 TTCCCCTGACCTTCGATTA

PARC_X9581 TGGTGACTCGGCATGTT PARC_X95819 GGTATAACGCATCGCAGCA

PARC_X9581 TGGTGACTCGGCATGTT PARC_X95819 TTCGGTATAACGCATCGCA

45_718672 TTATACCGGAAACTTCC 57257_7280 TAATGCGATACTGGCCTGC

45_737774 TGACATCCGGCTCACGT 574347462 TGTAAATTCCGCAAAGACT

PLAAF0539 PLA_AF05394 45_738274 TCCGGCTCACGTTATTA 574827502 TGGTCTGAGTACCTCCTTT

45_748175 TGCAAAGGAGGTACTCA 575397562 TATTGGAAATACCGGCAGC

RECA_AF251 RECA_AF2514 469_43_68_ TGGTACATGTGCCTTCA 69_140_163_ TTCAAGTGCTTGCTCACCA

RNASEP_BDP TGGCACGGCCATCTCCG RNASEP_BDP_ TCGTTTCACCCTGTCATGC

RNASEP_BKM TGCGGGTAGGGAGCTTG RNASEP_BKM_ TCCGATAAGCCGGATTCTG

RNASEP_BKM TCCTAGAGGAATGGCTG RNASEP_BKM_ TGCCGATAAGCCGGATTCT

RNASEP_BRM TACCCCAGGGAAAGTGC RNASEP_BRM_ TCTCTTACCCCACCCTTTC

RNASEP_BRM TAAACCCCATCGGGAGC RNASEP_BRM TGCCTCGTGCAACCCACCC

RNASEP_BRM TAAACCCCATCGGGAGC RNASEP_BRM_ TGCCTCGCGCAACCTACCC

RNASEP_BS GAGGAAAGTCCATGCTC RNASEP_BS_3 GTAAGCCATGTTTTGTTCC

RNASEP_BS_ GAGGAAAGTCCATGCTC RNASEP_EC_3 RNASEP_BS_ GAGGAAAGTCCATGCTC RNASEP_SA_3 ATAAGCCATGTTCTGTTCC

RNASEP_CLB TAAGGATAGTGCAACAG RNASEPCLB_ TTTACCTCGCCTTTCCACC

RNASEP_CLB TAAGGATAGTGCAACAG RNASEPCLB TGCTCTTACCTCACCGTTC

RNASEP_EC_ RNASEP_BS_3 GTAAGCCATGTTTTGTTCC

RNASEP_EC_ RNASEP_EC_3 RNASEP_EC_ RNASEP EC_3 RNASEP_EC_ RNASEP_SA_3 ATAAGCCATGTTCTGTTCC

RNASEP_RKP TCTAAATGGTCGTGCAG RNASEP_RKP_ TCTATAGAGTCCGGACTTT

RNASEP RKP TGGTAAGAGCGCACCGG RNASEP_RKP_ TCAAGCGATCTACCCGCAT

RNASEP_RKP TAAGAGCGCACCGGTAA RNASEPRKP_ TCAAGCGATCTACCCGCAT

RNASEP_RKP TGCATACCGGTAAGTTG RNASEP RKP_ TCAAGCGATCTACCCGCAT

RNASEP_RKP TCCACCAAGAGCAAGAT RNASEP_RKP_ TCAAGCGATCTACCCGCAT

RNASEP_SA_ GAGGAAAGTCCATGCTC RNASEP_BS_3 GTAAGCCATGTTTTGTTCC

RNASEPSA GAGGAAAGTCCATGCTC RNASEP_EC_3 RNASEP_SA_ GAGGAAAGTCCATGCTC RNASEP_SA_3 ATAAGCCATGTTCTGTTCC

RNASEP_SA_ GAGGAAAGTCCATGCTC RNASEP_SA_3 ATAAGCCATGTTCTGTTCC

RNASEP_VBC TCCGCGGAGTTGACTGG RNASEP_VBC_ TGACTTTCCTCCCCCTTAT

RPLB_EC_65 GACCTACAGTAAGAGGT RPLB_EC_739 TCCAAGTGCTGGTTTACCC

RPLBEC_65 067 9 TMOD TGACCTACAGTAAGAGG RPLB_EC_739 TTCCAAGTGCTGGTTTACC

RPLB_EC 66 TGTAATGAACCCTAATG RPLB_EC_735 CCAAGTGCTGGTTTACCCC
73 9 698 F~ ACCATCCACACGG 233 761 R ATGGAGTA 586 RPLB_EC_67 TAATGAACCCTAATGAC RPLB_EC_737 TCCAAGTGCTGGTTTACCC

RPLB_EC_68 CATCCACACGGTGGTGG RPLB_EC_736 GTGCTGGTTTACCCCATGG

RPLB_EC68 CATCCACACGGTGGTGG RPLB_EC_743 TGTTTTGTATCCAAGTGCT

RPLB_EC68 8 710 T_MOD TCATCCACACGGTGGTG RPLB_EC_736 TGTGCTGGTTTACCCCATG

RPLB_EC_69 TCCACACGGTGGTGGTG RPLB_EC_737 TGTGCTGGTTTACCCCATG

RPOBEC_13 GACCACCTCGGCAACCG RPOBEC_143 RPOB_EC_15 TCAGCTGTCGCAGTTCA RPOMEC163 TCGTCGCGGACTTCGAAGC

RPOB_EC_18 TATCGCTCAGGCGAACT RPOB_EC_190 GCTGGATTCGCCTTTGCTA

RPOBEC_18 RPOBEC190 45_1866_TM TTATCGCTCAGGCGAAC 9 1929 TMOD TGCTGGATTCGCCTTTGCT

RPOB_EC_20 TCGTTCCTGGAACACGA RPOB_EC_204 TTGACGTTGCATGTTCGAG

RPOB_EC_37 TCAACAACCTCTTGGAG RPOB_EC_383 TTTCTTGAAGAGTATGAGC

RPOB_EC_37 CTTGGAGGTAAGTCTCA RPOBEC_382 CGTATAAGCTGCACCATAA

RPOB_EC_37 TGGGCAGCGTTTCGGCG RPOB_EC_386 TGTCCGACTTGACGGTTAG

RPOB_EC_37 TGGGCAGCGTTTCGGCG RPOB_EC_386 TGTCCGACTTGACGGTCAG

RPOBEC37 GGGCAGCGTTTCGGCGA RPOBEC_386 GTCCGACTTGACGGTCAAC

RPOB_EC_37 TGGGCAGCGTTTCGGCG RPOB_EC_386 TGTCCGACTTGACGGTCAA

OD F R
RPOB_EC_38 CAGCGTTTCGGCGAAAT RPOB_EC386 CGACTTGACGGTTAACATT

RPOCEC_10 18_1045_2_ CAAAACTTATTAGGTAA RPOCEC109 TCAAGCGCCATCTCTTTCG

RPOC_EC_10 CAAAACTTATTAGGTAA RPOCEC109 TCAAGCGCCATTTCTTTTG

RPOC_EC_10 CGTGTTGACTATTCGGG RPOCEC_109 ATTCAAGAGCCATTTCTTT

RPOC_EC_12 ACCCAGTGCTGCTGAAC RPOCEC129 GTTCAAATGCCTGGATACC
227 56 1277 F CGTGC 251 51315_-R CA 613 RPOC_EC_13 CGCCGACTTCGACGGTG RPOC_EC_143 RPOC_EC_13 RPOC_EC_143 74 1393 TM TCGCCGACTTCGACGGT 7_1455_TMOD TGAGCATCAGCGTGCGTGC

RPOCEC_15 TGGCCCGAAAGAAGCTG RPOC_EC162 ACGCGGGCATGCAGAGATG

RPOC_EC_21 TCAGGAGTCGTTCAACT RPOC_EC_222 TTACGCCATCAGGCCACGC

RPOCEC_21 CAGGAGTCGTTCAACTC RPOC_EC_222 RPOCEC_21 RPOCEC222 46 2174 TM TCAGGAGTCGTTCAACT 7_2245_TMOD TACGCCATCAGGCCACGCA

RPOCEC_21 78_2196_2_ TGATTCCGGTGCCCGTG RPOC_EC_222 TTGGCCATCAGACCACGCA

RPOCEC_21 TGATTCTGGTGCCCGTG RPOC_EC222 TTGGCCATCAGGCCACGCA

RPOC_EC_22 18_22412_ CTTGCTGGTATGCGTGG RPOC_EC_231 CGCACCATGCGTAGAGATG

RPOC_EC_22 CTGGCAGGTATGCGTGG RPOC_EC_231 CGCACCGTGGGTTGAGATG

RPOC_EC_22 RPOC_EC231 182241_TM TCTGGCAGGTATGCGTG 32337 TMOD TCGCACCGTGGGTTGAGAT

RPOC_EC_22 TGGTATGCGTGGTCTGA RPOC_EC_232 TGCTAGACCTTTACGTGCA

RPOC EC 23 TGCTCGTAAGGGTCTGG RPOC_EC_238 TACTAGACGACGGGTCAGG

RPOCEC_80 CGTCGTGTAATTAACCG RPOCEC865 ACGTTTTTCGTTTTGAACG

RPOC_EC_80 CGTCGGGTGATTAACCG RPOC_EC_865 GTTTTTCGTTGCGTACGAT

RPOC_EC91 TATTGGACAACGGTCGT RPOC_EC_100 TTACCGAGCAGGTTCTGAC

RPOC_EC_91 TCTGGATAACGGTCGTC RPOCEC_100 TCCAGCAGGTTCTGACGGA

RPOC_EC_99 CAAAGGTAAGCAAGGAC RPOC_EC_103 CGAACGGCCAGAGTAGTCA

RPOC_EC_99 CAAAGGTAAGCAAGGTC RPOC_EC_103 CGAACGGCCTGAGTAGTCA

SP101_SPET AACCTTAATTGGAAAGA SP101SPET1 CCTACCCAACGTTCACCAA

SP101_SPET SP101_SPET1 11_1_29_TM TAACCTTAATTGGAAAG 1_92116_TM TCCTACCCAACGTTCACCA

SP101_SPET SP101_SPET1 SP101SPET SP101_SPET1 11_115411 TCAATACCGCAACAGCG 112511277 TGACCCCAACCTGGCCTTT

SP101_SPET
11_118147 GCTGGTGAAAATAACCC SP101_SPET1 TGTGGCCGATTTCACCACC

36_F
R

11131413 TCGCAAAAAAATCCAGC 1_14031431 TAAACTATTTTTTTAGCTA

SP101_SPET SP101_SPET1 11140814 CGAGTATAGCTAAAAAA 11486_1515 GGATAATTGGTCGTAACAA

SP101SPET SP101_SPET1 11_1408_14 TCGAGTATAGCTAAAAA 114861515 TGGATAATTGGTCGTAACA

SP101_SPET SP101_SPET1 11_1688_17 CCTATATTAATCGTTTA 117831808 ATATGATTATCATTGAACT

SP101_SPET SP101_SPET1 111688_17 TCCTATATTAATCGTTT 117831808 TATATGATTATCATTGAAC

SP101SPET SP101_SPET1 SP101_SPET SP101_SPET1 11 1711_17 TCTGGCTAAAACTTTGG 118081835 TGCGTGACGACCTTCTTGA
429 33~TMOD F CAACGGT 284 TMOD R ATTGTAATCA 639 SP101_SPET SP101_SPET1 SP101_SPET SP101_SPET1 11_1807_18 TATGATTACAATTCAAG 119011927 TTTGGACCTGTAATCAGCT

11_196719 TAACGGTTATCATGGCC 1206722083 ATTGCCCAGAAATCAAATC

SP101_SPET SP101_SPET1 11_1967_19 TTAACGGTTATCATGGC 120622083 TATTGCCCAGAAATCAAAT

SPIOl_SPET
11216243 AGCAGGTGGTGAAATCG SP101_SPET1 TGCCACTTTGACAACTCCT

11216_243 TAGCAGGTGGTGAAATC 1308_333_T TTGCCACTTTGACAACTCC

SP101_SPET SP101SPET1 11_226022 CAGAGACCGTTTTATCC 123752397 TCTGGGTGACCTGGTGTTT

SP101SPET SP101_SPET1 11_237523 TCTAAAACACCAGGTCA 1_2470_2497 AGCTGCTAGATGAGCTTCT

SP101_SPET SP101SPET1 11_2375_23 TTCTAAAACACCAGGTC 124702497 TAGCTGCTAGATGAGCTTC

SP101SPET SP101_SPET1 11_2468_24 ATGGCCATGGCAGAAGC 12543_2570 CCATAAGGTCACCGTCACC

SP101_SPET SP101_SPET1 11_246824 TATGGCCATGGCAGAAG 125432570 TCCATAAGGTCACCGTCAC

SP101_SPET
11266295 CTTGTACTTGTGGCTCA SP101_SPET1 GCTGCTTTGATGGCTGAAT

11266_295 TCTTGTACTTGTGGCTC 1355-380T TGCTGCTTTGATGGCTGAA

SP101_SPET SP101_SPET1 11_296129 ACCATGACAGAAGGCAT 1_3023_3045 GGAATTTACCAGCGATAGA

11_2961_29 TACCATGACAGAAGGCA 1_3023_3045 TGGAATTTACCAGCGATAG

SP101_SPET SP101_SPET1 113075_31 GATGACTTTTTAGCTAA 131683196 AATCGACGACCATCTTGGA

11_3075_31 ATGGTCAGGCAGC 131683196 AAAGATTTCTC

SP101_SPET SP101_SPET1 11_3085_31 TAGCTAATGGTCAGGCA 131703194 TCGACGACCATCTTGGAAA

SP101_SPET
11322344 GTCAAAGTGGCACGTTT SP101_SPET1 SP101_SPET SP101_SPET1 11_322_344 TGTCAAAGTGGCACGTT 1_423441T TATCCCCTGCTTCTGCTGC

SP101_SPET SP101_SPET1 11_338634 AGCGTAAAGGTGAACCT 134803506 CCAGCAGTTACTGTCCCCT

SP101_SPET SP101_SPET1 11_3386_34 TAGCGTAAAGGTGAACC 1_34803506 TCCAGCAGTTACTGTCCCC

SP101_SPET SP101_SPET1 98 35_F

SP101_SPET SP101_SPET1 441 35_TMOD _F

SP101_SPET
11358387 GGGGATTCAGCCATCAA SP101_SPET1 CCAACCTTTTCCACAACAG

SP101_SPET SP101_SPET1 11_358_387 TGGGGATTCAGCCATCA 1 448 473 T TCCAACCTTTTCCACAACA

11364385 TCAGCCATCAAAGCAGC SP101_SPET1 TACCTTTTCCACAACAGAA

SP101SPET SP101_SPET1 11_600_629 TCCTTACTTCGAACTAT 1_686714T TCCCATTTTTTCACGCATG

SP101_SPET

82 F_ ATAAGAAGAA 315 1 756 784 R TTTTCTAAAA 672 SP101_SPET SP101_SPET1 11658684 TGGGGATTGATATCACC 1756_784_T TGATTGGCGATAAAGTGAT

