AU2008255266A1 - Compositions for use in identification of bacteria - Google Patents

Description

S&F Ref: 780322D1

AUSTRALIA

PATENTS ACT 1990 COMPLETE SPECIFICATION FOR A STANDARD PATENT Name and Address of Applicant Actual Inventor(s): Address for Service: Invention Title: Ibis Biosciences, Inc., of 1896 Rutherford Road, Carlsbad, California, 92008, United States of America David J. Ecker Mark W. Eshoo Thomas A. Hall Christian Massire Rangarajan Sampath Spruson Ferguson St Martins Tower Level 31 Market Street Sydney NSW 2000 (CCN 3710000177) Compositions for use in identification of bacteria The following statement is a full description of this invention, including the best method of performing it known to me/us: 5845c(1890676_1) 00 1 CKI COMPOSITIONS FOR USE IN IDENTIFICATION OF BACTERIA CROSS-REFERENCE TO RELATED APPLICATIONS [0001] The present application claims the benefit of priority to: U.S. Provisional Application Serial No. 60/545,425 filed February 18,2004, U.S. Provisional Application Serial No.

S 60/559,754, filed April 5, 2004, U.S. Provisional Application Serial No. 60/632,862, filed tr~December 3, 2004, U.S. Provisional Application Serial No. 60/639,068, filed December 22, S 2004, and U.S. Provisional Application Serial No. 60/648,188, filed January 28, 2005, each of 00 C) which is incorporated herein by reference in its entirety.

STATEMENT OF GOVERNMENT SUPPORT [0002] This invention was made with United States Government support under DARPAISPO contract BAAOO-09. The United States Government may have certain rights in the invention.

SFIELD OF THE INVENTION [00031 The present invention relates generally to the field of genetic identification of bacteria and provides nucleic acid compositions and its useful for this purpose when combined with molecular mass analysis.

BACKGROUND OF THE INVENTION [0004] A problem in determining the cause of a natural infetious 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.

~sR. 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.

[00051 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 00bacteria, 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.

s [0006] A major conundrum in public health protection, biodefense, and agricultural safety and INO 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 00 isolation from other organisms and to obtain sufficient quantities of nucleic acid followed by S 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.

t 5 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.

[00081 There is a need for a method for identification of bioagents which is both specific and aC) rapid, and in which no culture or nucleic acid sequencing is required. Disclosed in U.S. Patent Application Serial Nos: 09/798,007, 09/891,793, 10/405,756, 10/418,514, 10/660,997, 10/660,122, 10/660,996, 10/728,486, 10/754,415 and 10/829,826, each of which is commonly owned and incorporated herein by reference in its entir .ety, are methods for identification of bioagents (any organism, cell, or virus, living or dead, or a nucleic acid derived from such an a 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.

00 00 -3- [00091 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 1c 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 is and does not require nucleic acid sequencing of the amplified target sequence for bioagent identification.

[00101 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) )O 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.

As [00111 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.

.2o 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), 00 S-4-

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S valyl-tRNA synthetase (valS), elongation factor EF-Tu (TufB), ribosomal protein L2 (rplB), dU 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.

N [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 ¢C comprising the same; an oligonucleotide primer 20 to 35 nucleobases in length comprising 00 S to 100% sequence identity with SEQ ID NO: 451, or a composition comprising the same; a C i o 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 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 )o 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 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 00 CN comprising the same; an oligonucleotide primer 19 to 35 nucleobases in length comprising U 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 S SEQ ID NO: 310, and a second oligonucleotide primer 19 to 35 nucleobases in length S comprising between 70% to 100% sequence identity of SEQ ID NO: 668.

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[0017] The present invention also provides an oligonucleotide primer 21 to 35 nucleobases in 00 length comprising 70% to 100% sequence identity with SEQ ID NO: 313, or a composition So comprising the same; an oligonucleotide primer 21 to 35 nucleobases in length comprising 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 i 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 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.

,aS [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 to 100% sequence identity with SEQ ID NO: 640, or a composition comprising the same; a ao 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.

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3 1 -6- [0020] The present invention also provides an oligonucleotide primer 21 to 35 nucleobases in S 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 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 q SEQ ID NO: 301, and a second oligonucleotide primer 21 to 35 nucleobases in length S 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 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 S 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, o 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 3s 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 00 o -7- 'N compositions or molecular masses identifies the unknown bacterium. The molecular mass can be U measured by mass spectrometry. In addition, the prrsence or absence of a particular clade, 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 S unknown bacterium in a sample. The sample is contacted with any of the compositions described N herein and a known quantity of a calibration polynucleotide comprising a calibration sequence.

S Concurrently, nucleic acid from the bacterium in the sample is amplified with any of the S compositions described herein and nucleic acid from the calibration polynucleotide in the sample io 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 S [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 3o number in the upper right hand corer 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 o -8- 0 C 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.

I [0030] Figure 5 is a represenataive mass spectrum of amplification products representing bioagent identifying amplicons of Streptococcus pyogenes, Neisseria meningitidis, and S Haemophilus influenzae obtained from amplification of nucleic acid from a clinical sample with 00 primer pair number 349 which targets 23S rRNA. Experimentally determined molecular masses 0 o 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 as 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 ao 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 00 CN functionality of producing, for example, bacterial bioagent identifying amplicons for general U identification of bacteria at the species level, for example, when contacted with bacterial nucleic acid under amplification conditions.

c-I [0035] 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 S (210) and analyzed (220). Primers are designed by selecting appropriate priming regions (230) V) which allows the selection of candidate primer pairs (240). The primer pairs are subjected to in 00 silico analysis by electronic PCR (ePCR) (300) wherein bioagent identifying amplicons are O 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 (240) 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).

[0036] 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.

[0037] 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 3o 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 00 C.1 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 6 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 00 for known bacterial bioagents. A match between the molecular mass or base composition of the c 1 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 IS 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.

[00391 In some embodiments, a bioagent identifying amplicon may be produced using only a )0 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 00 -11- N pair numbers 3, 10, 11, 14, 16, and 17 which consecutively correspond to SEQ ID NOs: 6:369, S 26:388, 29:391, 37:362, 48:404, and 58:414.

[0041] 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 D unambiguously identify a bacterial bioagent at the species level. These cases benefit from further Cl analysis of one or more bacterial bioagent identifying amplicons generated from at least one Vn additional broad range survey primer pair or from at least one additional "division-wide" primer 0 pair (vide infra). The employment of more than one bioagent identifying amplicon for D identification of a bioagent is herein referred to as "triangulation identification" (vide infra).

[0042] 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 and e-proteobacteria. In some embodiments, a division of bacteria comprises any grouping of bacterial genera with more than one genus represented. For example, the P-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 S identifying amplicon from the tufB gene of f-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 as protein chain initiation factor infB, chaperonins such as groL and dnaK, and cell division proteins such as peptidase fisH (hflB). In some embodiments, the division-wide primer pairs comprise oligonucleotides ranging in length from 13 to 35 nucleobases, each of which have from 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, 3o 349:711, 240:596, 246:602, 256:620, 344:700.

[0043] In other embodiments, the oligonucleotide primers are designed to enable the identification of bacteria at the clade 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 00 -12-

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C1 all of the descendants of that most recent common ancestor. The Bacillus cereus clade is an S example of a bacterial clade group. In some embodiments, the clade 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 ID NOs: i 322:686.

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[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 00 herein defined as genetic characteristics that provide the means to distinguish two members of 0 the same bacterial species. For example, Escherichia coli 0157:H7 and Escherichia coli K 2 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 (VNTRs). 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 o 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: 3o 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.

00 -13cI [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 traniscriptase are well known to those with ordinary skill in the art and can be routinely established without undue experimentation.

[00471 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 00 synthesis of a complementary nucleic acid strand in an amplification reaction. Moreover, a c-I primer may hybridize over one or more segments such that intervening or adjacent segments are not involved in the hybridization event 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 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.

[00481 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). lIn 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 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 therewitbin) sequence identity with the primer sequences specifically disclosed herein. Thus, for example, a primer may have between 70% and 00 -14s 100%, between 75% and 100%, between 80% and 100%, and between 95% and 100% sequence dU identity with SEQ ID NO: 26. Likewise, a primer may have similar sequence identity with any other primer whose nucleotide sequence is disclosed herein.

s [0050] One with ordinary skill is able to calculate percent sequence identity or percent sequence N 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 00 corresponding bioagent identifying amplicon.

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[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, 31, 32, 33, 34 or 35 nucleobases in length, or any range therewithin.

IS

[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 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 0o 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 3 r d position) in the conserved as 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 binds to U, C or A; guanine binds to U or C, and uridine binds to U or C. Other examples of universal nucleobases include nitroindoles Ssuch as 5-nitroindole or 3-nitropyrrole (Loakes et al., Nucleosides and Nucleotides, 1995, 14, 1001-1003), the degenerate nucleotides dP or dK (Hill et 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-j-D-ribofuranosyl)-imidazole-4-carboxamide (Sala et al., Nucl. Acids Res., 1996, 24, 3302-3306).

00 o C [0054] In some embodiments, to compensate for the somewhat weaker binding by the "wobble" d) 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 Snucleotide. 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 SU.S. Patent Nos. 5,645,985,5,830,653 and 5,484,908, each of which is commonly owned and incorporated herein by reference in its entirety. Propynylated primers are described in U.S Serial 00 0 No. 10/294,203 which is also commonly owned and incorporated herein by reference in entirety.