11_776_801 TTCGCCAATCAAAACTA 1 871 896 T TGCCCACCAGAAAGACTAG

SP101_SPET SP101SPET1 SP101_SPET SP101SPET1 11893_921 TGGGCAACAGCAGCGGA 1_988_1012 TCATGACAGCCAAGACCTC

SSPE BA 11 TCAAGCAAACGCACAAT SSPE_BA_196 TTGCACGTCTGTTTCAGTT

SSPE BA 11 TCAAGCAAACGCACAAC SSPE_BA_196 TTGCACGTU'C'GTTTCAGT
612 4 137P F 'U'AGAAGC 321 222P R TGCAAATTC 684 SSPE_BA_11 CAAGCAAACGCACAATC SSPE_BA_197 TGCACGTCTGTTTCAGTTG

SSPE_BA_11 513 7 TMOD TCAAGCAAACGCACAAT SSPE_BA_197 TTGCACGTCTGTTTCAGTT

SSPEBA12 SSPE_BA_197 TCTGTTTCAGTTGCAAATT

SSPE_BA_12 TGCACAATCAGAAGCTA SSPE BA 202 TTTCACAGCATGCACGTCT

SSPE_BA_14 TGCAAGCTTCTGGTGCT SSPE_BA_242 TTGTGATTGTTTTGCAGCT

SSPE_BA 15 TGCTTCTGGTGCTAGCA SSPE_BA_243 TGATTGTTTTGCAGCTGAT

SSPE_BA_15 TGCTTCTGGC'GU'C'AG SSPE_BA_243 TGATTGTTTTGU'AGU'TGA
610 0168P F UaATT 326 264P R CaCaGT 691 SSPE_BA_15 SSPE_BA 243 608 6 168P F TGGCaGUaCaAGUaATT 327 -255P R TGUaAGUaTGACaCaGT 690 SSPE_BA_63 TGCTAGTTATGGTACAG SSPE_BA_163 TCATAACTAGCATTTGTGC

SSPEBA_72 TGGTACAGAGTTTGCGA SSPE_BA_163 TCATTTGTGCTTTGAATGC

SSPEBA_72 TGGTAUaAGAGCaCaCaG SSPE_BA_163 TCATTTGTGCCaCaCaGAAC
611 89P F UaGAC 329 182P R 'GU'T 681 SSPE_BA_75 TAUaAGAGCaCaC CGUaG SSPE_BA_163 609 89P F AC 330 177P R TGTGCCaCaCaGAACaGUaT 680 TOXR_VBC 1 TCGATTAGGCAGCAACG TOXR'VBC_22 TTCAAAACCTTGCTCTCGC

TRPEAY094 TRPE_AY0943 355_10641 TCGACCTTTGGCAGGAA 55_1171119 TACATCGTTTCGCCCAAGA

355_12781 TCAAATGTACAAGGTGA 55_1392_141 TCCTCTTTTCACAGGCTCT

TRPE_AY094 TRPE_AY0943 355_1445_1 TGGATGGCATGGTGAAA 55_1551158 TATTTGGGTTTCATTCCAC

TRPEAY094 TRPE_AY0943 355_14671 ATGTCGATTGCAATCCG 551569159 TGCGCGAGCTTTTATTTGG

TRPE_AY094 TRPEAY0943 355_666_68 GTGCATGCGGATACAGA 55_769_791 TTCAAAATGCGGAGGCGTA

TRPE_AY094 TRPE_AY0943 35575777 TGCAAGCGCGACCACAT 55_864883 TGCCCAGGTACAACCTGCA

TUFB_EC_22 GCACTATGCACACGTAG TUFB_EC_284 TATAGCACCATCCATCTGA

TUFB_EC_23 TTGACTGCCCAGGTCAC TUFB_EC_283 GCCGTCCATTTGAGCAGCA

TUFB_EC 23 TAGACTGCCCAGGACAC TUFB_EC_283 GCCGTCCATCTGAGCAGCA
59 9 259 F~ GCTG 340 303-R CC 705 TUFB_EC_25 TGCACGCCGACTATGTT TUFB_EC_337 TATGTGCTCACGAGTTTGC

TUFBEC_27 TGATCACTGGTGCTGCT TUFB_EC_337 TGGATGTGCTCACGAGTCT

TUFB_EC_75 AAGACGACCTGCACGGG TUFB_EC_849 TUFB_EC_95 CCACACGCCGTTCTTCA TUFB_EC 103 GGCATCACCATTTCCTTGT

TUFB_EC_95 TUFB_EC_103 7 979 TMOD TCCACACGCCGTTCTTC 4_1058TMOD TGGCATCACCATTTCCTTG

TUFB_EC_97 AACTACCGTCCTCAGTT TUFB_EC_104 GTTGTCACCAGGCATTACC

TUFB_EC_97 AACTACCGTCCGCAGTT TUFB_EC_104 GTTGTCGCCAGGCATAACC

TUFB_EC_98 CCACAGTTCTACTTCCG TUFB_EC_103 TCCAGGCATTACCATTTCT

VALSEC_11 CGTGGCGGCGTGGTTAT VALS_EC_119 ACGAACTGGATGTCGCCGT

VALS_EC_11 CGTGGCGGCGTGGTTAT VALS_EC_119 CGGTACGAACTGGATGTCG

VALSEC_11 VALSEC_119 05_1124_TM TCGTGGCGGCGTGGTTA 5_1218_TMOD TCGGTACGAACTGGATGTC

VALSEC_11 TATGCTGACCGACCAGT VALSEC_123 TTCGCGCATCCAGGAGAAG

VALS_EC_18 CGACGCGCTGCGCTTCA VALS_EC_192 GCGTTCCACAGCTTGTTGC

VALSEC_19 CTTCTGCAACAAGCTGT VALSEC_194 TCGCAGTTCATCAGCACGA

VALS_EC_61 ACCGAGCAAGGAGACCA VALS_EC_705 TATAACGCACATCGTCAGG

WAAA_Z9692 TCTTGCTCTTTCGTGAG WAAA_Z96925 CAAGCGGTTTGCCTCAAAT

[0088] Primer pair name codes and reference sequences are shown in Table 2.
The primer name code typically represents the gene to which the given primer pair is targeted.
The primer pair name includes coordinates with respect to a reference sequence defined by an extraction of a section of sequence or defined by a GenBank gi number, or the corresponding complementary sequence of the extraction, or the entire GenBank gi number as indicated by the label "no extraction." Where "no extraction" is indicated for a reference sequence, the coordinates of a primer pair named to the reference sequence are with respect to the GenBank gi listing. Gene abbreviations are shown in bold type in the "Gene Name" column.
Table 2: Primer Name Codes and Reference Sequences Organism Extraction Primer Reference Extracted gene or entire name GenBank coordinates of gi gene code Gene Name gi number number SEQ ID NO:
16S rRNA (16S Escherichia 719 ribosomal RNA coli 16S EC gene) 16127994 4033120..4034661 23S rRNA (23S Escherichia 720 ribosomal RNA coli 23S EC gene) 16127994 4166220..4169123 capC (capsule Bacillus Complement 721 CAPC BA biosynthesis gene) anthracis 6470151 (55628..56074) cya (cyclic AMP Bacillus Complement 722 CYA BA gene) anthracis 4894216 (154288..156626) dnaK (chaperone Escherichia 723 DNAK EC dnaK gene) coli 16127994 12163..14079 groL (chaperonin Escherichia 724 GROL EC groL) coli 16127994 4368603..4370249 hflb (cell Escherichia 725 division protein coli Complement HFLB EC peptidase ftsH) 16127994 (3322645..3324576) infB (protein Escherichia 726 chain initiation coli Complement INFB EC factor infB gene) 16127994 (3310983..3313655) lef (lethal Bacillus Complement 727 LEE BA factor) anthracis 21392688 (149357..151786) pag (protective Bacillus 728 PAG BA antigen) anthracis 21392688 143779..146073 rplB (50S Escherichia 729 ribosomal protein coli RPLB EC L2) 16127994 3449001..3448180 rpoB (DNA-directed Escherichia 730 RNA polymerase coli Complement RPOB EC beta chain) 6127994 4178823..4182851 rpoC (DNA-directed Escherichia 731 RNA polymerase coli RPOC EC beta' chain) 16127994 4182928..4187151 SP101ET Concatenation _SPET1 comprising: Artificial 732 1 Sequence* - 15674250 gki (glucose partial gene Complement kinase) sequences of (1258294..1258791) Streptococcus gtr (glutamine pyogenes complement transporter (1236751..1237200) protein) murl (glutamate 312732..313169 racemase) mutS (DNA mismatch Complement repair protein) (1787602..1788007) xpt (xanthine 930977..931425 phosphoribosyl transferase) yqiL (acetyl-CoA- 129471..129903 acetyl transferase) tkt 1391844..1391386 (transketolase) sspE (small acid- 733 soluble spore Bacillus SSPE BA protein) anthracis 30253828 226496..226783 tufB (Elongation Escherichia 734 TUFB EC factor Tu) coli 16127994 4173523..4174707 valS (Valyl-tRNA Escherichia Complement 735 VALS EC synthetase) coli 16127994 (4481405..4478550) aspS (Aspartyl- Escherichia 16127994 complement(1946777.. 736 ASPS EC tRNA synthetase) coli 1948546) 2996286 No extraction - -CAF1 AF cafi (capsular Yersinia GenBank coordinates 053947 protein cafl) pestis used INVU22 Yersinia 1256565 74..3772 737 457 inv (invasin) pestis Y. pestis specific 16120353 No extraction - -chromosomal genes GenBank coordinates LLNCOO - difference Yersinia used 3143 region pestis 0381 BONTA X BoNT/A (neurotoxin Clostridium 4 77..3967 738 52066 type A) botulinum 2791983 No extraction - 739 MECAY1 mecA methicillin Staphylococcus GenBank, coordinates 4051 resistance gene aureus used trpE (anthranilate 20853695 No extraction -TRPE_AY synthase (large Acinetobacter GenBank coordinates 094355 component)) baumanii used 740 9965210 No extraction -RECA_AF recA (recombinase Acinetobacter GenBank coordinates 251469 A) baumanii used 741 4240540 No extraction -GYRA_AF gyrA (DNA gyrase Acinetobacter GenBank coordinates 100557 subunit A) baumanii used 742 4514436 No extraction -GYRB_AB gyrB (DNA gyrase Acinetobacter GenBank coordinates 008700 subunit B) baumanii used 743 waaA (3-deoxy-D- 2765828 No extraction -WAAA_Z9 manno-octulosonic- Acinetobacter GenBank coordinates 6925 acid transferase) baumanii used 744 Concatenation comprising:

Artificial Sequence* -partial gene tkt sequences of 1569415..1569873 (transketolase) Campylobacter CJST_CJ jejuni glyA (serine 367573..368079 hydroxymethyltrans ferase) 15791399 gltA (citrate complement synthase) (1604529..1604930) aspA (aspartate 96692..97168 ammonia lyase) 745 glnA (glutamine complement synthase) (657609..658085) pgm (phosphoglycerate 327773..328270 mutase) uncA (ATP 112163..112651 synthetase alpha chain) RNASEP_ RNase P Bordetella 33591275 Complement BDP (ribonuclease P) pertussis (3226720..3227933) 746 RNASEP RNase P Burkholderia 53723370 Complement BKM (ribonuclease P) mallei (2527296..2528220) 747 RNASEP_ RNase P Bacillus 16077068 Complement BS (ribonuclease P) subtilis (2330250..2330962) 748 RNASEP RNase P Clostridium 18308982 Complement CLB (ribonuclease P) perfringens (2291757..2292584) 749 RNASEP_ RNase P Escherichia 16127994 Complement EC (ribonuclease P) coli (3267457..3268233 750 RNASEP_ RNase P Rickettsia 15603881 complement(605276..6 RKP (ribonuclease P) prowazekii 06109) 751 RNASEP_ RNase P Staphylococcus 15922990 complement(1559869..
SA (ribonuclease P) aureus 1560651) 752 RNASEP_ RNase P Vibrio 15640032 complement(2580367..
VBC (ribonuclease P) cholerae 2581452) 753 icd (isocitrate Coxiella 29732244 complement(1143867..
ICD CXB dehydrogenase) burnetii 1144235) 754 multi-locus Acinetobacter 29732244 -IS1111A insertion baumannii ISIIIIA element No extraction ompA (outer Rickettsia 40287451 OMPA_AY membrane protein prowazekii 485227 A) No extraction 755 ompB (outer Rickettsia 15603881 OMPB_RK membrane protein prowazekii complement(881264..8 P B) 86195) 756 GLTA_RK g1tA (citrate Vibrio 15603881 complement(1062547..
P synthase) cholerae 1063857) 757 toxR Francisella 15640032 TOXR_VB (transcription tularensis complement(1047143..
C regulator toxR) 1048024) 758 asd (Aspartate Francisella 56707187 semialdehyde tularensis complement(438608..4 ASD FRT dehydrogenase) 39702) 759 GALE-FR galE (UDP-glucose Shigella 56707187 T 4-epimerase) flexneri 809039..810058 760 IPAHSG ipaH (invasion Campylobacter 30061571 F plasmid antigen) jejuni 2210775..2211614 761 Coxiella complement(849317..8 hupB (DNA-binding burnetii 15791399 49819) HUPB CJ protein Hu-beta) 762 Concatenation comprising: Artificial 763 Sequence* -partial gene sequences of Acinetobacter baumannii trpE (anthranilate synthase component I)) adk (adenylate Sequenced in-house AB MLST kinase) -mutt (adenine glycosylase) fumC (fumarate hydratase) efp (elongation factor p) ppa (pyrophosphate phospho-hydratase [0089] * Note: These artificial reference sequences represent concatenations of partial gene extractions from the indicated reference gi number. Partial sequences were used to create the concatenated sequence because complete gene sequences were not necessary for primer design.
The stretches of arbitrary residues "N"s were added for the convenience of separation of the partial gene extractions (10ON for SP101_SPET11 (SEQ ID NO: 732); 50N for CJST_CJ (SEQ
ID NO: 745); and 40N for AB_MLST (SEQ ID NO: 763)).

[0090] Example 2: DNA isolation and Amplification [0091] Genomic materials from culture samples or swabs were prepared using the DNeasy 96 Tissue Kit (Qiagen, Valencia, CA). All PCR reactions are assembled in 50 l reactions in the 96 well microtiter plate format using a Packard MPH liquid handling robotic platform and MJ
Dyad thermocyclers (MJ research, Waltham, MA). The PCR reaction consisted of 4 units of Amplitaq Gold , lx buffer II (Applied Biosystems, Foster City, CA), 1.5 mM
MgCl2, 0.4 M
betaine, 800 M dNTP mix, and 250 nM of each primer.

[0092] The following PCR conditions were used to amplify the sequences used for mass spectrometry analysis: 95C for 10 minutes followed by 8 cycles of 95C for 30 seconds, 48C for 30 seconds, and 72C for 30 seconds, with the 48C annealing temperature increased 0.9C after each cycle. The PCR was then continued for 37 additional cycles of 95C for 15 seconds, 56C for 20 seconds, and 72C for 20 seconds.