C Io Phenoxazines are described in U.S. Patent Nos. 5,502,177, 5,763,588, and 6,005,096, each of which is incorporated herein by reference in its entirety. G-clamps are described in U.S. Patent Nos. 6,007,992 and 6,028,183, each of which is incorporated herein by reference in its entirety.

[00551 In some embodiments, non-template primer tags are used to increase the melting temperature (Tm) 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 5 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 interucleoside linkage such as a phosphorothioate linkage, for example.

[0057] In some embodiments, the primers contain mass-modifying tags. Reducing the total so 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.

00 -16- C [0058] In some embodiments of the present invention, the mass modified nucleobase comprises U one or more of the following: for example, 7-deaza-2'-deoxyadenosine-5-triphosphate, 5-iodo-2'- 5-bromo-2'-deoxyuridine-5'-triphosphate, 5-bromo-2'- 5-iodo-2'-deoxycytidine-5'-triphosphate, 5-hydroxy-2'deoxyuridine-5'-triphosphate, 4-thiothymidine-5'-triphosphate, 5-aza-2'-deoxyuridine-5'- I 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 00 nucleobase comprises "N or "C or both "N and "C.

[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 from about 45 to about 200 linked nucleosides), from about 60 to about 150 nucleobases, from about 75 to about 125 nucleobases.

o 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 3o (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.

00 -17c-.I [00601 In some embodiments, bioagent identifying amplicons amenable to molecular mass

U

(1 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 I of providing a predictable fragmentation pattern of an amplification product include, but are not c.I 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.

00 o [0061] 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.

[0062] 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, 0 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: 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. Patent Serial No. 10/829,826 (incorporated herein by reference in its entirety) 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 0 0 00 -18-

O

S science prior to April, 2003 and since it was not known what bioagent (in this case a eU coronavirus) was present in the sample. On the other hand, if the method of U.S. Patent Serial No. 10/829,826 was to be employed subsequent to April 2003 to identify the SARS coronavirus in a clinical sample, only the first meaning of"unknown" bioagent would apply since the S SARS coronavirus became known to science subsequent to April 2003 and since it was not I known what bioagent was present in the sample.

IN

S [0064] The employment of more than one bioagent identifying amplicon for identification of a 0 bioagent is herein referred to as "triangulation identification." Triangulation identification is Si o 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 o 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 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 00 00- 19weight of the sample is several hundred, or in excess of one hundred thousand atomic mass units S (amu) or Daltons.

[0067] In some embodiments, intact molecular ions are generated from amplification products s using one of a variety of ionization techniques to convert the sample to gas phase. These S ionization methods include, but are not limited to, electrospray ionization matrix-assisted laser desorption ionization (MALDI) and fast atom bombardment (FAB). Upon ionization, C, 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 ,1 o 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.

.0 [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 T, C and 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 as 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 00

O

00 (O

O

oo

O-

^0 i ^s 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 o 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 ao 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 as 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 00 00 -21- S insertion within the variable region between the two priming sites. The amplified sample U 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 N 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 00 Scontacted 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 i6 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 jo 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 as 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 0 0 -22- S 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 N 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 C 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 C1 to 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 Lo 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), clade 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 00 -23- 23 0 CN particular bioagent. For example, a broad range survey primer kit may be used initially to q 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

C-I

thuringiensis from each other. A clade group primer kit may be used, for example, to identify an unknown bacterium as a member of the Bacillus cereus clade group. A drill-down kit may be N 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, clade group primers or drill-down primers, or any combination thereof, 0 0 for identification of an unknown bacterial bioagent.

C- 'O [0081] In some embodiments, the kit may contain standardized calibration polynucleotides for use as internal amplification calibrants. Internal calibrants are described in commonly owned U.S. Patent Application Serial No: 60/545,425 which is incorporated herein by reference in its entirety.