[0093] Example 3: Solution Capture Purification of PCR Products for Mass Spectrometry with Ion Exchange Resin-Magnetic Beads [0094] For solution capture of nucleic acids with ion exchange resin linked to magnetic beads, 25 l of a 2.5 mg/mL suspension of BioClon amine terminated supraparamagnetic beads were added to 25 to 50 l of a PCR reaction containing approximately 10 pM of a typical PCR
amplification product. The above suspension was mixed for approximately 5 minutes by vortexing or pipetting, after which the liquid was removed after using a magnetic separator. The beads containing bound PCR amplification product were then washed 3x with 50mM
ammonium bicarbonate/50% MeOH or 100mM ammonium bicarbonate/50% MeOH, followed by three more washes with 50% MeOH. The bound PCR amplicon was eluted with 25mM
piperidine, 25mM imidazole, 35% MeOH, plus peptide calibration standards.

[00951 Example 4: Mass Spectrometry and Base Composition Analysis [00961 The ESI-FTICR mass spectrometer is based on a Bruker Daltonics (Billerica, MA) Apex II 70e electrospray ionization Fourier transform ion cyclotron resonance mass spectrometer that employs an actively shielded 7 Tesla superconducting magnet. The active shielding constrains the majority of the fringing magnetic field from the superconducting magnet to a relatively small volume. Thus, components that might be adversely affected by stray magnetic fields, such as CRT monitors, robotic components, and other electronics, can operate in close proximity to the FTICR spectrometer. All aspects of pulse sequence control and data acquisition were performed on a 600 MHz Pentium II data station running Bruker's Xmass software under Windows NT 4.0 operating system. Sample aliquots, typically 15 l, were extracted directly from 96-well microtiter plates using a CTC HTS PAL autosampler (LEAP Technologies, Carrboro, NC) triggered by the FTICR data station. Samples were injected directly into a 10 l sample loop integrated with a fluidics handling system that supplies the 100 l /hr flow rate to the ESI source.
Ions were formed via electrospray ionization in a modified Analytica (Branford, CT) source employing an off axis, grounded electrospray probe positioned approximately 1.5 cm from the metalized terminus of a glass desolvation capillary. The atmospheric pressure end of the glass capillary was biased at 6000 V relative to the ESI needle during data acquisition. A counter-current flow of dry N2 was employed to assist in the desolvation process. Ions were accumulated in an external ion reservoir comprised of an rf-only hexapole, a skimmer cone, and an auxiliary gate electrode, prior to injection into the trapped ion cell where they were mass analyzed.
Ionization duty cycles > 99% were achieved by simultaneously accumulating ions in the external ion reservoir during ion detection. Each detection event consisted of 1M data points digitized over 2.3 s. To improve the signal-to-noise ratio (S/N), 32 scans were co-added for a total data acquisition time of 74 s.

[00971 The ESI-TOF mass spectrometer is based on a Bruker Daltonics MicroTOFTM. Ions from the ESI source undergo orthogonal ion extraction and are focused in a reflectron prior to detection. The TOF and FTICR are equipped with the same automated sample handling and fluidics described above. Ions are formed in the standard MicroTOFTM ESI
source that is equipped with the same off-axis sprayer and glass capillary as the FTICR ESI
source.
Consequently, source conditions were the same as those described above.
External ion accumulation was also employed to improve ionization duty cycle during data acquisition. Each detection event on the TOF was comprised of 75,000 data points digitized over 75 s.

[0098] The sample delivery scheme allows sample aliquots to be rapidly injected into the electrospray source at high flow rate and subsequently be electrosprayed at a much lower flow rate for improved ESI sensitivity. Prior to injecting a sample, a bolus of buffer was injected at a high flow rate to rinse the transfer line and spray needle to avoid sample contamination/carryover. Following the rinse step, the autosampler injected the next sample and the flow rate was switched to low flow. Following a brief equilibration delay, data acquisition commenced. As spectra were co-added, the autosampler continued rinsing the syringe and picking up buffer to rinse the injector and sample transfer line. In general, two syringe rinses and one injector rinse were required to minimize sample carryover. During a routine screening protocol a new sample mixture was injected every 106 seconds. More recently a fast wash station for the syringe needle has been implemented which, when combined with shorter acquisition times, facilitates the acquisition of mass spectra at a rate of just under one spectrum/minute.

[0099] Raw mass spectra were post-calibrated with an internal mass standard and deconvoluted to monoisotopic molecular masses. Unambiguous base compositions were derived from the exact mass measurements of the complementary single-stranded oligonucleotides.
Quantitative results are obtained by comparing the peak heights with an internal PCR
calibration standard present in every PCR well at 500 molecules per well for the ribosomal DNA-targeted primers and 100 molecules per well for the protein-encoding gene targets. Calibration methods are commonly owned and disclosed in U.S. Provisional Patent Application Serial No.
60/545,425.
[0100] Example 5: De Novo Determination of Base Composition of Amplification Products using Molecular Mass Modified Deoxynucleotide Triphosphates [0101] Because the molecular masses of the four natural nucleobases have a relatively narrow molecular mass range (A = 313.058, G = 329.052, C = 289.046, T = 304.046 - See Table 3), a persistent source of ambiguity in assignment of base composition can occur as follows: two nucleic acid strands having different base composition may have a difference of about 1 Da when the base composition difference between the two strands is G H A (-15.994) combined with C T (+15.000). For example, one 99-mer nucleic acid strand having a base composition of A27G30C21T21 has a theoretical molecular mass of 30779.058 while another 99-mer nucleic acid strand having a base composition of A26G3 1C22T20 has a theoretical molecular mass of 30780.052. A 1 Da difference in molecular mass may be within the experimental error of a molecular mass measurement and thus, the relatively narrow molecular mass range of the four natural nucleobases imposes an uncertainty factor.

[01021 The present invention provides for a means for removing this theoretical 1 Da uncertainty factor through amplification of a nucleic acid with one mass-tagged nucleobase and three natural nucleobases. The term "nucleobase" as used herein is synonymous with other terms in use in the art including "nucleotide," "deoxynucleotide," "nucleotide residue,"
"deoxynucleotide residue,"
"nucleotide triphosphate (NTP)," or deoxynucleotide triphosphate (dNTP).

[01031 Addition of significant mass to one of the 4 nucleobases (dNTPs) in an amplification reaction, or in the primers themselves, will result in a significant difference in mass of the resulting amplification product (significantly greater than 1 Da) arising from ambiguities arising from the G A combined with C T event (Table 3). Thus, the same the G A (-15.994) event combined with 5-Iodo-C H T (-110.900) event would result in a molecular mass difference of 126.894. If the molecular mass of the base composition A27G30 5-Iodo-C21T21 (33422.958) is compared with A26G315-Iodo-C22T20, (33549.852) the theoretical molecular mass difference is +126.894. The experimental error of a molecular mass measurement is not significant with regard to this molecular mass difference. Furthermore, the only base composition consistent with a measured molecular mass of the 99-mer nucleic acid is A27G305-Iodo-C21T21. In contrast, the analogous amplification without the mass tag has 18 possible base compositions.

Table 3: Molecular Masses of Natural Nucleobases and the Mass-Modified Nucleobase 5-Iodo-C and Molecular Mass Differences Resulting from Transitions Nucleobase Molecular Mass Transition A Molecular Mass A 313.058 A-->T -9.012 A 313.058 A-->C -24.012 A 313.058 A-->5-Iodo-C 101.888 A 313.058 A-->G 15.994 T 304.046 T-->A 9.012 T 304.046 T-->C -15.000 T 304.046 T-->5-Iodo-C 110.900 T 304.046 T-->G 25.006 C 289.046 C-->A 24.012 C 289.046 C-->T 15.000 C 289.046 C-->G 40.006 5-Iodo-C 414.946 5-Iodo-C-->A -101.888 5-Iodo-C 414.946 5-Iodo-C-->T -110.900 5-Iodo-C 414.946 5-Iodo-C-->G -85.894 G 329.052 G-->A -15.994 G 329.052 G-->T -25.006 G 329.052 G-->C -40.006 G 329.052 G-->5-Iodo-C 85.894 [0104] Example 6: Data Processing [0105] Mass spectra of bioagent identifying amplicons are analyzed independently using a maximum-likelihood processor, such as is widely used in radar signal processing. This processor, referred to as GenX, first makes maximum likelihood estimates of the input to the mass spectrometer for each primer by running matched filters for each base composition aggregate on the input data. This includes the GenX response to a calibrant for each primer.
[0106] The algorithm emphasizes performance predictions culminating in probability-of-detection versus probability-of-false-alarm plots for conditions involving complex backgrounds of naturally occurring organisms and environmental contaminants. Matched filters consist of a priori expectations of signal values given the set of primers used for each of the bioagents. A
genomic sequence database is used to define the mass base count matched filters. The database contains the sequences of known bacterial bioagents and includes threat organisms as well as benign background organisms. The latter is used to estimate and subtract the spectral signature produced by the background organisms. A maximum likelihood detection of known background organisms is implemented using matched filters and a running-sum estimate of the noise covariance. Background signal strengths are estimated and used along with the matched filters to form signatures which are then subtracted. the maximum likelihood process is applied to this "cleaned up" data in a similar manner employing matched filters for the organisms and a running-sum estimate of the noise-covariance for the cleaned up data.

[0107] The amplitudes of all base compositions of bioagent identifying amplicons for each primer are calibrated and a final maximum likelihood amplitude estimate per organism is made based upon the multiple single primer estimates. Models of all system noise are factored into this two-stage maximum likelihood calculation. The processor reports the number of molecules of each base composition contained in the spectra. The quantity of amplification product corresponding to the appropriate primer set is reported as well as the quantities of primers remaining upon completion of the amplification reaction.

[01081 Example 7: Use of Broad Range Survey and Division Wide Primer Pairs for Identification of Bacteria in an Epidemic Surveillance Investigation [01091 This investigation employed a set of 16 primer pairs which is herein designated the "surveillance primer set" and comprises broad range survey primer pairs, division wide primer pairs and a single Bacillus Glade primer pair. The surveillance primer set is shown in Table 4 and consists of primer pairs originally listed in Table 1. This surveillance set comprises primers with T modifications (note TMOD designation in primer names) which constitutes a functional improvement with regard to prevention of non-templated adenylation (vide supra) relative to originally selected primers which are displayed below in the same row. Primer pair 449 (non-T
modified) has been modified twice. Its predecessors are primer pairs 70 and 357, displayed below in the same row. Primer pair 360 has also been modified twice and its predecessors are primer pairs 17 and 118.
Table 4: Bacterial Primer Pairs of the Surveillance Primer Set Primer Forward Primer Name Forward Reverse Primer Name Reverse Target Gene Pair Primer Primer No. (SEQ ID (SEQ ID
NO:) NO:
346 16S_EC_713_732_TMOD_F 27 16S_EC_789_809_TMOD_R 389 16S rRNA
16S_EC_713_732_F 26 16SEC789809 388 16S rRNA
347 16S_EC_785_806_TMOD_F 30 16S_EC_880_897_TMOD_R 392 16S rRNA
11 16S EC 785 806 F 29 16S EC 880 897 R 391 16S rRNA
348 16S_EC 960_981_TMOD F 38 16S_EC_1054_1073_TMOD R 363 16S rRNA
14 16S EC 960 981 F 37 16S EC 1054 1073 R 362 16S rRNA
349 23S_EC_1826_1843_TM0D_F 49 23S_EC_1906_1924_T500_R 405 23S rRNA
16 23S EC 1826 1843 F 48 23S EC 1906 1924 R 404 23S rRNA
352 INFB EC 1365 1393 TMOD F 161 INFO EC 1439 1467 TMOD R 516 infB

34 INFB EC 1365 1393 F 160 INFB EC 1439 1467 R 515 infB
354 RPOCEC_2218_2241_TMOD_F 262 RPOC_EC_2313_2337_TMOD_R 625 rpoC
52 RPOC EC 2218 2241 F 261 RPOC EC 2313 2337 R 624 rpoC
355 SSPE_BA_115_137_TMOD_F 321 SSPE_BA 197_222_TMOD_R 687 sspE
58 SSPE BA 115 137 F 322 SSPE BA 197 222 R 686 sspE
356 RPLB EC 650 679 TMOD F 232 RPLB EC 739 762 TMOD R 592 rplB
66 RPLB EC 650 679 F 231 RPLB EC 739 762 R 591 rplB
358 VALS_EC_1105_1124_TMOD F 350 VALS_EC_1195_1218_TMOD_R 712 valS
71 VALS EC 1105 1124 F 349 VALS EC 1195 1218 R 711 valS
359 RPOB EC 1845 1866 TMOD F 241 RPOB EC 1909 1929 TMOD R 597 rpoB
72 RPOB EC 1845 1866 F 240 RPOB EC 1909 1929 R 596 rpoB
360 23S_EC_2646_2667_TM0D_F 60 23S_EC_2745_2765_TMOD_5 416 23S rRNA
118 235 EC 2646 2667 F 59 23S EC 2745 2765 R 415 23S rRNA
17 23S EC 2645 2669 F 58 23S EC 2744 2761 R 414 23S rRNA

361 16S EC 1090 1111 2 TMOD F 5 16S EC 1175 1196 TMOD R 370 16S rRNA
3 16S EC 1090 11112F 6 16S EC 1175 1196 R 369 16S rRNA
362 RPOB_EC 3799_3821_TMOD F 245 RPOB EC_3862_3888_TMOD R 603 rpoB

289 RPOB EC 3799 3821 F 246 RPOB EC 3862 3888 R 602 rpoB
363 RPOC_EC_2146_2174_TMOD F 257 RPOC EC 2227_2245_TMOD_R 621 rpoC
290 RPOC EC 2146 2174 F 256 RPOC EC 2227 2245_R 620 rpoC
367 TUFB EC 957 979 TMOD F 345 TUFB EC 1034 1058 TMOD R 701 tufB
293 TUFB EC 957 979F 344 TUFB EC 1034 1058 R 700 tufB
449 RPLB_EC_690_710_F 237 RPLB_EC_737_758_R 589 rplB
357 RPLB_EC_688_710_TMOD_F 236 RPLB_EC_736_757_TMOD_R 588 rplB
67 RPLB EC 688 710F 235 RPLB EC 736 757 R 587 rp1B