[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 ao 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, 0 -24- S 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 S [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 C' pairs of PCR primers would amplify products of about 45 to about 200 nucleotides in length and S distinguish species from each other by their molecular masses or base compositions. A typical i 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, which is incorporated herein by reference in its entirety) has been modified to include PCR parameters such as hybridization conditions, mismatches, and thermodynamic calculations (SantaLucia, Proc. Natl. Acad. Sci. 1998, 1460-1465, which is incorporated herein by reference in its entirety). 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 16S_EC_1077_1106 indicates that the primer hybridizes to residues 1077-1106 of the gene encoding 16S ribosomal RNA in an E. coli reference sequence represented by a sequence extraction of coordinates 4033120..4034661 from GenBank gi number 16127994 (as indicated in Table 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 In Table 1, Ua 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: 1 16S EC 107 GTGAGATGTTGGGTTAA 1 16S EC 1175 GACGTCATCCCCACCTTCC 368 7 1106 F GTCCCGTAACGAG 1195 R. TC 16SEC_1177 16S_-EC_108 ATGTTGGGTTAAGTCCC -1196_lOGil TGACGTCATGGCCACCTTC 266 2 1100F GC 2 1G R C 372 16S C_108 ATGTTGGGTTAAGTCCC 16SEC_1177 TGACGTCATGCCCACCTTC 265 2 1100 F GC 2 119;6 TOG R. C 373 163SEC_108 ATGTTGGGTTAAGTCCC lESEC_1177 TGACGTCATCCCCACCTTC 230 2 1100 F GC 2 1196 R C 374 16SEC_108 ATGTTGGGTTAAGTCCC 16SEC_1525 263 2 1100 F GC 2 .1541 R AAGGAGGTGATCCAGCC 382 16SEC_108 ATGTTGGGTTAAGTCCC 16SEC_1175 TTGACGTCATCCCCACCTT 2 2 1106 F GCAACGAG 3 1197 a CCTC 371 16SEC_109 TTAAGTCCCGCAACGAG 16SEC_1175 TGACGTCATCCCCACCTTC 278 10 1111 2 F CGCAA 4 1196 R. CTC- 369 16S_EC_109 16SEC_1175 0_1111_2 T TTTAAGTCCCGCAACGA 1196_'IMOD_ TTGACGTCATCCCCACCTT 361 MOD F CGCAA 5 1. CCTC 370 163_EC_109 TTAAGTCCCGCAACGAT 16SEC-1175 TGACGTCATCCCCACCTTC 3 0 1111 F CGCAA 6 1196 R CTC 1 369 163_SC_109 TAGTCCCGCAACGAGCG 16SSC 1174 GACGTCATCCCCACCTTCC 256 2 1109 F C 7 1195 R TCC 367 163_SC_110 16S_-EC_1174 159 0 1116 F CAACGAGCGCAACCCTT 8 1188 R. TCCCCACCTTCCTCC 366 163_EC_119 CAAGTCATCATGGCCCT 16sSEC 1525 247 5 121l3 F TA 9 1541 'i AAGGAGGTGATCCAGCC 382 16SEC_122 GCTACACACGTGCTACK 16SSC_1303 CGAGTTCCAGACTGCGATC 4 2 1-241-F ATG 10 1323 R. CG 376 16SEC_130 CGGATTGGAGTCTGCAA 163SEC_-1389 232 3 1323-F CTCG 11 1407 R GACGGGCGGTGTGTACAAG 378 16SEC_133 AAGTCGGAALTCGCTAGT 6E C_1389 2 13553-F AATCG 12 1407 9. GACGGGCTCTGTACAAG 378 163SEC_136 TACGGTGAATACGTTCC 16SEC_1485 ACCTTGTTACGACTTCACC 252 7 1387-F CGGG 13 1506 R. CCA 379 16S_EC_138 GCCTTGTACACACCTCC 16SSC 1494 CACGGCTACCTTGTTACGA 250 7 1407 F CGTC 14 11513 R. C 381 16SEC_138 CTTGTACACACCGCCCG 16SEC_-1525 231 9 1407 F 'IC 15 1541 R. AAGGAGGTGATCCAGCC 382 16S_SC_139 TTGTACACACCCCCGT 16SC_1486 CCTTGTTACGACTTCACCC 251 0 1411 F CTAC 16 1505 R C 380 16S_SC_30_ TGAACGCTGGTGGCATG 16S_SC 105_ TACGCATTACTCACCCGTC 6 54 F CTTAACAC 17 126 R. CCC 361 16SEC_314 CACTGGAACTGAGACAC 16SEC 556- CTTTACGCCCAGTAATTCC 243 332iF- GG 18 5759R G 385 16S_SC_38_ GTGGCATGCCTAATACA. 16SEC 101- TTACTCACCCCTCCGCCC 7 64 F TGCAAGTCG 19 120 R T 357 163S EC_405 TGAGTGATGAACGCCTT 16SSC 507 CGGCTGCTGGCACGAAGTT 279 432 F AGGGTTGTAAA 20 527 R. AG 384 16SEC 49 TAACACATGCAAGTCGA 16SEC 104 8 68 F ACG 21 120 R. TTACTCACCCGTCCGCC 359 16SEC 49- TAACACATGCAAGTCGA 16SEC_1061 275 68 F ACG 21 1078 R. ACGACACGAGCTGACGAC 364 16S_SC_49_ TAACACATGCAAGTCGA 16SEC 880 274 68 F ACG 21 894 9R CCTACTCCCCAGGCG 390 16SEC_518 CCAGCAGCCGCGGTAAT 16S SC 774- GTATCTAATCCTGTTTGCT 244 5 36 F- AC 22 795 R CCC 387 16SEC_556 CGGAATTACTGGCTA 16S -EC 683_ 226 575 F AAG_ 23 700 R9 CGCATTTCACCGCTACAC 386 165 EC 556 CGGAATTACTGGGCGTA 16S EC 774_ GTATCTAATCCTGTTTGCT 264 573 F- AAG 23 795-R CCC 387 16S SC_683 GTGTAGCGGTGAAATCC 16S EC 1303 CCAGTTGCAGACTCCGATC 273 706 F: G 24 13239.i CG 377 163SEC_683 GTGTAGCGGTGAAATGC 16SEC 774- GTATCTAATCCTGTTTGCT 9 700 F G 24 7959R CCC 387 16SS C_683 GTGTAGCGGTGAAATGC 16SSC 880- 158 700 G 24 894 R. CGTACTCCCCAGGCG 390 16SS C_683 GTGTAGCGGTGAAATGC 16SSC 967_ 245 700 F- G 24 985R r. GTAAGGTTCTTCGCGTTG 396 163SEC_7_3 GAGAGTTTGATCCTGGC 16SS C -101 TGTTACTCACCCGTCTCC 294 3 F TCAGAACGAA 25 122 R. ACT 358 163S EC_713 AGAACACCGATGGCGAA 16S SC 789- CCTGGACTACCAGGGTATC 732 1F GCC 26 8099R TA 388 163 SC_713 732i MOD_ TAGAACACCGATGGCGA 16S SC 789 TCGTGGACTACCAGGGTAT 346 F AGGC 27 809 Tf00 R CTA 369 228 163 EC 774 GGGAGCAAACAGCATTA 28 16S EC 880 CGTACTCCCCAGGCG 390 00 00 7-95 F GATAC 894 16SEC_785 GGATTAGAGACCCTGGT 16S EC 680 11 606 iF AGTCC 29 897-R GGCCGTACTCCCCAGGCG 391 16S EC_785 -806_THOD TGGATTAGAGACCCTGG 1iSSC 980 347 F TAGTCC 30 897 TfOD IC TGGCCGTACTCCCCAGGCG 392 16SEC_785 GGATTAGATACCCrGGT 165SEC_880- 12 810 AGTCCACGC 31 872R GGCCGTACTCCCCAGGCG 391 16SEC_789 TAGATACCCTGGTAGTC 16SEC 880 13 810 F CACGC 32 894-R CGTACTCCCCAGGCG 390 165_EC_789 TAGATACCCTGGTAGTC 16S-EC 882- 255 810 F CACGC 32 899 R GCGACCGTACTCCCCAGG 393 16sSEC_791 GATACCCTGGTAGTCCA 16SEC 886 254 812' F- CACCG 33 904- GCCTTGCGACCGTACTCCC 394 16sEC_6_2 AGAGTTTGATCATGGCT 16SEC_1525 248 7 F CAG 34 1541 R. AAGGAGGTGATCCAGCC 382 16sEC_8_2 AGAGTTTGATCATGGCT 16SSC 342- 242 7 F CAG 34 358 R ACTGCTGCCTCCCGTAG 383 16SEC_804 ACCACGCCGTAAACGAT 16SEC 909 CCCCCGTCAATTCCTTTGA 253 8 22 F GA 35 9295R- GT 395 16S_-EC_-937 AAGCGGTGGAGCATGTG 16SEC_1220 ATTGTAGCACGTGTGTAGC 246 954 F G 36 12405 CC cc 375 165 EC_960 TTCGATGCAACGCGAAG 16SEC_1054 ACGAGCTGACGACAGCCAT 14 981 F AACCT 37 1073 P. G 362 16S EC_960 16SEC_1054 -981_T-MOD- TTTCGATGCAACGCGAA 1073_Tt4OD_ TACGAGCTGACGACAGCCA 348 F GAACCTk 38 P. TG 363 165_SC_969 ACGCGAAGAACCTTA 165SC_1061 119 985 IP F UrC 39 1078 2P P. ACGACACGAGU'CGACGAC 364 165_EC_969 16SEC_1061 985 F ACGCGAAGAACCTTACC 39 1078 R. ACGAC-ACGAGCTGACGAC 364 16SEC_969 16S EC_1389 272 985 F- ACGCGAAGAACCTTACC 40 14067 PR GACGGGCGGTGTGTACAAG 378 16SEC_971 GCGAAGAACCTTACCAG 16SEC_1043 ACAACCATGCACCACCTGT 344 99 F- GTC 41 1 062 R C 360 165SEC_972 16SEC_1064 120 985 2P; F CGAAGAAUTTACC 42 1075 2P P. ACACGAGUCGAC 365 16S EC_972 165 EC_1064 121 985 CGAAGAACCTTACC 42 1075 R ACACGAGCTGAC 365 23S BRM_11 TGCGCGGAAGATGTAAC 23SURM_117 TCGCAGGCTTACAGAACGC 1073 10 1129 F GGG 43 6 1201 P.i TCTCCTA 397 23SBUN_51 TGCATACAAACAGTCGG 23SBUN_616 TCGGACTCGCTTTCGCTAC 1074 5 536 F AGCCT 44 635 R. G 398 23SBS AAACTAGATAACAGTAG 23SBS521 rA 241 68 -44 F ACATCAC 45 P. GTGCGCCCTTTCTAACTT 399 23SEC_160 TACCCCAAACCGACACA 23S EC_1686 235 2 16E20 F GG 46 17063 P. CCTTCTCCCGAAGTTACG 402 23SEC_168 CCGTAACTTCGGGAGAA 23SEC1828 236 5 173F G 47 182R CACCGGGCAGGCGTC 403 23SEC_182 CTGACACCTGCCCGGTG 23S EC_1906 16 -6 1843 F C 48 1924 P. GACCGTTATAGTTACGGCC 404 23SSC_182 23SSC_1906 6_1843_TMO TCTGACACCTGCCCGGT -1924_Tk4OD_ TGACCGTTATAGTTACGGC 349 gD F GC 49 R. C 405 23S EC_182 23SSC_1929 CCGACAAGGAATTTCGCTA 237 7 1643 F GACGCCTGCCCGGTGC 50 1949 R. cc 407 23S SC_183 ACCTGCCCAGTGCTGGA 23SEC_1919 249 1 1849 F AG 51 1936 R. TCGCTACCTTAGGACCGT 406 23 5SC_-187 GGGAACTGAAACATCTA 23SSC 242_ 234 207 F AGTA 52 256 P. TTCGCTCGCCGCTAC 408 23SSC 23- 23S5EC 115_ 233 37 F GGTGGATGCCTTGGC 53 130 P. GGGTTTCCCCATTCGG 401 235SEC_243 AAGGTACTCCGGGGATA 23SSC_2490 AGCCGACATCGAGGTGCCA 238 4 2456 F ACAGGC 54 2511 P. AAC 409 23SSC_258 TAGAACGTCGCGAGACA 23SEC_2658 AGTCCATCCCGGTCCTCTC 257 6 2607 F GTTCG 55 2677 P. G 411 235_EC_259 GACAGTTCGGTCCCTAT 23SSC_2653 239 9 2616 6F C 56 2669 "i CCGGTCCTCTCGTACTA 410 235SEC_264 CTGTCCCTAGTACGAGA 23SEC2751 GTTTCATGCTTAGATGCTT 18 5 2669 2 F GGACCGG 57 2767 P. TCAGC 417 23S SC_264 TCTGTCCCTAGTACGAG 23S EC 2744 17 5 26697F AGGACCGG 58 2761 P. TGCTTAGATGCTTTCAGC 414 23SSC_264 CTGTTCTTAGTACGAGA 23S SC_2745 TTCGTGCTTAGATGCTTTC 118 6 26g67 F GGACC 59 276g5 P. AG 1415 360 235 SC 264 TCTGTTCTTAGTACGAG 60 23S SC 2745 TTTCGTGCTTAGATGCTTT 41 -27- 6_2667_TMO AGGACC -2765_TM0D- CAG -DF 23SEC_265 CTAGTACGAGAGGACCG 23S C_-2741 ACTTAGATGCTTTCAGCGG 147 2 2669 F G 61 2760 R T 413 23SEC_265 23SEC 2737 TTAGATGCTTTCAGCACTT 240 -3 2669 F TAGTACGAGAGGACCGG 62 27598 R ATC 412 235SEC_493 GGGGAGTGAAAGAGATC 23S SC 551- ACAAAAGGCACGCCATCAC 518 2 F CTGAAACCG 63 571 2 R cc 418 23SEC_493 GGGGAGTGAAAGAGATC 23SEC 551- ACAAAAGGTACGCCGTCAC 19 518 F CTGAAACCG 63 571 R cc 419 23SEC_971 CGAGAGGGAAACAACCC 23SEC_1059 21 9 92 F- AGACC 64 1077 R TGGCTGCTTCTAAGCCAAC 400

ABNLST-

11-: ABMLST-11- 01F007_120 TCGTGCCCGCAATTTGC OI1F007_1266 TAATGCCGGGTAGTGCAAT 1158 2_1225 F ATAAAGC 65 1296 R CCATTCTTCTAG 420

ASMLST-

11-7 AB_MLST-11- 01F007_120 TCGTGCCCGCAATTTGC 01F007_1299 1159 2 1225-F ATAAAGC 65 1316 R TGCACCTr.CGGTCGAGCG 421

ASMLSTI-

11- AB k4LST-11- 01F007_123 TTGTAGCACAGCAAGGC 01F0O07_-1335 TGCCATCCATAATCACGCC 1160 4 1264 F AAATTTCCTGAAAC 66 1362 R ATACTGACG 422