[0110] The 16 primer pairs of the surveillance set are used to produce bioagent identifying amplicons whose base compositions are sufficiently different amongst all known bacteria at the species level to identify, at a reasonable confidence level, any given bacterium at the species level. As shown in Tables 6A-E, common respiratory bacterial pathogens can be distinguished by the base compositions of bioagent identifying amplicons obtained using the 16 primer pairs of the surveillance set. In some cases, triangulation identification improves the confidence level for species assignment. For example, nucleic acid from Streptococcus pyogenes can be amplified by nine of the sixteen surveillance primer pairs and Streptococcus pneumoniae can be amplified by ten of the sixteen surveillance primer pairs. The base compositions of the bioagent identifying amplicons are identical for only one of the analogous bioagent identifying amplicons and differ in all of the remaining analogous bioagent identifying amplicons by up to four bases per bioagent identifying amplicon. The resolving power of the surveillance set was confirmed by determination of base compositions for 120 isolates of respiratory pathogens representing 70 different bacterial species and the results indicated that natural variations (usually only one or two base substitutions per bioagent identifying amplicon) amongst multiple isolates of the same species did not prevent correct identification of major pathogenic organisms at the species level.
[0111] Bacillus anthracis is a well known biological warfare agent which has emerged in domestic terrorism in recent years. Since it was envisioned to produce bioagent identifying amplicons for identification of Bacillus anthracis, additional drill-down analysis primers were designed to target genes present on virulence plasmids of Bacillus anthracis so that additional confidence could be reached in positive identification of this pathogenic organism. Three drill-down analysis primers were designed and are listed in Tables 1 and 5. In Table 5 the drill-down set comprises primers with T modifications (note TMOD designation in primer names) which constitutes a functional improvement with regard to prevention of non-templated adenylation (vide supra) relative to originally selected primers which are displayed below in the same row.
Table 5: Drill-Down Primer Pairs for Confirmation of Identification of Bacillus anthracis Primer Forward Primer Name Forward Reverse Primer Name Reverse Target Gene Pair Primer Primer No. (SEQ ID (SEQ ID
NO:) NO:) 350 CAPC BA 274 303 TMOD F 98 CAPC BA 349 376 TMOD R 452 capC
24 CAPC BA 274 303 F 97 CAPC BA 349 376 R 451 capC
351 CYA BA 1353 1379 TMOD F 128 CYA BA 1448 1467 TMOD R 483 cyA
30 CYA BA 1353 1379 F 127 CYA BA 1448 1467 R 482 cyA
353 LEF BA 756 781 TMOD F 175 LEE BA 843 872 TMOD R 531 lef 37 LEF BA 756 781 F 174 LEF BA 843 872 R 530 lef [0112] Phylogenetic coverage of bacterial space of the sixteen surveillance primers of Table 4 and the three Bacillus anthracis drill-down primers of Table 5 is shown in Figure 3 which lists common pathogenic bacteria. Figure 3 is not meant to be comprehensive in illustrating all species identified by the primers. Only pathogenic bacteria are listed as representative examples of the bacterial species that can be identified by the primers and methods of the present invention. Nucleic acid of groups of bacteria enclosed within the polygons of Figure 3 can be amplified to obtain bioagent identifying amplicons using the primer pair numbers listed in the upper right hand corner of each polygon. Primer coverage for polygons within polygons is additive. As an illustrative example, bioagent identifying amplicons can be obtained for Chlamydia trachomatis by amplification with, for example, primer pairs 346-349, 360 and 361, but not with any of the remaining primers of the surveillance primer set. On the other hand, bioagent identifying amplicons can be obtained from nucleic acid originating from Bacillus anthracis (located within 5 successive polygons) using, for example, any of the following primer pairs: 346-349, 360, 361 (base polygon), 356, 449 (second polygon), 352 (third polygon), 355 (fourth polygon), 350, 351 and 353 (fifth polygon). Multiple coverage of a given organism with multiple primers provides for increased confidence level in identification of the organism as a result of enabling broad triangulation identification.

[0113] In Tables 6A-E, base compositions of respiratory pathogens for primer target regions are shown. Two entries in a cell, represent variation in ribosomal DNA operons.
The most predominant base composition is shown first and the minor (frequently a single operon) is indicated by an asterisk (*). Entries with NO DATA mean that the primer would not be expected to prime this species due to mismatches between the primer and target region, as determined by theoretical PCR.

Table 6A - Base Compositions of Common Respiratory Pathogens for Bioagent Identifying Amplicons Corresponding to Primer Pair Nos: 346, 347 and 348 Primer 346 Primer 347 Primer 348 Organism Strain [A G C T] [A G C T] [A G C T]
Klebsiella [29 32 25 13] [23 38 28 26] [26 32 28 30]
pneumoniae MGH78578 [29 31 25 13]* [23 37 28 261* [26 31 28 301*
CO-92 Biovar [29 30 28 29]
Yersinia pestis Orientalis [29 32 25 13] [22 39 28 26] [30 30 27 293*
KIM5 P12 (Biovar Yersinia pestis Mediaevalis) [29 32 25 13] [22 39 28 26] [29 30 28 29]
[29 30 28 29]
Yersinia pestis 91001 [29 32 25 13] [22 39 28 26] [30 30 27 291*
Haemophilus influenzae KW20 [28 31 23 17] [24 37 25 27] [29 30 28 29]
Pseudomonas [26 36 29 24]
aeruginosa PA01 [30 31 23 15] [27 36 29 231* [26 32 29 29]
Pseudomonas fluorescens Pf0-1 [30 31 23 15] [26 35 29 25] [28 31 28 29]
Pseudomonas putida KT2440 [30 31 23 15] [28 33 27 27] [27 32 29 28]
Legionella pneumophila Philadelphia-1 [30 30 24 151 [33 33 23 27] [29 28 28 31]
Francisella tularensis schu 4 [32 29 22 16] [28 38 26 26] [25 32 28 31]
Bordetella pertussis Tohama [30 29 24 16] [23 37 30 24] [30 32 30 26]
Burkholderia [27 36 31 24]
cepacia J2315 [29 29 27 14] [27 32 26 29] [20 42 35 191*
Burkholderia pseudomallei K96243 [29 29 27 14] [27 32 26 29] [27 36 31 24]
Neisseria FA 1090, ATCC
gonorrhoeae 700825 [29 28 24 18] [27 34 26 283 [24 36 29 27]
Neisseria meningitides MC58 (serogroup B) [29 28 26 16] [27 34 27 271 [25 35 30 26]
Neisseria meningitides serogroup C, FAM18 [29 28 26 16] [27 34 27 27] [25 35 30 26]
Neisseria meningitides Z2491 (serogroup A) [29 28 26 16] [27 34 27 27] [25 35 30 26]
Chiamydophila pneumoniae TW-183 (31 27 22 19] NO DATA [32 27 27 29]
Chiamydophila pneumoniae AR39 [31 27 22 19] NO DATA [32 27 27 29]
Chiamydophila pneumoniae CWL029 [31 27 22 19] NO DATA (32 27 27 29]
Chlamydophila pneumoniae J138 [31 27 22 19] NO DATA [32 27 27 29]
Corynebacterium diphtheriae" NCTC13129 [29 34 21 15] [22 38 31 25] (22 33 25 34]
Mycobacterium avium k10 [27 36 21 15] [22 37 30 28] [21 36 27 30]
Mycobacterium avium 104 [27 36 21 15] [22 37 30 28] [21 36 27 30]
Mycobacterium tuberculosis CSU#93 [27 36 21 15] [22 37 30 28] [21 36 27 30]
Mycobacterium tuberculosis CDC 1551 [27 36 21 15] [22 37 30 28] (21 36 27 30]
Mycobacterium tuberculosis H37Rv (lab strain) [27 36 21 15] [22 37 30 28] [21 36 27 30]
Mycoplasma pneumoniae M129 [31 29 19 20] NO DATA NO DATA
Staphylococcus (30 29 30 29]
aureus MRSA252 [27 30 21 21] [25 35 30 26] [29 31 30 291*
Staphylococcus [30 29 30 29]
aureus MSSA476 [27 30 21 21] [25 35 30 26] [30 29 29 301*
Staphylococcus [30 29 30 29]
aureus COL [27 30 21 21] [25 35 30 26] [30 29 29 301*
Staphylococcus [30 29 30 29]
aureus Mu50 [27 30 21 21] [25 35 30 26] [30 29 29 301*
Staphylococcus [30 29 30 29]
aureus MW2 [27 30 21 21] [25 35 30 26] [30 29 29 301*

Staphylococcus [30 29 30 29]
aureus N315 [27 30 21 21] [25 35 30 26] [30 29 29 301*
Staphylococcus [25 35 30 26] [30 29 30 29]
aureus NCTC 8325 [27 30 21 21] [25 35 31 261* [30 29 29 30]
Streptococcus [24 36 31 251 agalactiae NEM316 [26 32 23 18] [24 36 30 261* [25 32 29 30]
Streptococcus equi NC 002955 [26 32 23 18] [23 37 31 25] [29 30 25 32]
Streptococcus pyogenes MGAS8232 [26 32 23 181 [24 37 30 25] [25 31 29 31]
Streptococcus pyogenes MGAS315 [26 32 23 18] [24 37 30 25] [25 31 29 31]
Streptococcus pyogenes SSI-1 [26 32 23 18] [24 37 30 25] [25 31 29 31]
Streptococcus pyogenes MGAS10394 [26 32 23 18] [24 37 30 25] [25 31 29 31]
Streptococcus pyogenes Manfredo (M5) [26 32 23 18] [24 37 30 25] [25 31 29 31]
Streptococcus pyogenes SF370 (Ml) [26 32 23 18] [24 37 30 25] [25 31 29 31]
Streptococcus pneumoniae 670 [26 32 23 18] [25 35 28 28] [25 32 29 30]
Streptococcus pneumoniae R6 [26 32 23 181 [25 35 28 28] [25 32 29 30]
Streptococcus pneumoniae TIGR4 [26 32 23 18] [25 35 28 28] [25 32 30 29]
Streptococcus gordonii NCTC7868 [25 33 23 18] [24 36 31 25] [25 31 29 31]
Streptococcus [25 32 29 30]
mitis NCTC 12261 [26 32 23 18] [25 35 30 26] [24 31 35 291*
Streptococcus mutans UA159 [24 32 24 19] [25 37 30 24] [28 31 26 31]

Table 6B - Base Compositions of Common Respiratory Pathogens for Bioagent Identifying Amplicons Corresponding to Primer Pair Nos: 349, 360, and 356 Primer 349 Primer 360 Primer 356 Organism Strain [A G C T] [A G C TI [A G C T]
Kiebsiella pneumoniae MGH78578 [25 31 25 22] [33 37 25 27] NO DATA
CO-92 Biovar [25 31 27 20]
Yersinia pestis Orientalis [25 32 26 20]* [34 35 25 28] NO DATA
KIM5 P12 (Biovar [25 31 27 20]
Yersinia pestis Mediaevalis) [25 32 26 201* [34 35 25 28] NO DATA
Yersinia pestis 91001 [25 31 27 20] [34 35 25 28] NO DATA
Haemophilus influenzae KW20 [28 28 25 20] [32 38 25 27] NO DATA
Pseudomonas [31 36 27 27]
aeruginosa PA01 [24 31 26 20] [31 36 27 281* NO DATA
Pseudomonas [30 37 27 28]
fluorescens Pf0-1 NO DATA [30 37 27 28] NO DATA
Pseudomonas putida KT2440 [24 31 26 20] [30 37 27 28] NO DATA
Legionella pneumophila Philadelphia-1 [23 30 25 23] [30 39 29 24] NO DATA
Francisella tularensis schu 4 [26 31 25 19] [32 36 27 27] NO DATA
Bordetella pertussis Tohama I [21 29 24 18] [33 36 26 27] NO DATA
Burkholderia cepacia J2315 [23 27 22 20] (31 37 28 26] NO DATA
Burkholderia pseudomallei K96243 [23 27 22 20] [31 37 28 26] NO DATA
Neisseria gonorrhoeae FA 1090, ATCC 700825 [24 27 24 17] [34 37 25 26] NO DATA
Neisseria meningitidis MC58 (serogroup B) [25 27 22 18] [34 37 25 26] NO DATA
Neisseria meningitidis serogroup C, FAM18 [25 26 23 18] [34 37 25 26] NO DATA
Neisseria Z2491 (serogroup A) [25 26 23 18] [34 37 25 26] NO DATA

meningitidis Chlamydophila pneumoniae TW-183 [30 28 27 18] NO DATA NO DATA
Chlamydophila pneumoniae AR39 [30 28 27 18] NO DATA NO DATA
Chlamydophila pneumoniae CWL029 [30 28 27 18] NO DATA NO DATA
Chlamydophila pneumoniae J138 [30 28 27 18] NO DATA NO DATA
Corynebacterium diphtheriae NCTC13129 NO DATA [29 40 28 25] NO DATA
Mycobacterium avium k10 NO DATA [33 35 32 22] NO DATA
Mycobacterium avium 104 NO DATA [33 35 32 22] NO DATA
Mycobacterium tuberculosis CSU#93 NO DATA [30 36 34 22] NO DATA
Mycobacterium tuberculosis CDC 1551 NO DATA [30 36 34 22] NO DATA
Mycobacterium tuberculosis H37Rv (lab strain) NO DATA [30 36 34 22] NO DATA
Mycoplasma pneumoniae M129 [28 30 24 19] [34 31 29 28] NO DATA
Staphylococcus aureus MRSA252 [26 30 25 20] [31 38 24 29] [33 30 31 27]
Staphylococcus aureus MSSA476 [26 30 25 20] [31 38 24 29] [33 30 31 27]
Staphylococcus aureus COL [26 30 25 20] [31 38 24 29] [33 30 31 27]
Staphylococcus aureus Mu50 [26 30 25 20] [31 38 24 29] [33 30 31 27]
Staphylococcus aureus MW2 [26 30 25 20] [31 38 24 29] [33 30 31 27]
Staphylococcus aureus N315 [26 30 25 20] [31 38 24 29] [33 30 31 27]
Staphylococcus aureus NCTC 8325 [26 30 25 20] [31 38 24 29] (33 30 31 27]
Streptococcus agalactiae NEM316 [28 31 22 20] [33 37 24 28] [37 30 28 26]
Streptococcus equi NC 002955 [28 31 23 19] [33 38 24 27] [37 31 28 25]
Streptococcus pyogenes MGAS8232 [28 31 23 19] [33 37 24 28] [38 31 29 23]
Streptococcus pyogenes MGAS315 [28 31 23 19] [33 37 24.28] [38 31 29 23]
Streptococcus pyogenes SSI-1 [28 31 23 19] [33 37 24 28] [38 31 29 23]
Streptococcus pyogenes MGAS10394 [28 31 23 19] [33 37 24 28] [38 31 29 23]
Streptococcus pyogenes Manfredo (M5) [28 31 23 19] [33 37 24 28] [38 31 29 23]
Streptococcus [28 31 23 19]
pyogenes SF370 (141) [28 31 22 20]* [33 37 24 28] [38 31 29 23]
Streptococcus pneumoniae 670 [28 31 22 20] [34 36 24 28] [37 30 29 25]
Streptococcus pneumoniae R6 [28 31 22 20] [34 36 24 28] [37 30 29 25]
Streptococcus pneumoniae TIGR4 [28 31 22 20] [34 36 24 28] [37 30 29 25]
Streptococcus gordonii NCTC7868 [28 32 23 20] [34 36 24 28] [36 31 29 25]
Streptococcus [28 31 22 20]
mitis NCTC 12261 [29 30 22 20]* [34 36 24 28] [37 30 29 25]
Streptococcus mutans UA159 [26 32 23 22] [34 37 24 27] NO DATA

Table 6C - Base Compositions of Common Respiratory Pathogens for Bioagent Identifying Amplicons Corresponding to Primer Pair Nos: 449, 354, and 352 Primer 449 Primer 354 Primer 352 Organism Strain [A G C T] [A G C T] [A G C T]
Klebsiella pneumoniae MGH78578 NO DATA [27 33 36 26] NO DATA
CO-92 Biovar Yersinia pestis Orientalis NO DATA [29 31 33 29] [32 28 20 25]
KIM5 P12 (Biovar Yersinia pestis Mediaevalis) NO DATA [29 31 33 29] [32 28 20 25]
Yersinia pestis 91001 NO DATA [29 31 33 29] NO DATA
Haemophilus influenzae KW20 NO DATA [30 29 31 32] NO DATA
Pseudomonas aeruginosa PA01 NO DATA [26 33 39 24] NO DATA
Pseudomonas fluorescens Pf0-1 NO DATA [26 33 34 29] NO DATA
Pseudomonas putida KT2440 NO DATA [25 34 36 27] NO DATA
Legionella pneumophila Philadelphia-1 NO DATA NO DATA NO DATA
Francisalla tularensis schu 4 NO DATA [33 32 25 32] NO DATA
Bordetella pertussis Tohama I NO DATA [26 33 39 24] NO DATA
Burkholderia cepacia J2315 NO DATA [25 37 33 27] NO DATA
Burkholderia pseudomallei K96243 NO DATA [25 37 34 26] NO DATA
Neisseria gonorrhoeae FA 1090, ATCC 700825 [17 23 22 10] [29 31 32 30] NO DATA
Neisseria meningitidis MC58 (serogroup B) NO DATA [29 30 32 31] NO DATA
Neisseria meningitidis serogroup C, FAM18 NO DATA [29 30 32 31] NO DATA
Neisseria meningitidis Z2491 (serogroup A) NO DATA [29 30 32 31] NO DATA
Chlamydophila pneumoniae TW-183 NO DATA NO DATA NO DATA
Chlamydophila pneumoniae AR39 NO DATA NO DATA NO DATA
Chlamydophila pneumoniae CWL029 NO DATA NO DATA NO DATA
Chlamydophila pneumoniae J138 NO DATA NO DATA NO DATA
Corynebacterium diphtheriae NCTC13129 NO DATA NO DATA NO DATA
Mycobacterium avium k10 NO DATA NO DATA NO DATA
Mycobacterium avium 104 NO DATA NO DATA NO DATA
Mycobacterium tuberculosis CSU#93 NO DATA NO DATA NO DATA
Mycobacterium tuberculosis CDC 1551 NO DATA NO DATA NO DATA
Mycobacterium tuberculosis H37Rv (lab strain) NO DATA NO DATA NO DATA
Mycoplasma pneumoniae M129 NO DATA NO DATA NO DATA
Staphylococcus aureus MRSA252 [17 20 21 17] [30 27 30 35] [36 24 19 26]
Staphylococcus aureus MSSA476 [17 20 21 17] [30 27 30 35] [36 24 19 26]
Staphylococcus aureus COL [17 20 21 17] [30 27 30 35] [35 24 19 27]
Staphylococcus aureus Mu50 [17 20 21 17] [30 27 30 35] [36 24 19 26]
Staphylococcus aureus MW2 [17 20 21 17] [30 27 30 351 [36 24 19 26]

Staphylococcus aureus N315 [17 20 21 17] [30 27 30 35] [36 24 19 26]
Staphylococcus aureus NCTC 8325 [17 20 21 17] [30 27 30 35] [35 24 19 27]
Streptococcus agalactiae NEM316 [22 20 19 14] [26 31 27 38] [29 26 22 28]
Streptococcus equi NC 002955 [22 21 19 13] NO DATA NO DATA
Streptococcus pyogenes MGAS8232 [23 21 19 12] [24 32 30 36] NO DATA
Streptococcus pyogenes MGAS315 [23 21 19 12] [24 32 30 361 NO DATA
Streptococcus pyogenes SSI-1 [23 21 19 12] [24 32 30 36] NO DATA
Streptococcus pyogenes MGAS10394 [23 21 19 12] [24 32 30 361 NO DATA
Streptococcus pyogenes Manfredo (M5) [23 21 19 12] [24 32 30 36] NO DATA
Streptococcus pyogenes SF370 (M1) [23 21 19 12] [24 32 30 361 NO DATA
Streptococcus pneumoniae 670 [22 20 19 14] [25 33 29 35] [30 29 21 251 Streptococcus pneumoniae R6 [22 20 19 14] [25 33 29 35] (30 29 21 25]
Streptococcus pneumoniae TIGR4 [22 20 19 14] [25 33 29 351 [30 29 21 25]
Streptococcus gordonii NCTC7868 [21 21 19 14] NO DATA [29 26 22 28]
Streptococcus mitis NCTC 12261 [22 20 19 14] [26 30 32 34] NO DATA
Streptococcus mutans UA159 NO DATA NO DATA NO DATA

Table 6D - Base Compositions of Common Respiratory Pathogens for Bioagent Identifying Amplicons Corresponding to Primer Pair Nos: 355, 358, and 359 Primer 355 Primer 358 Primer 359 Organism Strain [A G C T] [A G C T] [A G C T]
Klebsiella pneumoniae MGH78578 NO DATA [24 39 33 20] [25 21 24 17]
CO-92 Biovar Yersinia pestis Orientalis NO DATA [26 34 35 21] [23 23 19 22]
KIM5 P12 (Biovar Yersinia pestis Mediaevalis) NO DATA [26 34 35 21] [23 23 19 22]
Yersinia pestis 91001 NO DATA [26 34 35 21] [23 23 19 22]
Haemophilus influenzae KW20 NO DATA NO DATA NO DATA
Pseudomonas aeruginosa PAO1 NO DATA NO DATA NO DATA
Pseudomonas fluorescens Pf0-1 NO DATA NO DATA NO DATA
Pseudomonas putida KT2440 NO DATA [21 37 37 21] NO DATA
Legionella pneumophila Philadelphia-1 NO DATA NO DATA NO DATA
Francisella tularensis schu 4 NO DATA NO DATA NO DATA
Bordetella pertussis Tohama I NO DATA NO DATA NO DATA
Burkholderia cepacia J2315 NO DATA NO DATA NO DATA
Burkholderia pseudomallei K96243 NO DATA NO DATA NO DATA
Neisseria gonorrhoeae FA 1090, ATCC 700825 NO DATA NO DATA NO DATA
Neisseria meningitidis MC58 (serogroup B) NO DATA NO DATA NO DATA
Neisseria meningitidis serogroup C, FAM18 NO DATA NO DATA NO DATA

Neisseria meningitidis Z2491 (serogroup A) NO DATA NO DATA NO DATA
Chlamydophila pneumoniae TW-183 NO DATA NO DATA NO DATA
Chlamydophila pneumoniae AR39 NO DATA NO DATA NO DATA
Chlamydophila pneumoniae CWL029 NO DATA NO DATA NO DATA
Chlamydophila pneumoniae J138 NO DATA NO DATA NO DATA
Corynebacterium diphtheriae NCTC13129 NO DATA NO DATA NO DATA
Mycobacterium avium k10 NO DATA NO DATA NO DATA
Mycobacterium avium 104 NO DATA NO DATA NO DATA
Mycobacterium tuberculosis CSU#93 NO DATA NO DATA NO DATA
Mycobacterium tuberculosis CDC 1551 NO DATA NO DATA NO DATA
Mycobacterium tuberculosis H37Rv (lab strain) NO DATA NO DATA NO DATA
Mycoplasma pneumoniae M129 NO DATA NO DATA NO DATA
Staphylococcus aureus MRSA252 NO DATA NO DATA NO DATA
Staphylococcus aureus MSSA476 NO DATA NO DATA NO DATA
Staphylococcus aureus COL NO DATA NO DATA NO DATA
Staphylococcus aureus Mu50 NO DATA NO DATA NO DATA
Staphylococcus aureus MW2 NO DATA NO DATA NO DATA
Staphylococcus aureus N315 NO DATA NO DATA NO DATA
Staphylococcus aureus NCTC 8325 NO DATA NO DATA NO DATA
Streptococcus agalactiae NEM316 d NO DATA NO DATA NO DATA
Streptococcus equi NC 002955 NO DATA NO DATA NO DATA
Streptococcus pyogenes MGAS8232 NO DATA NO DATA NO DATA
Streptococcus pyogenes MGAS315 NO DATA NO DATA NO DATA
Streptococcus pyogenes SSI-1 NO DATA NO DATA NO DATA
Streptococcus pyogenes MGAS10394 NO DATA NO DATA NO DATA
Streptococcus pyogenes Manfredo (M5) NO DATA NO DATA NO DATA
Streptococcus pyogenes SF370 (M1) NO DATA NO DATA NO DATA
Streptococcus pneumoniae 670 NO DATA NO DATA NO DATA
Streptococcus pneumoniae R6 NO DATA NO DATA NO DATA
Streptococcus pneumoniae TIGR4 NO DATA NO DATA NO DATA
Streptococcus gordonii NCTC7868 NO DATA NO DATA NO DATA
Streptococcus mitis NCTC 12261 NO DATA NO DATA NO DATA
Streptococcus mutans UA159 NO DATA NO DATA NO DATA

Table 6E - Base Compositions of Common Respiratory Pathogens for Bioagent Identifying Amplicons Corresponding to Primer Pair Nos: 362, 363, and 367 Primer 362 Primer 363 Primer 367 Organism Strain [A G C T] [A G C T] [A G C T]
Klebsiella pneumoniae MGH78578 [21 33 22 16] [16 34 26 26] NO DATA
CO-92 Biovar Yersinia pestis Orientalis [20 34 18 20] NO DATA NO DATA
KIM5 P12 (Biovar Yersinia pestis Mediaevalis) [20 34 18 20] NO DATA NO DATA, Yersinia pestis 91001 [20 34 18 20] NO DATA NO DATA
Ha emophilus influenzae KW20 NO DATA NO DATA NO DATA
Pseudomonas aeruginosa PAO1 [19 35 21 17] [16 36 28 22] NO DATA
Pseudomonas fluorescens Pf0-1 NO DATA [18 35 26 23] NO DATA
Pseudomonas putida KT2440 NO DATA [16 35 28 23] NO DATA
Legionella pneumophila Philadelphia-1 NO DATA NO DATA NO DATA
Francisella tularensis schu 4 NO DATA NO DATA NO DATA
Bordetella pertussis Tohama I (20 31 24 17] [15 34 32 21] [26 25 34 19]
Burkholderia cepacia J2315 [20 33 21 18] [15 36 26 25] [25 27 32 20]
Burkholderia pseudomallei K96243 [19 34 19 203 [15 37 28 22] [25 27 32 20]
Neisseria , gonorrhoeae FA 1090, ATCC 700825 NO DATA NO DATA NO DATA
Neisseria meningitidis MC58 (serogroup B) NO DATA NO DATA NO DATA
Neisseria meningitidis serogroup C, FAM18 NO DATA NO DATA NO DATA
Neisseria meningitidis Z2491 (serogroup A) NO DATA NO DATA NO DATA
Chlamydophila pneumoniae TW-183 NO DATA NO DATA NO DATA
Chlamydophila pneumoniae AR39 NO DATA NO DATA NO DATA
Chlamydophila pneumoniae CWL029 NO DATA NO DATA NO DATA
Chlamydophila pneumoniae J138 NO DATA NO DATA NO DATA
Corynebacterium diphtheriae NCTC13129 NO DATA NO DATA NO DATA
Mycobacterium avium k10 [19 34 23 16] NO DATA [24 26 35 19]
Mycobacterium avium 104 [19 34 23 161 NO DATA [24 26 35 19]
Mycobacterium tuberculosis CSU#93 [19 31 25 17] NO DATA [25 25 34 201 Mycobacterium tuberculosis CDC 1551 [19 31 24 18] NO DATA [25 25 34 20]
Mycobacterium tuberculosis H37Rv (lab strain) [19 31 24 18] NO DATA [25 25 34 20]
Mycoplasma pneumoniae M129 NO DATA NO DATA NO DATA
Staphylococcus aureus MRSA252 NO DATA NO DATA NO DATA
Staphylococcus aureus MSSA476 NO DATA NO DATA NO DATA
Staphylococcus aureus COL NO DATA NO DATA NO DATA
Staphylococcus aureus Mu50 NO DATA NO DATA NO DATA
Staphylococcus aureus MW2 NO DATA NO DATA NO DATA
Staphylococcus N315 NO DATA NO DATA NO DATA

aureus Staphylococcus aureus NCTC 8325 NO DATA NO DATA NO DATA
Streptococcus agalactiae NEM316 NO DATA NO DATA NO DATA
Streptococcus equi NC 002955 NO DATA NO DATA NO DATA
Streptococcus pyogenes MGAS8232 NO DATA NO DATA NO DATA
Streptococcus pyogenes MGAS315 NO DATA NO DATA NO DATA
Streptococcus pyogenes SSI-1 NO DATA NO DATA NO DATA
Streptococcus pyogenes MGAS10394 NO DATA NO DATA NO DATA
Streptococcus pyogenes Manfredo (M5) NO DATA NO DATA NO DATA
Streptococcus pyogenes SF370 (M1) NO DATA NO DATA NO DATA
Streptococcus pneumoniae 670 NO DATA NO DATA NO DATA
Streptococcus pneumoniae R6 [20 30 19 23] NO DATA NO DATA
Streptococcus pneumoniae TIGR4 [20 30 19 23] NO DATA NO DATA
Streptococcus gordonii NCTC7868 NO DATA NO DATA NO DATA
Streptococcus mitis NCTC 12261 NO DATA NO DATA NO DATA
Streptococcus mutans UA159 NO DATA NO DATA NO DATA

[0114] Four sets of throat samples from military recruits at different military facilities taken at different time points were analyzed using the primers of the present invention. The first set was collected at a military training center from November 1 to December 20, 2002 during one of the most severe outbreaks of pneumonia associated with group A Streptococcus in the United States since 1968. During this outbreak, fifty-one throat swabs were taken from both healthy and hospitalized recruits and plated on blood agar for selection of putative group A Streptococcus colonies. A second set of 15 original patient specimens was taken during the height of this group A Streptococcus -associated respiratory disease outbreak. The third set were historical samples, including twenty-seven isolates of group A Streptococcus, from disease outbreaks at this and other military training facilities during previous years. The fourth set of samples was collected from five geographically separated military facilities in the continental U.S.
in the winter immediately following the severe November/December 2002 outbreak.

[0115] Pure colonies isolated from group A Streptococcus-selective media from all four collection periods were analyzed with the surveillance primer set. All samples showed base compositions that precisely matched the four completely sequenced strains of Streptococcus pyogenes. Shown in Figure 4 is a 3D diagram of base composition (axes A, G and C) of bioagent identifying amplicons obtained with primer pair number 14 (a precursor of primer pair number 348 which targets 16S rRNA). The diagram indicates that the experimentally determined base compositions of the clinical samples closely match the base compositions expected for Streptococcus pyogenes and are distinct from the expected base compositions of other organisms.

[0116] In addition to the identification of Streptococcus pyogenes, other potentially pathogenic organisms were identified concurrently. Mass spectral analysis of a sample whose nucleic acid was amplified by primer pair number 349 (SEQ ID NOs: 49 and 405) exhibited signals of bioagent identifying amplicons with molecular masses that were found to correspond to analogous base compositions of bioagent identifying amplicons of Streptococcus pyogenes (A27 G32 C24 T18), Neisseria meningitidis (A25 G27 C22 T18), and Haemophilus influenzae (A28 G28 C25 T20) (see Figure 5 and Table 6B). These organisms were present in a ratio of 4:5:20 as determined by comparison of peak heights with peak height of an internal PCR
calibration standard as described in commonly owned U.S. Patent Application Serial No:
60/545,425.