AB_MLST-

11- ABMLST-11- 0EF007_132 TAGGTTTACGTCAGTAT 01F00O7_1422 TGCCAGTTTCCACATTTCA 1161 7 1356 F GGCGTGATTATGG 67 1448 Ri CGTTCGTG 423 AS_k4LST- 11- AS _MLST-11- 01F007_134 TCGTGATTATGGATGGC OIF007_1470 TCGCTTIGAGTGTAGTCATG 1162 5 1369-F AACGTGAA 68 1494 R ATTGCG 424

ASMLST-

11-: AB_?4LST-11- 01F007_135 TTATGGATGGCAACGTG OIT007_1470 TCGCTTGAGTGTAGTCATG 1163 1 1375 F AAACGCGT 69 1494 R ATTGCG 424 AB_?4LST- 11-: ABMLST-11- 01F007 138 TCTTTGCCATTGAAGAT 01-f007_1470 TCGCTTGAGTGTAGTCATG 1164 7 1412 F GACTTAAGC 70 1494 R ATTGCG 424

ASHLST-

11- ABMLST-11- OIF007 154 TACTAGCGGTAAGCTTA OIF007 1656 TGAGTCGGGTTCACTTTAC 1165 2 1569 F AACAAGATTGC 71 1680 R. CTGGCA 425

ASNLST-

11- ABMLST-11- 0IF007_156 TTGCCAATGATATTCGT 01F007_1656 TGAGTCGGGTTCACTTTAC 1166 6 1593 F TGGTTAGCAAG 72 1680 R. CTGGk 425 AS _MLST- 11- AS MLST-11- 0IF007_161 TCGGCGAAATCCGTATT 01f007_1731 TACCGGAAGCACCAGCGAC 1167 1 1638 F CCTGAAAATGA 73 1757 R ATTAATAG 427

ASMLST-

11- ABMLST-11- 01F007_172 TACCACTATTAATGTCG OIF007_1790 TGCAACTGAATAGATTGCA 1168 6 1752 F CTGGTGCTTC 74 1821 9. GTAAGTTATAAGC 428

ASMLST-

11- TTATAACTTACTGCAAT AB MLST-11- 01F007_179 CTATTCAGTTGCTTGGT 01F007_1876 TGAATTATGCAAGAAGTGA 1169 2 1826 F G 75 1909 R. TCAATTTTCTCACGA 429 ASBl4LST- 11- TTATAACTTACTGCAAT ABMLST-11- OIF007_179 CTATTCAGTTGCTTGGT OIFOD71895 TGCCGTAACTAACATAAGA 1170 2 1826 F G 75 1927 R GAATTATGCAAGAA 430

ABMLST-

11- ASXLST-11- 0IF007 185 TATTGTTTCAAATGTAC 01F007 291- TCACAGGTTCTACTTCATC 1152 214 F AAGGTGAAGTGCG 76 324 R. AATAATTTCCATTGC 432

AS_-MLST-

11- AS_!4LST-11- 0IF007_197 TGGTTATGTACCAAATA OIF007_2097 TGACGGCATCGATACCACC 1171 0 2002 F CTTTGTCTGAAGATGG 77 2118 R. CTC 431

AS_MLST-

11- ASNLST-11- 01FOOI_206 TGAAGTGCGTGATGATA OIF007 318 TCCGCCAAAAACTCCCCTT 1154 239 E: TCGATGCACTTGATGTA 78 344 R TTCACAGG 433 28 ABMST ABNMLST-11- 01F007_260 TGGAACGTTATCAGGTG 01F007_364 TTGCAATCGACATATCCAT 113 289 F CCCCAAAAATTCG 79 393 R TTCACCATGCC 434 AB MLST- 11-: ABMLST-11- 0IF007_522 TCGGTTTAGTAAAAGAA 01F007_587_ TTCTGCTTGAGGAATAGTG 1155 552 F CGTATTGCTCAACC 80 610 R CGTGG 435 ASN LST- 11- AS MLST-11- OIF007_547 TCAACCTGACTGCGTGA 01F007 656 TACGTTCTACGATTTCTTC 1156 571 F- ATGGTTGT 81 686 R. ATCAGGTACATC 436

AS_MLST-

11- ABNLST-11- OIF007_601 TCAAGCAGAAGCTTTGG 01F0077 10 TACAACGTGATAAACACGA 1157 627 F AAGAAGAAGG 82 736 R. CCAGAAGC 437 AB_?4LST- 11-: ABMLST-11- OIF007 62 TGAGATTGCTGAACATT 01F007 169 TTGTACATTTGAAACAATA 1151 91 F TAATGCTGATTGA 83 -203 R. TGCATGACATGTGAAT 426 ASDFRT 1 TTGCTTAAAGTTGGTTT ASD_FRT-86- TGAGATGTCGAAAAAAACG 1100 29 Fi TATTGGTTGGCG 84 1116 R. TTGGCAAP.ATAC 439 ASDFRT_43 TCAGTTTTAA~TGTCTCG ASD_FP.T_129 TCCATAT1TGTTGCATAAAA 1101 76 F TATGATCGAATCAAAAG 85 156 R CCTGTTGGC 438 ASPS EC_40 GCACAACCTGCGGCTGC ASPS_-EC_-521 291 5 4 22 F G 86 538 R. ACGGCACGAGGTAGTCGC 440 BONTA X520 66_450_473 TCTAGTAATAATAGGAC BONTAX520 6 TAACCATTTCGCGTAAGAT 485 F CCTCAGC 87 6 517 539 R TCAA 441 BONTAX520 T*Lt*CAGTAATAATAG BONTAX5206 66_450_ 473 GAU*L*U0*C'*U'AG 6_517_539P TAACCA*CI* C*Ca*UaGC 486 P F C 87 R. GTAAGA*C*C*UAA 441 BONTAx520 66_538_552 BONTA x5206 481 F TATGGCTCTACTCAA 88 6 647 660 R TGTTACTGCTGGAT 443 BONTAX520 BONTA -X5206 66_538_ 552 TA*C*GGC*Ca*W*CA I 6_647_660P_ TG*C'*CA*a*CG*U'*C 482 P F *UA 88 R. GGAT 443 BONTA_X520 66_591_620 TGAGTCACTTGAAGTTG BONTAX5206 TCATGTGCTAATGTTACTG 487 F ATACAAATCCTCT 89 6 644 671 R CTGGATCTG 442 BONTA X520 66_701_720 GALATAGCAATTAATCCA BONTA-X5206 483 F: AAT 90 6 759 775 R. TTACTTCTAACCCACTC 444 BONTAX520 BONTAX5206 66_701_720 GAA*CAG*O*AA*C'*C 6_759_775F_ TTA*Ua*Ca*Ca*U.*CAA* 484 P F oAA*C*Ua*AAAT 90 R. U.*US*U A*t*C*C 444 CAFlAF053 CAFlAF0539 947 33407 TCAGTTCCGTTATCGCC 47 33494_33 TGCGGGCTGGTTCAACAAG 774 33430 F- ATTGCAT 91 514 P. AG '445 CAFlAF053 CAFIAF0539 947_33435 TGGAACTATTGCAACTG 47_33499_33 776 33457 F CTAATG 92 517 P. TGATGCGGGCTGGTTCAAC 446 CAFlAF053 CAFlAF0539 947 33515 TCACTCTTACATATAAG 47 33595_33 TCCTGTTTTATAGCCGCCA 44' 775 33541 F GAAGGCGCTC 93 621 P. AGAGTAAG44 CAFIAF053 CAFlAF0539 947J3687 TCAGGATGGAAATAACC 47_33755_33 TCAAGGTTCTCACCGTTTA 48 777 33716 F ACCAATTCACTAC 94 782 R. CCTTAGGAG48 GTTATTTAGCACTCGTT CAPCBA_180 TGAATCTTGAAACACCATA 49 22 4 131 F TTTAATCAGCC 95 205 R. CGTAACG49 CAPC_BA 11 ACTCGTTTTTAATCAGC CAPCBA-185 TGAATCTTGAAACACCATA 23 4 133 F CCG 96 205PR CG 450 CAPC_BA_27 GATTATTGTTATCCTGT CAPC-BA-349 GTAACCCTTGTCTTTGAAT 24 4 303 F TATGCCATTTG.AG 97 376 P. TGTATTTGC 451 CAPCBA_27 4_303_THOD TGATTATTGTTATCCTG CAPCBA_349 TGTAACCCTTGTCTTTGAA 350 F TTATGCCATTTGAG 98 376 TiOD R. TTGTATTTGC 452 CAPCBA 27 TTATTGTTATCCTGTTA CAPC_-BA -358 GGTAACCCTTGTCTTTGAA 6 29-6 1 TGCC 99 377 R T 453 CAPCBA-28 GTTATCCTGTTATGCCA CAPCBA_361 26 1 301 F TTTG 100 378 R. TGGTAACCCTTGTCTTTG 454 CAPCBA_31 CCGTGGTATTGGAGTTA CAPC_-BA_-361 27 5 334 Fr TTG 101 378 R. TGGTAACCCTTGTCTTTG 454 1053 CJST CJ 10 TTGAGGGTATGCACCGT 102 CJST CJ 116 TCCCCTCATGTTTAAATGA 456; 29- 860 1110F CTTTTTGATTCTTT 6 1198 R TCAGGATAAAAAGC CJSTCJ_12 AGTTATAAACACGGCTT CJSTCa 134 TCGGTTTAAGCTCTACATG 1063 66 1299-F TCCTATGGCTTATCC 103 -9 1379 R ATCGTAAGGATA 457 CJSTCJ_12 TGGCTTATCCAAATTTA CJST CJ 140 TTTGCTCATGATCTGCATG 1050 90 1320 F GATCGTGGTTTTAC 104 6 143i3 P. AAGCATAAA 458 CJST_-CJ_-16 TTATCGTTTGTGGAGCT CJSTCJ 172 TGCAATGTGTGCTAtGTCA 1056 43 1670 F AGTGCTTATGC 105 4 172R C.AAA 459 CJSTCJ_16 TGCTCGAGTGATTGACT CJST _a177 TGAGCGTGTGGAAAAGGAC 1045 68 1700 F TTGCTAAATTTAGAGA 106 4 17*99 R TTGGATG 460 CJSTCa_16 TGATTTTGCTAAATTTA CJST-CJ-179 TATGTGTAGTTGAGCTTAC 1064 80 1713-F GAGAAATTGCGGATGAA 107 5 1822 R. TACATGAGC 461 CJST-CJ-18 TCCCAATTAATTCTGCC CJSTCJ 196 TGGTTCTTACTTGCTTTGC 1056 80 1910 F ATTTTTCCAGGTAT 108 1 2011 R ATAAACTTTCCA 462 TCCCGGACTTAATATCA CJ3STCJ-214 TCGATCCGCATCACCATCA 1054 60 2090 F ATGAAAATTGTGGA 109 18 2174 R. AAAGCAAA -463 CJSTCJ_21 TGCGGATCGTTTGGTGG CJSTCJ 224 TCC-ACACTGGATTGTAATT 1059 65 2194-F TTGTAGATGAAAA 110 7 2278 P. TACCTTGTTCTTT 464 CJSTCJ_21 TCGTTTGGTGGTGGTAG CJST-CJ-228 TCTCTTTCAAAGCACCATT 1046 71 2197 F ATGAAAAAGG ill 3 2313 R GCTCATTATAGT 465 CaSTCa_21 TAGATGAAAACGGCGAA CJSTCJ_228 TGAATTCTTTCAAAGCACC 1057 85 2i22F GTGGCTAATGG 112 3, 2 316 P. ATTGCTCATTATAGT 466 CJSTCa_26 TGCCTAGAAGATCTTAA CJSTCJ_275 TTGCTGCCATAGCAAAGCC 1049 36 26g68 F AAATTTCCGCCAACTT 113 13 2777 R. TACAGC 467 CJSTCa_26 TCCCCAGGACACCCTGA CJSTCa 276 TGTGCTTTTTTTGCTGCCA 1062 178 2703 F AATTTCAAC 114 0 2787 R TAGCAAAGC 468 CJST_Ca_28 TGGCATTTCTTATGAAG CaST_-CJ_296 TGCTTCAAAACGCATTTTT 1065 57 2887 F CTTGTTCTTTAGCA 115 5 2998 -R ACATTTTCGTTAAAG 469_ 1CJSTCJ_2B TGAAGCTTGTTCTTTAG CJSTCa 297 TCCTCCTTGTGCCTCAAAA 1055 69 2895-F CAGGACTTCA 116 .9 3 007 P9 CGCATTTTTA 470 101 CJST_Ca_32 TTTGATTTTACGCCGTC CJSTCJ 335 TCAAAGAACCCGCACCTAA 101 67 3293 F CTCCAGGTCG 117 6 3385 R. TTC-ATCATTTA 471 101 CJST_ca_36 TCCTGTTATCCCTGAAG CJST_-CJ_-443