[0117] Since certain division-wide primers that target housekeeping genes are designed to provide coverage of specific divisions of bacteria to increase the confidence level for identification of bacterial species, they are not expected to yield bioagent identifying'amplicons for organisms outside of the specific divisions. For example, primer pair number 356 (SEQ ID
NOs: 232:592) primarily amplifies the nucleic acid of members of the classes Bacilli and Clostridia and is not expected to amplify proteobacteria such as Neisseria meningitidis and Haeinophilus influenzae. As expected, analysis of the mass spectrum of amplification products obtained with primer pair number 356 does not indicate the presence of Neisseria meningitidis and Haemophilus influenzae but does indicate the presence of Streptococcus pyogenes (Figures 3 and 6, Table 6B). Thus, these primers or types of primers can confirm the absence of particular bioagents from a sample.

[0118] The 15 throat swabs from military recruits were found to contain a relatively small set of microbes in high abundance. The most common were Haemophilus influenza, Neisseria meningitides, and Streptococcus pyogenes. Staphylococcus epidermidis, Moraxella cattarhalis, Corynebacterium pseudodiphtheriticum, and Staphylococcus aureus were present in fewer samples. An equal number of samples from healthy volunteers from three different geographic locations, were identically analyzed. Results indicated that the healthy volunteers have bacterial flora dominated by multiple, commensal non-beta-hemolytic Streptococcal species, including the viridans group streptococci (S. parasangunis, S. vestibularis, S. mitis, S.
oralis and S.
pneumoniae; data not shown), and none of the organisms found in the military recruits were found in the healthy controls at concentrations detectable by mass spectrometry. Thus, the military recruits in the midst of a respiratory disease outbreak had a dramatically different microbial population than that experienced by the general population in the absence of epidemic disease.

[0119] Example 8: Drill-down Analysis for Determination of emm-Type of Streptococcus pyogenes in Epidemic Surveillance [0120] As a continuation of the epidemic surveillance investigation of Example 7, determination of sub-species characteristics (genotyping) of Streptococcus pyogenes, was carried out based on a strategy that generates strain-specific signatures according to the rationale of Multi-Locus Sequence Typing (MLST). In classic MLST analysis, internal fragments of several housekeeping genes are amplified and sequenced (Enright et al. Infection and Immunity, 2001, 69, 2416-2427).
In classic MLST analysis, internal fragments of several housekeeping genes are amplified and sequenced. In the present investigation, bioagent identifying amplicons from housekeeping genes were produced using drill-down primers and analyzed by mass spectrometry.
Since mass spectral analysis results in molecular mass, from which base composition can be determined, the challenge was to determine whether resolution of emm classification of strains of Streptococcus pyogenes could be determined.

[0121] An alignment was constructed of concatenated alleles of seven MLST
housekeeping genes (glucose kinase (gki), glutamine transporter protein (gtr), glutamate racemase (murl), DNA mismatch repair protein (mutS), xanthine phosphoribosyl transferase (xpt), and acetyl-CoA
acetyl transferase (yqiL)) from each of the 212 previously emm-typed strains of Streptococcus pyogenes. From this alignment, the number and location of primer pairs that would maximize strain identification via base composition was determined. As a result, 6 primer pairs were chosen as standard drill-down primers for determination of einm-type of Streptococcus pyogenes.
These six primer pairs are displayed in Table 7. This drill-down set comprises primers with T
modifications (note TMOD designation in primer names) which constitutes a functional improvement with regard to prevention of non-templated adenylation (vide supra) relative to originally selected primers which are displayed below in the same row.

Table 7: Group A Streptococcus Drill-Down Primer Pairs Primer Forward Primer Name Forward Primer Reverse Primer Name Reverse Primer Target Gene Pair No. (SEQ ID NO:) (SEQ ID NO:) SP101_SPET11358_387_ SP101_SPET11_448_ 442 TMOD F 311 473_TMOD_R 669 gki 80 SP101_SPET11_358_387_ 310 SP101_SPET11_448_ 668 gki SP101_SPET11_600_629_ SP101_SPET11_686_ 443 TMOD F 314 714_TMOD R 671 gtr 81 SP101_SPET11_600_629_ 313 SP101_SPET11_686_ 670 gtr SP101_SPET11_1314_133 SP101_SPET11_1403 426 6 TMOD F 278 1431_TMOD_R 633 murl 86 SP101SPET11_1314_133 277 SP101_SPET111403 632 murl SP101SPET11_1807_183 SP101_SPET11_1901 430 5 TMOD F 286 _1927_TMOD_R 641 mutS
90 SP101_SPET11_1807_183 285 SP101SPET111901 640 mutS

SP101_SPET11_3075_310 SP101_SPET11_3168 438 3 TMOD F 302 3196_TMOD_R 657 xpt 96 SP101SPET11_3075_310 301 SP101_SPET11_3168 656 xpt SP101SPET11_3511_353 SP101SPET113605 441 5 TMOD F 309 3629_TMOD_R 664 yqiL
98 SP101_SPET11_3511_353 308 SP101_SPET11_3605 663 yqiL

[01221 The primers of Table 7 were used to produce bioagent identifying amplicons from nucleic acid present in the clinical samples. The bioagent identifying amplicons which were subsequently analyzed by mass spectrometry and base compositions corresponding to the molecular masses were calculated.

[01231 Of the 51 samples taken during the peak of the November/December 2002 epidemic (Table 8A-C rows 1-3), all except three samples were found to represent emm3, a Group A
Streptococcus genotype previously associated with high respiratory virulence.
The three outliers were from samples obtained from healthy individuals and probably represent non-epidemic strains. Archived samples (Tables 8A-C rows 5-13) from historical collections showed a greater heterogeneity of base compositions and emm types as would be expected from different epidemics occurring at different places and dates. The results of the mass spectrometry analysis and emm gene sequencing were found to be concordant for the epidemic and historical samples.

Table 8A: Base Composition Analysis of Bioagent Identifying Amplicons of Group A
Streptococcus samples from Six Military Installations Obtained with Primer Pair Nos. 426 and 430 emm-type by murl mutS
Instances # of Mass SequGeneencing (sample) Location Year (Primer Pair (Primer Pair Spectrometry Sequencing 426) No. 430) 48 3 3 MCRD San A39 G25 C20 T34 A38 G27 C23 T33 2 6 6 Diego 2002 A40 G24 C20 T34 A38 G27 C23 T33 (Cultured) 3 5,58 5 A40 G24 C20 T34 A38 G27 C23 T33 6 6 6 NHRC San A40 G24 C20 T34 A38 G27 C23 T33 1 11 11 Diego- A39 G25 C20 T34 A38 G27 C23 T33 3 12 12 Archive 2003 A40 G24 C20 T34 A38 G26 C24 T33 1 22 22 (Cultured) A39 G25 C20 T34 A38 G27 C23 T33 3 25,75 75 A39 G25 C20 T34 A38 G27 C23 T33 4 44/61,82,9 44/61 A40 G24 C20 T34 A38 G26 C24 T33 2 53,91 91 A39 G25 C20 T34 A38 G27 C23 T33 1 6 6 Ft. A40 G24 C20 T34 A38 G27 C23 T33 11 25 or 75 75 Woodard 2003 A39 G25 C20 T34 A38 G27 C23 T33 25,75, 33, 1 34,4,52,84 75 (Cultured) A39 G25 C20 T34 A38 G27 C23 T33 44/61 or 82 1 or 9 44/61 A40 G24 C20 T34 A38 G26 C24 T33 2 5 or 58 5 A40 G24 C20 T34 A38 G27 C23 T33 2 3 3 Ft. Sill 2003 A39 G25 C20 T34 A38 G27 C23 T33 1 4 4 (Cultured) A39 G25 C20 T34 A38 G27 C23 T33 1 11 11 Ft. A39 G25 C20 T34 A38 G27 C23 T33 Benning 1 13 94** 2003 A40 G24 C20 T34 A38 G27 C23 T33 44/61 or 82 (Cultured) 1 or 9 82 A40 G24 C20 T34 A38 G26 C24 T33 1 5 or 58 58 A40 G24 C20 T34 A38 G27 C23 T33 1 78 or 89 89 A39 G25 C20 T34 A38 G27 C23 T33 2 5 or 58 Lackland A40 G24 C20 T34 A38 G27 C23 T33 1 81 or 90 ND 2003 A40 G24 C20 T34 A38 G27 C23 T33 1 78 (Throat Swabs) A38 G26 C20 T34 A38 G27 C23 T33 3*** No detection No detection No detection 1 3 ND MCRD San No detection A38 G27 C23 T33 1 3 ND Diego 2002 No detection No detection 1 3 ND (Throat No detection No detection 2 3 ND Swabs) No detection A38 G27 C23 T33 3 No detection ND No detection No detection Table 8B: Base Composition Analysis of Bioagent Identifying Amplicons of Group A
Streptococcus samples from Six Military Installations Obtained with Primer Pair Nos. 438 and 441 # of e1 3 -type by emm-Gene Location xpt yqiL
Instances Mass Sequencing (sample) Year (Primer Pair (Primer Pair Spectrometry No. 438) No. 441) 48 3 3 MCRD San A30 G36 C20 T36 A40 G29 C19 T31 2 ,6 6 Diego 2002 A30 G36 C20 T36 A40 G29 C19 T31 1 28 28 (Cultured) A30 G36 C20 T36 A41 G28 C18 T32 3 5,58 5 A30 G36 C20 T36 A40 G29 C19 T31 6 6 6 NHRC San A30 G36 C20 T36 A40 G29 C19 T31 1 11 11 Diego- A30 G36 C20 T36 A40 G29 C19 T31 3 12 12 Archive 2003 A30 G36 C19 T37 A40 G29 C19 T31 1 22 22 (Cultured) A30 G36 C20 T36 A40 G29 C19 T31 3 25,75 75 A30 G36 C20 T36 A40 G29 C19 T31 4 44/61,82,9 44/61 A30 G36 C20 T36 A41 G28 C19 T31 2 53,91 91 A30 G36 C19 T37 A40 G29 C19 T31 1 6 6 Ft. A30 G36 C20 T36 A40 G29 C19 T31 11 25 or 75 75 Leonard 2003 A30 G36 C20 T36 A40 G29 C19 T31 25,75, 33, 1 34,4,52,84 75 (Cultured) A30 G36 C19 T37 A40 G29 C19 T31 44/61 or 82 1 or 9 44/61 A30 G36 C20 T36 A41 G28 C19 T31 2 5 or 58 5 A30 G36 C20 T36 A40 G29 C19 T31 2 3 3 Ft. Sill 2003 A30 G36 C20 T36 A40 G29 C19 T31 1 4 4 (Cultured) A30 G36 C19 T37 A41 G28 C19 T31 1 11 11 Ft. A30 G36 C20 T36 A40 G29 C19 T31 Benning 1 13 94** 2003 A30 G36 020 T36 A41 G28 C19 T31 44/61 or 82 (Cultured) 1 or 9 82 A30 G36 C20 T36 A41 G28 C19 T31 1 5 or 58 58 A30 G36 C20 T36 A40 G29 C19 T31 1 78 or 89 89 A30 G36 C20 T36 A41 G28 C19 T31 2 5 or 58 A30 G36 C20 T36 A40 G29 C19 T31 Lackland 1 81 or 90 ND 2003 A30 G36 C20 T36 A40 G29 C19 T31 1 78 (Throat Swabs) A30 G36 C20 T36 A41 G28 C19 T31 3*** No detection No detection No detection 1 3 ND MCRD San A30 G36 C20 T36 A40 G29 C19 T31 1 3 ND Diego 2002 A30 G36 C20 T36 No detection 1 3 ND (Throat No detection A40 G29 C19 T31 2 3 ND Swabs) A30 G36 C20 T36 A40 G29 C19 T31 3 No detection ND No detection No detection Table 8C: Base Composition Analysis of Bioagent Identifying Amplicons of Group A
Streptococcus samples from Six Military Installations Obtained with Primer Pair Nos. 438 and 441 emm-type by gki gtr # of emm-Gene Location Instances Mass Sequencing (sample) Year (Primer Pair ((Primer Pair Spectrometry No. 442) No. 443) 48 3 3 MCRD San A32 G35 C17 T32 A39 G28 C16 T32 2 6 6 Diego 2002 A31 G35 C17 T33 A39 G28 C15 T33 1 28 28 (Cultured) A30 G36 C17 T33 A39 G28 C16 T32 3 5,58 5 A30 G36 C20 T30 A39 G28 C15 T33 6 6 6 NHRC San A31 G35 C17 T33 A39 G28 C15 T33 1 11 11 Diego- A30 G36 C20 T30 A39 G28 C16 T32 3 12 12 Archive 2003 A31 G35 C17 T33 A39 G28 C15 T33 1 22 22 (Cultured) A31 G35 C17 T33 A38 G29 C15 T33 3 25,75 75 A30 G36 C17 T33 A39 G28 C15 T33 4 44/61,82,9 44/61 A30 G36 C18 T32 A39 G28 C15 T33 2 53,91 91 A32 G35 C17 T32 A39 G28 C16 T32 1 6 6 Ft. A31 G35 C17 T33 A39 G28 C15 T33 11 25 or 75 75 Leonard 2003 A30 G36 C17 T33 A39 G28 C15 T33 25,75, 33, 1 34,4,52,84 75 (Cultured) A30 G36 C17 T33 A39 G28 C15 T33 44/61 or 82 1 or 9 44/61 A30 G36 C18 T32 A39 G28 C15 T33 2 5 or 58 5 A30 G36 C20 T30 A39 G28 C15 T33 2 3 3 Ft. Sill 2003 A32 G35 C17 T32 A39 G28 C16 T32 1 4 4 (Cultured) A31 G35 C17 T33 A39 G28 C15 T33 1 11 11 Ft. A30 G36 C20 T30 A39 G28 C16 T32 Benning 1 13 94** 2003 A30 G36 C19 T31 A39 G28 C15 T33 44/61 or 82 (Cultured) 1 or 9 82 A30 G36 C18 T32 A39 G28 C15 T33 1 5 or 58 58 A30 G36 C20 T30 A39 G28 C15 T33 1 78 or 89 89 A30 536 C18 T32 A39 G28 C15 T33 2 5 or 58 Lackland A30 G36 C20 T30 A39 G28 C15 T33 1 81 or 90 ND 2003 A30 G36 C17 T33 A39 G28 C15 T33 1 78 (Throat Swabs) A30 G36 C18 T32 A39 G28 C15 T33 3*** No detection No detection No detection 1 3 ND MCRD San No detection No detection 1 3 ND Diego 2002 A32 G35 C17 T32 A39 G28 C16 T32 1 3 ND (Throat A32 G35 C17 T32 No detection 2 3 ND Swabs) A32 G35 C17 T32 No detection 3 No detection ND No detection No detection [01241 Example 9: Design of Calibrant Polynucleotides based on Bioagent Identifying Amplicons for Identification of Species of Bacteria (Bacterial Bioagent Identifying Amplicons) [01251 This example describes the design of 19 calibrant polynucleotides based on bacterial bioagent identifying amplicons corresponding to the primers of the broad surveillance set (Table 4) and the Bacillus anthracis drill-down set (Table 5).

[01261 Calibration sequences were designed to simulate bacterial bioagent identifying amplicons produced by the T modified primer pairs shown in Table 4 (primer names have the designation "TMOD"). The calibration sequences were chosen as a representative member of the section of bacterial genome from specific bacterial species which would be amplified by a given primer pair. The model bacterial species upon which the calibration sequences are based are also shown in Table 9. For example, the calibration sequence chosen to correspond to an amplicon produced by primer pair no. 361 is SEQ ID NO: 722. In Table 9, the forward (_F) or reverse (-R) primer name indicates the coordinates of an extraction representing a gene of a standard reference bacterial genome to which the primer hybridizes e.g.: the forward primer name 16S EC 713 732 TMOD F indicates that the forward primer hybridizes to residues 713-732 of the gene encoding 16S ribosomal RNA in an E. coli reference sequence (in this case, the reference sequence is an extraction consisting of residues 4033120-4034661 of the genomic sequence of E. coli K12 (GenBank gi number 16127994). Additional gene coordinate reference information is shown in Table 10. The designation "TMOD" in the primer names indicates that the 5' end of the primer has been modified with a non-matched template T
residue which prevents the PCR polymerase from adding non-templated adenosine residues to the 5' end of the amplification product, an occurrence which may result in miscalculation of base composition from molecular mass data (vide supra).

[01271 The X19 calibration sequences described in Tables 9 and 10 were combined into a single calibration polynucleotide sequence (SEQ ID NO: 741 - which is herein designated a "combination calibration polynucleotide") which was then cloned into a pCR -Blunt vector (Invitrogen, Carlsbad, CA). This combination calibration polynucleotide can be used in conjunction with the primers of Table 9 as an internal standard to produce calibration amplicons for use in determination of the quantity of any bacterial bioagent. Thus, for example, when the combination calibration polynucleotide vector is present in an amplification reaction mixture, a calibration amplicon based on primer pair 346 (16S rRNA) will be produced in an amplification reaction with primer pair 346 and a calibration amplicon based on primer pair 363 (rpoC) will be produced with primer pair 363. Coordinates of each of the 19 calibration sequences within the calibration polynucleotide (SEQ ID NO: 783) are indicated in Table 10.
Table 9: Bacterial Primer Pairs for Production of Bacterial Bioagent Identifying Amplicons and Corresponding Representative Calibration Sequences Primer Forward Primer Name Forward Reverse Primer Name Reverse Calibration Calibration Pair No. Primer Primer Sequence Model Sequence (SEQ ID (SEQ ID Species (SEQ ID
NO:) NO:) NO:) 361 16S_EC1090_1111_2_T 5 16S_EC_1175_1196_TM0D_R 370 Bacillus 764 MOD F anthracis 346 16S EC 713 732 TMOD 27 16S EC 789_809_TMOD_R 389 Bacillus 765 F - - _ anthracis 347 16S EC 785 806 TMOD 30 16S_EC_880_897_TMOD_R 392 Bacillus 766 F - anthracis 348 16S EC 960 981 TMOD 38 16S EC_1054_1073_TMOD_R 363 Bacillus 767 F - - - - anthracis 349 23S_EC1826_1843_TMO 49 23S_EC_1906_1924_TMOD_R 405 Bacillus 768 D F anthracis 360 23S_EC_2646_2667_TMO 60 23S_EC_2745 2765_TMOD_R 416 Bacillus 769 D F _ anthracis 350 CAPC BA 274 303 TMOD 98 CAPC_BA_349_376_TMOD_R 452 Bacillus 770 F anthracis 351 CYABA_1353_1379_TMO 128 CYA_BA 1448_1467_TMOD_R 483 Bacillus 771 D F- anthracis 352 INFBEC_1365_1393_TM 161 INFB_EC_1439_1467_TMOD_ 516 Bacillus 772 OD F R anthracis 353 LEF BA 756 781 TMOD 175 LEF_BA 843_872_TMOD_R 531 Bacillus 773 F - - anthracis 356 RPLB EC 650 679 TMOD 232 RPLB_EC_739_762_TMOD R 592 Clostridium 774 F - - botulinum 449 RPLB EC 690 710 F 237 RPLB_EC_737_758_R 589 Clostridium 775 botulinum 359 RPOBEC_1845_1866_TM 241 RPOB EC_1909_1929_TMOD_ 597 Yersinia 776 OD F_ R _ Pestis 362 RPOBEC3799_3821 TM 245 RPOB_EC_3862_3888_TMOD_ 603 Burkholderia 777 OD F_ _ R mallei 363 1 RPOCEC_21462174_TM 257 RPOCEC 22272245_TMOD_ 621 Burkholderia 778 OD F_ R mallei 354 RPOC_EC_2218_2241_TM 262 RPOCEC23132337_TMOD_ 625 Bacillus 779 OD F R anthracis 355 SSPE BA_115 137 TMOD 321 SSPE_BA 197_222_TMOD_R 687 Bacillus 780 F anthracis 367 TUFB_EC_957_979_TMOD 345 TUFB_EC_1034_1058_TMOD_ 701 Burkholderia 781 F R mallei 358 VALS_EC_1105_1124_TM 350 VALS_EC_1195_1218_TMOD_ 712 Yersinia 782 OD F R Pestis Table 10: Primer Pair Gene Coordinate References and Calibration Polynucleotide Sequence Coordinates within the Combination Calibration Polynucleotide Bacterial Gene Gene Extraction Coordinates Reference GenBank GI No. of Primer Pair Coordinates of Calibration and Species of Genomic or Plasmid Sequence Genomic (G) or Plasmid (P) No.
Sequence in Combination Sequence Calibration Polynucleotide (SEQ
ID NO: 783) 16S E. coli 4033120. .4034661 16127994 (G) 346 16..109 16S E. coli 4033120. .4034661 16127994 (G) 347 83..190 16S E. coli 4033120. .4034661 16127994 (G) 348 246. .353 16S E. coli 4033120. .4034661 16127994 (G) 361 368. .469 23S E. coli 4166220. .4169123 16127994 (G) 349 743. .837 23S E. coli 4166220. .4169123 16127994 (G) 360 865..981 rpoB E. 4178823. .4182851 16127994 (G) 359 1591. .1672 coli. (complement strand) rpoB B. coli 4178823..4182851 16127994 (G) 362 2081. .2167 (complement strand) rpoC E. coli 4182928..4187151 16127994 (G) 354 1810. .1926 rpoC B. coli 4182928..4187151 16127994 (G) 363 2183. .2279 infB E. coli 3313655..3310983 16127994 (G) 352 1692. .1791 (complement strand) tufB E. coli 4173523..4174707 16127994 (G) 367 2400. .2498 rplB B. coli 3449001..3448180 16127994 (G) 356 1945. .2060 rp1B B. coli 3449001..3448180 16127994 (G) 449 1986. .2055 valS B. coli I 4481405..4478550 16127994 (G) 358 1462..1572 (complement strand) caps 56074..55628 (complement 6470151 (P) 350 2517..2616 B. anthracis strand) cya 156626..154288 4894216 (P) 351 1338..1449 B. anthracis (complement strand) lef 127442..129921 4894216 (P) 353 1121..1234 B. anthracis sspE 226496..226783 30253828 (G) 355 1007-1104 B. anthracis [0128] Example 10: Use of a Calibration Polynucleotide for Determining the Quantity of Bacillus Anthracis in a Sample Containing a Mixture of Microbes [0129] The process described in this example is shown in Figure 7. The capC
gene is a gene involved in capsule synthesis which resides on the pX02 plasmid of Bacillus anthracis. Primer pair number 350 (see Tables 9 and 10) was designed to identify Bacillus anthracis via production of a bacterial bioagent identifying amplicon. Known quantities of the combination calibration polynucleotide vector described in Example 3 were added to amplification mixtures containing bacterial bioagent nucleic acid from a mixture of microbes which included the Ames strain of Bacillus anthracis. Upon amplification of the bacterial bioagent nucleic acid and the combination calibration polynucleotide vector with primer pair no. 350, bacterial bioagent identifying amplicons and calibration amplicons were obtained and characterized by mass spectrometry. A mass spectrum measured for the amplification reaction is shown in Figure 8).
The molecular masses of the bioagent identifying amplicons provided the means for identification of the bioagent from which they were obtained (Ames strain of Bacillus anthracis) and the molecular masses of the calibration amplicons provided the means for their identification as well. The relationship between the abundance (peak height) of the calibration amplicon signals and the bacterial bioagent identifying amplicon signals provides the means of calculation of the copies of the pX02 plasmid of the Ames strain of Bacillus anthracis.
Methods of calculating quantities of molecules based on internal calibration procedures are well known to those of ordinary skill in the art.

[0130] Averaging the results of 10 repetitions of the experiment described above, enabled a calculation that indicated that the quantity of Ames strain of Bacillus anthracis present in the sample corresponds to approximately 10 copies of pX02 plasmid.

[0131] Example 11: Drill-down Genotyping of Campylobacter Species [0132] A series of drill-down primers were designed as described in Example 1 with the objective of identification of different strains of Campylobacterjejuni. The primers are listed in Table 11 with the designation "CJST CJ." Housekeeping genes to which the primers hybridize and produce bioagent identifying amplicons include: tkt (transketolase), glyA
(serine hydroxymethyltransferase), gitA (citrate synthase), aspA (aspartate ammonia lyase), glnA
(glutamine synthase), pgm (phosphoglycerate mutase), and uncA (ATP synthetase alpha chain).
Table 11: Campylobacter Drill-down Primer Pairs Primer Forward Primer Name Forward Primer Reverse Primer Name Reverse Primer Target Gene Pair (SEQ ID NO:) (SEQ ID NO:) No.
1053 CJST CJ 1080 1110 F 102 CJST CJ 1166 1198 R 456 gitA
1064 CJST CJ 1680 1713 F 107 CJST CJ 1795 1822 R 461 91yA
1054 CJST CJ 2060 2090 F 109 CJST CJ 2148 2174 R 463 pgm 1049 CJST CJ 2636 2668 F 113 CJST CJ 2753 2777 R 467 tkt 1048 CJST CJ 360 394 F 119 CJST Cl 442 476 R 472 aspA
1047 CJST CJ 584 616 F 121 CJST CJ 663 692 R 474 ginA

[0133] The primers were used to amplify nucleic acid from 50 food product samples provided by the USDA, 25 of which contained Campylobacter jejuni and 25 of which contained Campylobacter coli. Primers used in this study were developed primarily for the discrimination of Campylobacter jejuni clonal complexes and for distinguishing Campylobacter jejuni from Campylobacter coli. Finer discrimination between Campylobacter coli types is also possible by using specific primers targeted to loci where closely-related Campylobacter coli isolates demonstrate polymorphisms between strains. The conclusions of the comparison of base composition analysis with sequence analysis are shown in Tables 12A-C.
Table 12A - Results of Base Composition Analysis of 50 Campylobacter Samples with Drill-down MLST Primer Pair Nos: 1048 and 1047 Base Base MLST type or Composition of Composition of Clonal MLST Type or Clonal Bioagent Bioagent Isolate Complex by Identifying Identifying Group Species origin Base Complex by Strain Amplicon Amplicon Composition Sequence Obtained with Obtained with analysis analysis Primer Pair No: Primer Pair 1048 (aspA) No: 1047 (glnA) J-1 C. Goose ST 690 ST 991 RM3673 A30 G25 C16 T46 A47 G21 C16 T25 jejuni /692/707/991 ST 356C Complex , J-2 jejuni Human 206/48/353 complex RM4192 A30 G25 C16 T46 A48 G21 C17 T23 J-3 C. Human Complex ST 436 RM4194 A30 G25 C15 T47 A48 G21 C18 T22 jejuni 354/179 ST 257, J-4 C. Human Complex 257 complex RM4197 A30 G25 C16 T46 A48 G21 C18 T22 ejuni 257 ejuni RM4277 A30 G25 C16 T46 A48 G21 C17 T23 J-5 C. Human Complex 52 complex 52, ex 52 ST 51, R844275 A30 525 C15 T47 A48 521 C17 T23 j J-6 Human Complex 443 complex ejuni 443 RM4279 A30 G25 C15 T47 A48 G21 C17 T23 J-7 C. Human Complex 42 ST 604, RM1864 A30 G25 C15 T47 A48 G21 C18 T22 jejuni complex 42 ST 362, J-8 C. Human Complex complex RM3193 A30 G25 C15 T47 A48 G21 C18 T22i ejuni 42/49/362 362 J-9 C. Human Complex ST 147, RM3203 A30 G25 C15 T47 A47 G21 C18 T23 jejuni 45/283 Complex 45 C.
Human Consistent ST 828 RM4183 A31 G27 C20 T39 A48 G21 C16 T24 jejuni with 74 ST 832 RM1169 A31 G27 C20 T39 A48 G21 C16 T24 closely related ST 1056 RM1857 A31 G27 C20 T39 A48 G21 C16 T24 sequence types (none ST 889 RM1166 A31 G27 C20 T39 A48 G21 C16 T24 belong to a clonal ST 829 RM1182 A31 G27 C20 T39 A48 G21 C16 T24 complex) Poultry C-1 C. coli ST 860 RM1840 A31 G27 C20 T39 A48 G21 C16 T24 Swine ST 1069 RM3231 A31 G27 C20 T39 A48 G21 C16 T24 Unknown C-2 C. coli Human ST 895 ST 895 RM1532 A31 G27 C19 T40 A48 G21 C16 T24 Consistent ST 1064 RM2223 A31 G27 C20 T39 A48 G21 C16 T24 with 63 closely ST 1082 RM1178 A31 G27 C20 T39 A48 G21 C16 T24 Poultry related C-3 C. coli sequence ST 1054 RM1525 A31 G27 C20 T39 A48 G21 C16 T24 types (none belong to a ST 1049 RM1517 A31 G27 C20 T39 A48 G21 C16 T24 clonal Marmoset complex) ST 891 RM1531 A31 G27 C20 T39 A48 G21 C16 T24 Table 12B - Results of Base Composition Analysis of 50 Campylobacter Samples with Drill-down MLST Primer Pair Nos: 1053 and 1064 Base Base MLST type or Composition of Composition of Clonal MLST Type Bioagent Bioagent or Clonal Isolate Complex by Identifying Identifying Group Species origin Base Complex by Strain pmplicon Amplicon Sequence Composition Obtained with Obtained with analysis analysis Primer Pair Primer Pair No: 1053 (gltA) No: 1064 (glyA) J-1 jejuni Goose /692/007/991 ST 991 RM3673 ST 356, A24 025 C23 T47 A40 029 C29 T45 J-2 je Human 206/4Complex8/353 complex RM4192 j uni 353 J-3 C. Human Complex ST 436 RM4194 A24 G25 C23 T47 A40 G29 C29 T45 jejuni 354/179 ST 257, A24 G25 C23 T47 A40 G29 C29 T45 C jejuni Human Complex 257 complex RM4197 J-5 C. Human Complex 52 ST 52, RM4277 A24 G25 C23 T47 A39 G30 C26 T48 jejuni complex 52 ST 51, RM4275 A24 G25 C23 T47 A39 G30 C28 T46 J-6 jejuna Human Complex 443 complex 4279 A24 G25 C23 T47 A39 G30 C28 T46 J-7 C. Human Complex 42 ST 604, RM1864 jejuni complex 42 Complex 362, A24 G25 C23 T47 A38 G31 C28 T46 J-8 jejuni Human 42/49/362 complex RM3193 C. Complex ST 147, A24 G25 C23 T47 A38 G31 C28 T46 J-9 jejuni Human 45/283 Complex 45 RM3203 C. ST 828 RM4183 jejuni Human ST 832 RM1169 A23 G24 C26 T46 A39 G30 C27 T47 Consistent with 74 ST 1055 RM1527 A23 G24 C26 T46 A39 G30 C27 T47 Poultry closely related ST 1017 RM1529 A23 G24 C26 T46 A39 G30 C27 T47 sequence C-1 C. coli types (none ST 860 RM1840 A23 G24 C26 T46 A39 G30 C27 T47 belong to a clonal ST 1063 RM2219 A23 G24 C26 T46 A39 G30 C27 T47 complex) ST 1066 RM2241 A23 G24 C26 T46 A39 G30 C27 T47 Swine ST 1069 RM3231 A23 G24 C26 T46 NO DATA