TACAACTGGTTCAAAAACA

101 0 393 F TAGTTAATCAAGTTTGT 118 477 a TTAAGCTGTAATTGTC 473

TCCTGTTATCCCTGAAG

CJSTCa_36 TAGTTAATCAAGTTTGT CJST Ca 442 TCALACTGGTTCAAAAACAT 1048 0 394 F- T 119 476 fR TAAGTTGTAATTGTCC 472

TAGGCGAAGATATACAA

CJST_-CJ -5 AGAGTATTAGAAGCTAG CJST-Ca_104 TCCCTTATTTTTCTTTCTA 1052 39 F A 120 137 R CTACCTTCGGATAAT 455 CJSTI CJ_ 58 TCCAGGACP.AATGTATG CJST_-CJ 663 TTCATTTTCTGGTCCAAAG 1047 4 61j F- AAAAATGTCCAAGAAG 121 692 a TAAGCAGTATC 474 100 CJST_-Ca_59 TGAAAAATGTCCAAGAA CJSTCa_711

TCCCGAACAATGAGTTGTA

100 9 632 F- GCATAGCAAAAAAAGCA 122 743 R. TCAACTATTTTTAC 475 CTXAVBIC_1 TCTTATGCCAAGAGGAC CTXA-VBC-19 TGCCTAACAAATCCCGTCT 1096 17 142 F AGAGTGAGT 123 4 218'. GAGTTC 476 CTXA_VBC_3 TGTATTAGGGGCATACA CTXA_-VBC_44 TGTCATCAAGCACCCCAAA 1097 51 377 F GTCCTCATCC 124 1 466 a ATGAACT 477 CYA_BA_105 GAAAGAGTTCGGATTGG CYA-BA_1112 28 5 1072 F G 125 1130 R. TGTTGACCATGCTTCTTAG 479 CYA_BA_134 ACAACGAAGTACAATAC CYABA-1426 CTTCTACATTTTTAGCC-AT 27?7 9 1370 F AAGAC 126 1447 Rt CAC 480 CYA BA_135 CGAAGTACAATACAAGA CYABA-1448 TGTTAACGGCTTCAAGACC 3 137 97F CAAAAGAAGG 127 1467 R. C 482 CYA BA 135 CYABA 1448 3_137 9'T4 TCGAAGTACAATACAAG 147_IMOD_ TTGTTAACGGCTTCAAGAC 351 D F ACAAAAGAAGG 128 R cc 483 CYABA_135 ACAATACAAGACAAAAG CYA-BA-1447 31 9 1379 F AAGG 129 1461 R. CGGCTTCAAG.ACCCC 461 CYA-BA-914 CP.GGTTTAGTACCAGAA CYABA 999 ACCACTTTTAATAAGGTT 32 937 F CATGCAG t 130 10269 1- GTAGCTAAC 484 CYABA_916 GGTTTAGTACCAGAACA CYABA_1003 CCACTTTTAATAAGGTTTG 33 935 F- TGC 131 1025 P. TAGC 478 D14AKEC_42 CGGCGTACTTCAACGAC DNAKEC_503 CGCGGTCGGCTCGTTGATG 115 8 449 F AGCCA 132 522 P. A 485 GAEFRT_1 TTIATCAGCTAGACCTTT GALEFR.T_24 TCACCTACAGCTTTAAAGC 1102 68 199 F TAGGTAAAGCTAAGC 133 1 269 R. CAGCAAAATG 486 GALE_-FUT 3 TCCAAGGTACACTAAAC GALEFR.T_39 TCTTCTGTAAAGGGTGGTT 1104 08 339 F- TTACTTGAGCTAATG 134 0 422 P. TATTATTCATCCCA 487 GALE FRT_8 TCAAAAAGCCCTAGGTA GALEFRT_90 TAGCCTTGGCAACATCAGC 1103 34 865 F AAGAGATTCCATATC 135 1 925 R. AAAACT 488 GLTARKP_1 TCCGTTCTTACAAATAG GLTA RKP 11 TTGGCGACGGTATACCCAT 1092 023 1055 F CAATAGAACTTGAAGC 136 29 1156 R. AGCTTTATA 489 GLTA_RKP_1 043_-1072_2 TGGAGCTTGAAGCTATC GLTARKP 11 TGAACATTTGCGACGGTAT 1093 F GCTCTTAAAGATG. 137 38 1162 ACCCAT 490 GLTARI(P_1 043_1072_3 TGGAACTTGAAGCTCTC GLTARKP_11 TGTGAACATrTGCGACGGT 1094 F GCTCTTAAAGATG 138 38 1164 R ATACCCAT 492 GLTARI(P_1 TGGGACTTGAAGCTATC GLTA RKP_11 TGAACATTTGCGACGGTAT 1090 043 '10-72-F GCTCTTAAAGATG 139 38 1162 R ACCCAT 491 GLTARKP_4 TCTTCTCATCCTATGCC GLTARKP_49 TGGTGG7GTATCTTAGCAAT 1091 100 420 F TATTATGCTTGC 140 9 529 R CATTCTAATAGC 493 GLTA RKP_4 TCTTCTCATCCTATGGC GLTARRP_50 TGCGATGGTAGGTATCTTA 1095 00 428 F- TATTATGCTTGC 140 5 534 RH GCAATCATTCT49 GROLEC 21 GGTGAAAGAAGTTGCCT GROLEC_328 TTCAGGTCCATCGGGTTCA 224 9 2 42 E; CTAAAGC 141 350 R TGCC 496 GROL -EC_-49 ATGGACAAGGTTGGCAA GROLEC_577 TAGCCGCGGTCGAATTGCA 280 6 518 F GGAAGG 142 596 R T 498 GROLEC-51 AAGGAAGGCGTGATCAC GROLEC_571 CCGCGGTCGAATTGCATGC 281 11 536 F CGTTGAAGA 143 593 R -CTTC 497 GROL EC_94 TGGAAGATCTGGGTCAG GROLEC_103 CAATCTGCTGACGGATCTG 220 1 959FE; GC 144 19 1060 R AGC 495 GYRAAF1005 GYRAAF100 TCTGCCCGTGTCGTTGG 57_119 142 TCGAACCGAAGTTACCCTG 924 557 4 23 F TGA 145 R ACCAT 499 GYRAAF100 GYRAAF1005 557_*70 94 TCCATTGTTCGTATGGC 57_176 201 TGCCAGCTTAGTCATACGG 925 F TCAAGACT 146 R ACTTC 500 GYRE B 0O8 GYRBAB0087 700_19 40 TCAGGTGGCTTACACGG 00_111 140_ TATTGCGGATCACCATGAT 926 F CGTAG 147 R GATATTCTTGC 501 GYRBABOO8 GYRBAB0087 700_265_29 TCTTTCTTGAATGCTGG 00_369 395- TCGTTGAGATGGTTTTTAC 927 2 F TGTACGTATCG 148 R CTTCGTTG 502 GYRB ABO08 GYREAB0087 100_568_39 TCAACGAAGGTAAAAAC 00_4*6494_ TTTGTGAAACAGCGAACAT 928 4 F CATCTCAACG 149 R TTTCTTGGTA 503 GYRB_ABOO8 GYRBAB0087 700_477_50 TGTTCGCTGTTTCACAA 00_611_632_ TCACGCGCATCATCACCAG 929 4 F- ACAACATTCCA 150 R T-CA 504 GYRBABOOB GYREAB0087 700_760_78 TACTTACTTGAGAATCC 00_66g2 888 TCCTGCAATATCTAATGCA 949 7 F ACAAGCTGCAA 151 2 R CTCTTACG 505 GYRBABOO8 GYRB AB0087 700_760_78 TACTTACTTGAGAATCC 00_6692 888_ ACCTGCAATATCTAATGCA 930 7 FACAAGCTGCAA 151 R. 7CTCTTACG 506 TGGCGAACCTGGTGAAC HFLEEC_114 CTTTCGCTTTCTCGAACTC 222 62 1102 F' GAAGC 152 4 1168 R AACCAT 507 HOPECJ_11 TAGTTGCTCAAACAGCT HUPBCJ_157 TCCCTAATAGTAGAAATAA 1128 3 134 F GGGCT 153 -188-R CTGCATCAGTAGC 509 HOPBCJ_76 TCCCGGAGCTTTTATGA HOPE_-CJ 114 TAGCCCAGCTGTTTGAGCA 1130 102 F CTAAAGCAGAT 154 135 R ACT 508 HUPB CJ 76 TCCCGGAGCTTTTATGA HUPBCJ_157 TCCCTAATAGTAGAAATAA 1129 102 F CTAAAGCAGAT 154 188 R CTGCATCAGTAGC 510 ICD-CXB-17 