Unknown ST 901 RM1505 A23 G24 C26 T46 A39 G30 C27 T47 C-2 C. coli Human ST 895 ST 895 RM1532 A23 G24 C26 T46 A39 G30 C27 T47 Consistent ST 1064 RM2223 A23 G24 C26 T46 A39 G30 C27 T47 with 63 closely ST 1082 RM1178 A23 G24 C26 T46 A39 G30 C27 T47 Poultry related A23 G24 C25 T47 A39 G30 C27 T47 C-3 C. coli sequence ST 1054 RM1525 types (none belong to a ST 1049 RM1517 A23 G24 C26 T46 A39 G30 C27 T47 clonal A23 G24 C26 T46 A39 G30 C27 T47 Marmoset complex) ST 891 RM1531 t Table 12C - Results of Base Composition Analysis of 50 Campylobacter Samples with Drill-down MLST Primer Pair Nos: 1054 and 1049 Base Base MIST type or Composition of Composition of Clonal MLST Type or Clonal Bioagent Bioagent Isolate Complex by Identifying Identifying Group Species origin Base Complex by Strain Amplicon Amplicon Composition Sequence Obtained with Obtained with analysis analysis Primer Pair No: Primer Pair 1054 (pgm) No: 1049 (tkt) J-1 jejuni Goose /692/007/991 ST 991 9143673 A26 933 C18 T38 A41 G28 C35 T38 ST 356, C. Complex J-2 jejuni Human 206/48/353 complex RM4192 A26 G33 C19 T37 A41 G28 J-3 C. Human Complex ST 436 RM4194 jejuni 354/179 A27 G32 C19 T37 A42 G28 C36 T36 C 257, J-4 jejuni Human Complex 257 complex RM4197 A27 G32 C19 T37 A41 G29 C35 T37 J-5 jejuni Human Complex 52 cST 52, omplex 52 RM4277 A26 G33 C18 T38 A41 G28 C. ST 51, RM4275 A27 G31 C19 T38 A41 G28 C36 T37 J-6 jejuni Human Complex 443 complex J-7 C. Human Complex 42 ST 604, RM1864 A27 G32 C19 T37 A42 G28 C35 T37 jejuni complex 42 ST 362, J-8 jejuni Human 42/49/362 complex RM3193 A26 G33 C19 T37 A42 G28 C35 T37 C. Complex ST 147, J-9 jejuni Human 45/283 Complex 45 RM3203 A28 G31 C19 T37 A43 G28 C36 T35 C.
jejuni ST 828 RM4183 A27 G30 C19 T39 A46 G28 C32 T36 Human ST 832 RM1169 Consistent ST 1055 RM1527 with 74 A27 G30 C19 T39 A46 G28 C32 T36 Poultry closely related ST 1017 RM1529 A27 G30 C19 T39 A46 G28 C32 T36 sequence C-1 C. coli types (none ST 860 RM1840 belong to a A27 G30 C19 T39 A46 G28 C32 T36 clonal ST 1063 RM2219 complex) A27 G30 C19 T39 A46 G28 C32 T36 Swine ST 1069 RM3231 A27 G30 C19 T39 A46 G28 C32 T36 Unknown C-2 C. coli Human ST 895 ST 895 RM1532 Consistent A27 530 C19 T39 A45 G29 C32 T36 with 63 closely ST 1082 RM1178 A27 G30 C19 T39 A45 G29 C32 T36 Poultry related C-3 C. coli sequence ST 1054 RM1525 types (none A27 G30 C19 T39 A45 G29 C32 T36 belong to a ST 1049 RM1517 clonal A27 G30 C19 T39 A45 G29 C32 T36 complex) Marmoset ST 691 RM1531 [0134] The base composition analysis method was successful in identification of 12 different strain groups. Campylobacter jejuni and Campylobacter coli are generally differentiated by all loci. Ten clearly differentiated Campylobacter jejuni isolates and 2 major Campylobacter coli groups were identified even though the primers were designed for strain typing of Campylobacter jejuni. One isolate (RM4183) which was designated as Campylobacter jejuni was found to group with Campylobacter coli and also appears to actually be Campylobacter coli by full MLST sequencing.

[0135] Example 12: Identification of Acinetobacter baumannii Using Broad Range Survey and Division-Wide Primers in Epidemiological Surveillance [0136] To test the capability of the broad range survey and division-wide primer sets of Table 4 in identification of Acinetobacter species, 183 clinical samples were obtained from individuals participating in, or in contact with individuals participating in Operation Iraqi Freedom (including US service personnel, US civilian patients at the Walter Reed Army Institute of Research (WRAIR), medical staff, Iraqi civilians and enemy prisoners). In addition, 34 environmental samples were obtained from hospitals in Iraq, Kuwait, Germany, the United States and the USNS Comfort, a hospital ship.

[0137] Upon amplification of nucleic acid obtained from the clinical samples, primer pairs 346-349, 360, 361, 354, 362 and 363 (Table 4) all produced bacterial bioagent amplicons which identified Acinetobacter baumannii in 215 of 217 samples. The organism Klebsiella pneumoniae was identified in the remaining two samples. In addition, 14 different strain types (containing single nucleotide polymorphisms relative to a reference strain of Acinetobacter baumannii) were identified and assigned arbitrary numbers from 1 to 14. Strain type 1 was found in 134 of the sample isolates and strains 3 and 7 were found in 46 and 9 of the isolates respectively.

[0138] The epidemiology of strain type 7 of Acinetobacter baumannii was investigated. Strain 7 was found in 4 patients and 5 environmental samples (from field hospitals in Iraq and Kuwait).
The index patient infected with strain 7 was a pre-war patient who had a traumatic amputation in March of 2003 and was treated at a Kuwaiti hospital. The patient was subsequently transferred to a hospital in Germany and then to WRAIR. Two other patients from Kuwait infected with strain 7 were found to be non-infectious and were not further monitored. The fourth patient was diagnosed with a strain 7 infection in September of 2003 at WRAIR. Since the fourth patient was not related involved in Operation Iraqi Freedom, it was inferred that the fourth patient was the subject of a nosocomial infection acquired at WRAIR as a result of the spread of strain 7 from the index patient.

[0139] The epidemiology of strain type 3 of Acinetobacter baumannii was also investigated.
Strain type 3 was found in 46 samples, all of which were from patients (US
service members, Iraqi civilians and enemy prisoners) who were treated on the USNS Comfort hospital ship and subsequently returned to Iraq or Kuwait. The occurrence of strain type 3 in a single locale may provide evidence that at least some of the infections at that locale were a result of a nosocomial infections.

[0140] This example thus illustrates an embodiment of the present invention wherein the methods of analysis of bacterial bioagent identifying amplicons provide the means for epidemiological surveillance.

[0141] Example 13: Selection and Use of MLST Acinetobacter baumanii Drill-down Primers [0142] To combine the power of high-throughput mass spectrometric analysis of bioagent identifying amplicons with the sub-species characteristic resolving power provided by multi-locus sequence typing (MLST) such as the MLST methods of the MLST Databases at the Max-Planck Institute for Infectious Biology (web.mpiib-berlin.mpg.de/mist/dbs/Mcatarrhalis/
documents/primersCatarrhalis html), an additional 21 primer pairs were selected based on analysis of housekeeping genes of the genus Acinetobacter. Genes to which the drill-down MLST analogue primers hybridize for production of bacterial bioagent identifying amplicons include anthranilate synthase component I (trpE), adenylate kinase (adk), adenine glycosylase (mutt), fumarate hydratase (fumC), and pyrophosphate phospho-hydratase (ppa).
These 21 primer pairs are indicated with reference to sequence listings in Table 13.
Primer pair numbers 1151-1154 hybridize to and amplify segments of trpE. Primer pair numbers 1155-hybridize to and amplify segments of adk. Primer pair numbers 1158-1164 hybridize to and amplify segments of mutY. Primer pair numbers 1165-1170 hybridize to and amplify segments of fumC. Primer pair number 1171 hybridizes to and amplifies a segment of ppa.
The primer names given in Table 13 indicates the coordinates to which the primers hybridize to a reference sequence which comprises a concatenation of the genes TrpE, efp (elongation factor p), adk, mutT, fumC, and ppa. For example, the forward primer of primer pair 1151 is named AB MLST-11-OIF007_62_91 F because it hybridizes to the Acinetobacter MLST
primer reference sequence of strain type 11 in sample 007 of Operation Iraqi Freedom (OIF) at positions 62 to 91.

Table 13: MLST Drill-Down Primers for Identification of Sub-species characteristics (Strain Type) of Members of the Bacterial Genus Acinetobacter primer Forward Primer Name Forward Reverse Primer Name Reverse Pair Primer Primer No. (SEQ ID NO:) (SEQ ID NO:) [0143] Analysis of bioagent identifying amplicons obtained using the primers of Table 13 for over 200 samples from Operation Iraqi Freedom resulted in the identification of 50 distinct strain type clusters. The largest cluster, designated strain type 11(STI 1) includes 42 sample isolates, all of which were obtained from US service personnel and Iraqi civilians treated at the 28th Combat Support Hospital in Baghdad. Several of these individuals were also treated on the hospital ship USNS Comfort. These observations are indicative of significant epidemiological correlation/linkage.

[0144] All of the sample isolates were tested against a broad panel of antibiotics to characterize their antibiotic resistance profiles. As an example of a representative result from antibiotic susceptibility testing, ST1 I was found to consist of four different clusters of isolates, each with a varying degree of sensitivity/resistance to the various antibiotics tested which included penicillin, extended spectrum penicillin, cephalosporins, carbipenem, protein synthesis inhibitors, nucleic acid synthesis inhibitors, anti-metabolites, and anti-cell membrane antibiotics.
Thus, the genotyping power of bacterial bioagent identifying amplicons, particularly drill-down bacterial bioagent identifying amplicons, has the potential to increase the understanding of the transmission of infections in combat casualties, to identify the source of infection in the environment, to track hospital transmission of nosocomial infections, and to rapidly characterize drug-resistance profiles which enable development of effective infection control measures on a time-scale previously not achievable.

[0145] Various modifications of the invention, in addition to those described herein, will be apparent to those skilled in the art from the foregoing description. Such modifications are also intended to fall within the scope of the appended claims.

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Claims (10)

1. An oligonucleotide primer pair composition selected from the group consisting of a forward primer 26 to 36 nucleobases in length comprising 85% to 100% sequence identity with the forward primer of Primer Pair No. 350 SEQ ID NO: 98, and a reverse primer 24 to 34 nucleobases in length comprising 85% to 100% sequence identity with the reverse primer of Primer Pair No. 350 SEQ ID NO: 452; a forward primer 23 to 33 nucleobases in length comprising 85% to 100% sequence identity with the forward primer of Primer Pair No. 351 SEQ
ID NO: 128, and a reverse primer 17 to 25 nucleobases in length comprising 85%
to 100%
sequence identity with the reverse primer of Primer Pair 351 SEQ ID NO: 483; a forward primer 25 to 35 nucleobases in length comprising 85% to 100% sequence identity with the forward primer of Primer Pair No. 352 SEQ ID NO: 161, and a reverse primer 21 to 31 nucleobases in length comprising 85% to 100% sequence identity with the reverse primer of Primer Pair No.
352 SEQ ID NO: 516; a forward primer 22 to 32 nucleobases in length comprising 85% to 100%
sequence identity with the forward primer of Primer Pair No. 353 SEQ ID NO:
175, and a reverse primer 18 to 26 nucleobases in length comprising 85% to 100% sequence identity with the reverse primer of Primer Pair No. 353 SEQ ID NO: 531; and a forward primer 20 to 28 nucleobases in length comprising 85% to 100% sequence identity with the forward primer of Primer Pair No. 355 SEQ ID NO: 321, and a reverse primer 22 to 32 nucleobases in length comprising 85% to 100% sequence identity with the reverse primer of Primer Pair No. 355 SEQ
ID NO: 687.

2. A composition comprising two or more of the oligonucleotide primer pair compositions of claim 1.

3. The composition of claim 1 or 2, wherein one of said primers comprises at least one modified nucleobase comprising: 7-deaza-2'-deoxyadenosine-5-triphosphate, 5-iodo-2'-deoxyuridine-5'-triphosphate, 5-bromo-2'-deoxyuridine-5'-triphosphate, 5-bromo-2'-deoxycytidine-5 '-triphosphate, 5-iodo-2'-deoxycytidine-5'-triphosphate, 5-hydroxy-2'-deoxyuridine-5'-triphosphate, 4-thiothymidine-5'-triphosphate, 5-aza-2'-deoxyuridine-5'-triphosphate, 5-fluoro-2'-deoxyuridine-5'-triphosphate, 06-methyl-2'-deoxyguanosine-5'- triphosphate, N2-methyl-2'-deoxyguanosine-5'-triphosphate, 8-oxo-2'-deoxyguanosine-5'- triphosphate, thiothymidine-5'-triphosphate, or a nucleobase comprising 15N or 13C.

4. The composition of claim 1 or 2, wherein both of said forward primer and said reverse primer comprise at least one modified nucleobase comprising: 7-deaza-2'-deoxyadenosine-5-triphosphate, 5-iodo-2'- deoxyuridine-5'-triphosphate, 5-bromo-2'-deoxyuridine-

5'-triphosphate, 5-bromo-2'-deoxycytidine-5'-triphosphate, 5-iodo-2'-deoxycytidine-5'-triphosphate, 5-hydroxy-2'- deoxyuridine-5'-triphosphate, 4-thiothymidine-5'-triphosphate, 5-aza-2'-deoxyuridine-5'-triphosphate, 5-fluoro-2'-deoxyuridine-5'-triphosphate, 06-methyl-2'-deoxyguanosine-5'-triphosphate, N2-methyl-2'-deoxyguanosine-5'-triphosphate, 8-oxo-2'-deoxyguanosine-5'-triphosphate, thiothymidine-5'-triphosphate, or a nucleobase comprising 15N or 13C.

5. The composition of claim 1 or 2, wherein one of said primers comprises a non-templated T
residue on the 5 '-end.

6. The composition of claim 1 or 2, wherein both of said forward primer and said reverse primer comprise a non-templated T residue on the 5 '-end.

7. The composition of claim 1 or 2, wherein one of said primers comprises at least one non-template tag.

8. The composition of claim 1 or 2, wherein both of said forward primer and said reverse primer comprise at least one non-template tag.

9. The composition of any one of claims 1 to 8, wherein one of said primers comprises at least one molecular mass modifying tag.

10. The composition of any one of claims 1 to 8, wherein both of said forward primer and said reverse primer comprise at least one molecular mass modifying tag.