TCGCCGTGGAAAAATCC ICD-CXB-224 TAGCCTTTTCTCCGGCGTA 1079 6 198 F TACGCT 155 247 R GATCT :512 ICD CXB 92 TTCCTGACCGACCCATT ICD-CXB-172 TAGGATTTTTCCACGGCGG 1078 120 F ATTCCCTTTATC 156 194 R CATC 510 ICD CXB 93 TCCTGACCGACCCATTA ICD CXB 172 TAGGATTTTTCCACGGCGG 1077 120o F TTCCCTTTATC 157 194i R CATC 511 INFBEC_11 GTCGTGAAAACGAGCTG INFBEC_117 221 03 1124 F GAAGA 158 4 11791 Hi CATGATGGTCACAACCGG 513 INFB-EC_13 TGCGTTTACCGCAATGC INFB _EC_141 964 47 1367 F GTGC 159 4 1432 Ri TCGGCATCACGCCGTCGTC 514 INFB EC_13 TGCTCGTGGTGCACAAG INFBEC_143 TGCTGCTTTCGCATGGTTA 34 65 1393-F TAACGGATATTA 160 9 1467 -R ATTGCTTCAA 515 INFBEC_13 INFB EC_143 65_1393_TM TTGCTCGTGGTGC-ACAA 9_14697_TMOD TTGCTGCTTTCGCATGGT 352 OD F GTAACGGATATTA 161 R AATTGCTTCAA 516 INFBEC_19 CGTCAGGGTAAATTCCG INFE-EC_203 AACTTCGCCTTCGGTCATG 223 69 1994-F TGPIAGTTAA 162 8 2058 R TT 517 INVu22457 1558_1581 TGGTAACAGAGCCTTAT INV 0 22457_ TTGCGTTGCAGATTATCTT 781 F AGGCGCA 163 1619 1643 R TACCAA 518 INV_022457 TGGCTCCTTGGTATGAC XINV 022457- TGTTAAGTGTGTTGCGGCT 778 515 539 F TCTGCTTC 164 571 598 R GTCTTTATT 51 INVU22457 TGCTGAGGCCTGGACCG INV 022457 TCACGCGACGAGTGCC 779 699 724 F ATTATTTAC 165 753-776 R CATTG 520 INV_022457 TTATTTACCTGCACTCC INV U22457_ TGACCCAAAGCTGAAAGCT 780 834 858 F CACAACTG 166 942 966 R TTACTG 521 -31- IPAHSGF 1 TCCTTGACCGCCTTTCC IPAHSGF_17 TTTTCCAGCCATGCAGCGA 1106 13 1354 EF' GATAC 167 2 191 Rt _c 522 XPAHSGF 2 TGAGGACCGTGTCGCGC IPAHSGF_30 TCCTTCtGATGCCTGATG 1105 58 277 1F TCA 168 1 327 R ACCAGGAG 523 IPAHSGF 4 TCAGACCATGCTCGCAG IPAHISGF_52 1107 62 486 AGAAACTT 169 2 540 R TGTCACTCCCGACACGCCA 524 IS1111A NC IS11llANCO 002971_686 TCAGTATGTATCCACCG 02971 6928 TAAACGTCCGATACCAATG 1080 6 6891 F -TAGCCAGTC 170 6954 Rt GTTCGCTC 525 IS1111ANC IS1111ANCO 002971_745 TGGGTGACATTCATCAA 02971 75929 TCAACAACACCTCCTTATT 1081 6 7483 F TTTCATCGTTC 171 7554 ii CCCACTC 526 LEF BA_103 LEFBA_1119 3 105s2-F TCAAGAAGAAAAAGAGC 172 1135 Rt GAATATCAATTTGTAGC 527 LEF BA_103 CAAGAAGAAAAAGAGCT LEFBA_1119 AGATAAAGAATCACGAATA 36 6 106 1 TCAAAG A 17 119 Rt TCAATTTGTAGC 528 LEFBA_756 AGCTTTTGCATATTATA LEFa -843 TCTTCCAAGGATAGATTTA 37 781l F- TCGAGCCAC 174 872 fR TTTCTTGTTCG 530 LEFBA_756 -781_TMOD_ TAGCT2TTTGCATATTAT LEF_BA -843 TTCTTCCAAGGATAGATTT 353 F ATCGAGCCAC 175 872 T14OD Rt ATTTCTTGTTCG 531 LEFBA_758 CTTTTGCATATTATATC LEFBA 843- AGGATAGATTTATTTCTTG 38 778 F GAGC 176 865 ft TTCG 529 LEFBA_795 TTTACAGCTTTATGCAC LEF_-BA 883 39 18 13 CG 177 900 Rt TCTTGACAGCATCCGTTG 532 LEF_BA_663 LEFBA 939- CAGATAAAGAATCGCTCCA 899 F CAACGGATGCTGGCAAG 178 958 Rt G 533 LLNC00314 LL NCO03143 3 2i366996 TGTAGCCGCTAAGCACT 23567073_23 TCTCATCCCGATATTACCG 782 2367019 F ACCATCC 179 67097 R CCATGA 534 LL-NC00314 LLNC003143 3 2367172 TGGACGGCATCACGATT 2367249_23 TGGCAACAGCTCAACACCT 783 23 67194 F- CTCTAC 180 67271 Rft TTGG 535 MECA Y1405 MECA Y14051 1 3645_367 TGAAGTAGAAATGACTG 3690 3719 TGATCCTGAATGTTTATAT 878 O-F AACGTCCGA 181 ft CTTTAACGCCT 536 1_3754_380 TAAAACAAACTACGGTA 3828_3854_ TCCCAATCTAACTTCCACA 8 77 2-F ACATTGATCGCA 182 ft TACCATCT 537 MECAY1405 MECAY14051 1_4507_453 TCAGGTACTGCTATCCA 455534581 TGGATAGACGTCATATGAA 879 0 F CCCTCAA '183 Rt GGTGTGCT 538 rMECA _Y1405 MECA _Y14051 14510_453 TGTACTGCTATCCACCC 45864610 TATTCTTCGTTACTCATGC 880 O F TCAA 184 Rt CATACA 539 MECAY1405 MECAY14051 1_4520_453 4590_4600P 882 OP F TUOUAUUUC'UAA 165 Rf CAUC'0AC'GUUA 540 MECA-Yl405 I4ECA Y14051 1_4520_453 4600_4610P 88 3 OP F TUUAUU'U'C*AA 185 Rt C.ACCUCN-COGC'T 541 MTEA Y14 05 MECA_-Y14051 1_469_469 TCACCAGGTTCAACTCA 4765 4793 TAACCACCCCAAGATTTAT 881 8-F AAAAATATTAACA 186 Rt CTTTTTGCCA 542 MECIA Y140 MECIA-Y1405 51_3315_33 TTACACATATCGTGAGC 1_3367_3393 TGTGATATGGAGGTGTA .GA 876 41F F AATGAACTGA 187 ft AGGTGTTA 543 OMPAAY485 OMPA_AY4852 227_272_30 TTACTCCATTATTGCTT 27_364388 GAGCTGCGCCAACGAATAA 914 1 F GGTTACACTTTCC 188 R ATCGTC 544 OMPA AY4 85 OMPAAY4852 227_311_33 TACACAACAATGGCGGT 27_424 453 TACGTCGCCTTTAACTTGG 916 5 F' AAAGATGG 109 Rft TTATATTCAGC 545 OMPA AY485 OMPAAY48521 227_379_40 TGCGCAGCTCTTGGTAT 27_492 519 TGCCGTAACATAGAAGTTA 915 1 F CGAGTT 190 Rft CCGTTGATT 546 OMPA AY485 OMPA_-AY4852 227_4Z15_44 TGCCTCGAAGCTGAATA 27514 546 TCGGGCGTAGTTTTTAGTA 917 1 F- TAACCAAGTT 191 ft ATTAAATCAGAAGT 547 OMPA,_AY485 OMPAAY4852 227_494_52 TCAACGGTAACTTCTAT 27_569 596 TCGTCGTATTTATAGTGAC 918 0 F GTTACTTCTG 192 Rt CAGCACCTA 548 OMPAAY485 OMPA AY4852 227_551_57 TCAAGCCGTACGTATTA 27_658 680 TTTAAGCGCCAGAAAGCAC 919 7 F- TTAGGTGCTG 193 R CAAC 550 00

IN

00 -32- -OMPAAY485 OMPAAY4 852 227_55_58 TCCGTACGTATTATTAG 27_635 662 TCAACACCAGCGTTACCTA 920 1 1F GTGCTGGTCA 194 R. AAGTACCTT 549 OMPA-AY4 85 ONIPA-AY4852 227556_58 TCGTACGTATTATTAGG 27_659 683 TCGTTTAAGCGCCAGAAAG 921 3 F TGCTGGTCACT 195 R -CACCAA 551 OMPAAY485 OMPAAY4852 227_657_67 TGTTGGTGCTTTCTGGC 27_739 765 TAAGCCAGCAAGAGCTGTA 922 -9-F GCTTAA 196 R TAGTTCCA 552 OMPA-AY4 85 OMPA-AY48 52 227_660_68 TGGTGCTTTCTGGCGCT 27_786 807 TACAGGAGCAGCAGGCTTC 923 3 F TAAACGA 197 p. AAG 553 OMPB RKP_1 TCTACTGATTTTGGTAA OMPBRI(P_12 TAGCAGCAAAAGTTATCAC 1088 192 1221 F TCTTGCAGCACAG 198 88 1315 R. ACCTGCAGT 554 OMPB RKP_-3 7GCAAGTGGTACTTCAA OM4PBRKP_35 TGGTTGTAGTTCCTGTAGT 1089 417 3i440 F CATGGGG 199 20 3550 R. TGTTGCATTAAC 555 OMPE RRP_8 'ITACAGGAAGTTTAGGT OMPBRKP_97 TCCTGCAGCTCTACCTGCT 1087 160 890 F GGTAATCTAAAAGG 200 2 996 R. CCATTA 556 PAGBA_-122 CAGAATCAAGTTCCCAG PAGBA 190- CCTGTAGTAGAAGAGGThA 41 142iF GGG 201 209 R C 558 PAGBA_123 AGAATCAAGTTCCCAGG PAGBA 187 CCCTGTAGTAGAAGAGGTA 42 145 F7 GGTTAC 202 210 p. ACCAC 557 PAGBA_269 AATCTGCTATTTGGTCA PAGBA_326_ 43 287 F GG203 344 R. TGATTATCAGCGGAAGTAG 559 PAG BA_655 GAAGGATATACGGTTGA PAGBA 755- 44 675 F TGTC 204 772 R. CCGTGCTCCATTTTTCAG 560 PAGBA_753 TCCTGAAAAATGGAGCA PAGBA 849 TCGGATAAGCTGCCACAAG 77i2F CGG 205 868 R G 561 PAGBA_763 TGGAGCACGGCTTCTGA PAGBA 849- TCGGATAAGCTGCCACAAG 46 781 F TC 206 868 R. G 562 1?ARCX9581 9_123S 141 GGCTC.AGCCATTTAGTT PARC X95619 TCGCTCAGCAATAATTCAC 912 F ACCGCTAT 207 232 260 R. TATAAGCCGA 566 PARCX9581 TCAGCGCGTACAGTGGG PARCX95819 TTCCCCTGACCTTCGATTA 913 9 43 63 F TGAT 208 143 170 p. AAGGATAGC 563 PARC X9581 TGGTGACTCGGCATGTT PARC X95819 GGTATAACGCATCGCAGCA 911 9 87 110 F ATGAAGC 209 192 219 R. AAAGATTTA 564 PARCX9581 TGGTGACTCGGCATGTT PARCX95819 TTCGGTATAACGCATCGCA 910 9 87 110 F ATGAAGC 209 201 222 R. GCA 565 PLAAF0539 PLAAF05394 45_7 186_72 TTATACCGGAAACTTCC 5_7257_7280 TAATGCGATACTGGCCTGC 773 11-F CGAAAGGAG 210 ii AAGTC 567 PLAAF0539 PLAAF05394 45_75377_74 TGACATCCGGCTCACGT 5_7434_7462 TGTAAATTCCGCAAAGACT 770 02 F TATTATGGT 211 -R TTGGCATTAG 568 PLA AF0539 PLA AF05394 45_7 382 -74 TCCGGCTCACGTTATTA 5_7482_7502 TGGTCTGAGTACCTCCTTT 771 04 F TGGTAC 212 R GC 569 PLAAF0539 PLA AF05394 45_7481_75 TGCAAAGGAGGTACTCA 5_7539_7562 TATTGGAAATACCGGC-AGC 772 03 F GACCAT 213 ii ATCTC 570 RECAAF251 RECAAF2514 469_1 69_-19 TGACATGCTTGTCCGTT 69_277_300_ TGGCTCATAAGACGCGCTT 909 0 F CAGGC 214 R GTAGA 572 RECAAF251 RECAAF2514 46943b 68- TGG;TACATGTGCCTTCA 69140O 163 TTCAAGTGCTTGCTCACCA 908 F TTGATGCTG 215 P. T'rGTC 571 EXASPBDP TGGCACGGCCATCTCCG RNASEP BDP TCGTTTCACCCTGTCATGC 1072 R574 592 F TG 216 616 635 p.R CG 573 RNASEPBR14 TGCGGGTAGGGAGCTTG RNASEP 5KM- TCCGATAAGCCGGATTCTG 1070 580 599 F AGC 217 665 686 R TGC 574 BZ4ASEP_BEN TCCTAGAGGAATGGCTG RNASEP 8KM- TGCCGATAAGCCGGATTCT 1071 616 637 F CCACG 218 665 687 P. GTGC 575 RAEP -BR?4 TACCCCAGGGAAAGTGC P14ASEP_BRM_ TCTCTTACCCCACCCTTTC 1112 R325 347 F CACAGA 219 402 428 p. ACCCTTAC 576 RNASEP 5514 TAAACCCCATCGGGAGC RNASEP 8514 TGCCTCGTGCAACCCACCC 1172 461 4868 F AAGACCGAATA 220 542 561 2 p. G 577 RNASEPERM TAAACCCCATCGGGAGC RNASEP BRM- TGCCTCGCGCAACCTACCC 1111 461 488 F AAGACCGAATA 220 542 561 p.R G 578 RNASEPBS_ GAGGAAAGTCCATGCTC RNASEPES_3 GTAAGCCATGT'ITTGTTCC 258 43 61 Fi GC 221 63 384- p. ATC 579 RNASEPES_ GAGGAAAGTCCATGCTC RNASEPES_3 GTAAGCCATGTTTTGTTCC 259 43 61 F GC 221 63 384PR ATC 578 RNASEPES_ GAGGAAAGTCCATGCTC RNASEPEC_3 258 43 61 F GC 221 45_362 p. ATAAGCCGGGTTCTGTCG 581 00 00 -33 RNASEP_-BS_- GAGGAAAGTCCATGCTC P.NASEPSA_3 ATAAGCCATGTTCTGTTCC 2 58 43 61 F GC 221 58 379 R ATC 584 RNASEPCLB TAAGGA'IAGTGCAACAG RNASEPCLE TTTACCTCGCCTTTCCACC 1016 459 48 7 F AGATATACCGCC 222 498 522 R CTTACC 579 RNASEPCLB TAAGGATAGTGCAACAG R1NASPCLB- TGCTCTTACCTCACCGTTC 1075 459 467 F AGATATACCGCC 222 498 526 R CACCCTTACC 580 RNASEPEC- RNASEPBS_3 GTAAGCCATGTTTTGTTCC 258 61 77 F GAGGAAAGTCCGGGCTC 223 63 384 PR ATC 578 P.NASEPEC -RNASEPSC_3 256 61 7-7 F; GAGGAAAGTCCGGGCTC 223' 45 362 R. ATAAGCCGGGTTCTGTCG 583.

RNASEP SC- RNASEPEC_3 260 61 77 F GAGGAAAGTCCGGGCTC 223 45 362 R. ATAAGCCGGGTTCTGTCG 581 PNASSPSC PNASEPSA_3 ATAAGCCATGCGTC 258 61 77 F GAGGAAAGTCCGGGCTC 223 58 379 R ATC 584 RNASEP RP.( TCTAAATGGTCGTGCAG RI4ASEP 9.9.9 TCTATAGAGTCCGGACTTT 1085 264 287 F TTGCGTG 224 295 321 R. CCTCGTGA 582 P.NASEPRKP TGGTAAGAGCGCACCGG RIIASEPPKP TCAAGCGATCTACCCGCAT 1082 419 448 F TAAGTTGGTAAC-A 225 542 565 R. TACAA 583 RNASEPRKP TAALGAGCGCACCGGTAA P.NASEPRP- TCAAGCGATCTACCCGCAT 1083 422 443 F GTTGG 226 542 565 R. TACAA 583 108 RASEPREIIP TGCATACCGGTAAGTTG P.NASP_RP_

TCAAGCGATCTACCCGCAT

106 426 448 F GCAACA 227 542 565 P. TACAA 583 P.NASEPRP9 TCCACCAAGAGCAAGAT R14ASEP RP. TCAAGCGATCTACCCGCAT 1084 466 491 F CAAATAGGC 228 542 565 R. TACAA 583 RNIASEP_SA_ GAGGAAAGTCCATGCTC RNASEP BS 3 GTAAGCCATGTTTTGTTCC 258 31 49 F AC 229 63 384 R ATC 578 RNASEP_SA_ GAGGAAAGTCCATGCTC PNASEPSC_3 258 31 49 F AC 229 45 362 P. ATAAGCCGGGTTCTGTCG 581 RNASEP_SA_ GAGGAAAGTCCATGCTC RNASE3PSA_3 ATAAGCCATGTTCTGTTCC 258 31 49 F AC 229 58 379 ATC 584 RNASEPSA_ GAGGAAAGTCCATGCTC P.NASEP SA 3 ATAAGCCATGTTCTGTTCC 262 31 49 F AC 229 58 379 R ATC 584 loe RNASEP_VBC TCCGCGGAGTTGACTGG RNASEP VEC TGACTTTCCTCCCCCTTAT 108 331 349 F GT 230 388 414 P.R CAGTCTCC 585 RPLBEC -65 GACCTACAGTAAGAGGT PPLB_EC_739 TCCAAGTGCTGGTTTACCC 66 0 679; F TCTGTP.ATGAACC 231 762 P. CATGG 591 P.PLBS C_65 0_6'79_TMOD TGACCTACAGTAAGAGG R.PLBEC-739 TTCCAAGTGCTGGTTTACC 356 F TTCTGTAATGAACC 232 762 TMOD P. CCATGG 592 RPLB SC_66 TGTAATGAACCCTAATG RPLB_-EC_-735 CCAAGTGCTGGTTTACCCC '73 9 69W F ACCATCCACACGG 233 761 P. ATGGAGTA 586 RPLBEC -67 TAATGAACCCTAATGAC R.PLBEC_737 TCCAAGTGCTGGTTTACCC 74 1 -70 F' CATCCACACGGTG 234 762 R. CATGGAG 590 R.PLBEC_68 CATCCACACGGTGGTGG R.PLBEC-736 GTGCTGGTTTACCCCATGG 67 8 710 F TGAAGG 235 757 P. AGT 587 R.PLBEC_68 CATCC-ACAcGGTG.GTGG RPLBEC_743 TGTTTTGTATCCAAGTGCT 8 710 F TGAAGG 235 771 R. GGTTTACCCC 593 RPLB EC_68 8_710_TiMOD TCATCCAC-ACGGTGGTG RPLBEC_736 TGTGCTGGTTTACCCCATG 357 FGTGAAGG 236 757-THOD P. GAGT 588 R.PLBEC_69 TCCACACGGTGGTGGTG RPLBS C_-737 TGTGCTGGTTTACCCCATG 449 0 710 F AAGG 237 '7589R. GAG 589 P9B-EC_13 GACCACCTCGGCAACCG R.POBEC-143 113 36 1353 F T 238 1455 R. TTCGCTCTCGGCCTGGCC 594 TCAGCTGTCGCAGTTCA PP0 _C_163 TCGTCGCGGACTTCGAAGC 963 27 1549WF TGGACC 239 0 1649 R. C 595 P.POBEC_18 TATCGCTCAGGCGAACT RPOBEC_190 GCTGGATTCGCCTTTGCTA 72 45 1866 F CCAAC 240 9 1929 R. CG 596 RPOBEC_18 RLPOB-EC_190 45_1866_TM TTATCGCTCAGGCGAAC 9_1929_TM40D TGCTGGATTCGCCTTTGCT 359 OD F TCCAAC 241 P. ACG 597 TCGTTCCTGGAACACGA RP90BEC-204 TTGACGTTGCATGTTCGAG 962 05 2027 F TGACGC 242 1 2064 PR CCCAT 598 P.POBEC_-37 TCAACAACCTCTTGGAG P.POBEC-383 TTTCTTGAAGAGTATGAGC 69 62 3790 F GTAAAGCTCAGT 243 6 3865 R TGCTCCGTAAG 600 RPOBEC_37 CTTGGAGGTAAGTCTCA POB09EC_382 CGTATAAGCTGCACCATAA ill 75 3803 F TTTTGGTGG.GCA 244 9 38598 P. GCTTGTAATGC 599 P.POB EC_37 TGGGCAGCGTTTCGGCG RPOBEC_386 TGTCCGACTTGACGGTAG 940 98E 321 F AAATGGA 245 2 3889 2-P. CATTTCCTG 604 RPOB S C_-37 TGGGCAGCGTTTCGGCG 9.90BEC_386 TGTCCGACTTGACGGTCAG 939 98 321 F AAATGGA 245 2 3889 P. CATTTCCTG 605 RPOB-EC_37 GGGCAGCGTTTCGGCGA RPOB_-EC 386 GTCCGACTTGACGGTCAAC 289 99 3821 F AAGGA 246 2 3888 ii ATTTCCTG 602 362 POBS C_-37 TGGGCAGCGTTTCGGCG j PPOBEC_386 TGTCCGACTTGACGGTCAA 32 99 821 TM AAATGGA 245 2 3888 TMOD CATTTCCTG60 00 F -R RPOBEC_38 CAGCGTTTCGGCGAAAT RPOBEC_386 CGACTTGACGGTTAACAT'r 286 02 3821-F GGA 247 2 3 885 ft TCCTG 601 18_1045 2 CAAAACTTATTAGGTAA RPOCEC_109 TCAAGCGCCATCTCTTTCG 48 F GCGTGTTGACT 248 5 1124 2i R GTAATCCACAT 610 CAAAACTTATTAGGTAA RPOC-EC_109 flCAAGCGCCATTTCTTTTG 47 118 1045SF GCGTGTTGACT 248 15 1124 R GTAAACCACAT 611 CGTGTTGACTATTCGGG ftPOCEC_109 ATTCAAGAGCCATTTCTTT 68 36 10660-F GCGTTCAG 249 7 112i6 ft TGGTAAACCAC 612 RPOCEC_11 TAAGAAGCCCGAAACCA RPOCEC_213 GGCGCTTGTACTTACCGCA 49 4 140 E; TCAACTACCG 250 232 ft C 617 RPOCEC 12 ACCCAGTGCTGCTGAAC RPOCEC_129 GTTCAAATGCCTGGATACC 227 56 1277-F CGTGC 251 5 1315 R CA 613 RPOCEC_13 CGCCGACTTCGACGGTG RPOCEC_143 292 174 1393 F ACC 252 7 1455 ft GAGCATCAGCGTGCGTGCT 614 RPOC_EC_13 RPOCEC-143 74_1393_TM TCGCCGACTTCGACGGT 7_1455TMOD TGAGCATCAGCGTGCGTGC 364 CDF GACC 253 Rt T 615 TGGCCCGAAAGAAGCTG RPOCEC_162 ACGCGGGCATGCAGAGATG 229 84 1604-F AGCG 254 3 1643 Rt cc 616 RPOCEC_21 TCAGGAGTCGTTCAACT ftPOCEC_222 TTACGCCATCAGGCCACGC 978 45 2175-F CGATCTACATGATG 255 8 224i7 ft -A 622 RPOC EC_21 CAGGAGTCGTTCAACTC RPOCEC_222 290 46 2174 F GATCTACATGAT 256 7 224i5 R ACGCCATCAGGCCACGCAT 620 RPOCEC_21 FLPOC EC_222 46_2174_TM TCAGGAGTCGTTCAACT 7_2245TMOD TACGCCATC-AGGCCACGCA 363 00 F CGATCTACATGAT 257 Rt T 621 RPOCEC_21 78_21962 TGATTCCGGTGCCCGTG P.POC-EC_222 TTGGCCATCAGACCACGCA 51 F GT 258 5 2246 2R TAC 618 RPOCEC 21 TGATTCTGGTGCCCGTG RPOCEC_222 TTGGCCATCAGGCCACGCA 78 2196?F GT 259 5 2246 ft TAC 619 RPOCEC_22 18_22i41 2 CTTGCTGGTATGCGTGG RPOCEC_231 CGCACCATGCGTAGAG.ATG 53 F TCTGATG 260 3 2337 2 Rt AAGTAC 623 RPOC_EC_22 CTGGCAGGTATGCGTGG RPOCEC-231 CGCACCGTGGGTTGAGATG 52 18 2241 F TCTGATG 261 3 2337 R AAGTAC 624 PCEC_22 RPOCEC_231 18_22 41_TM TCTGGCAGGTATGCGTG 3_2337_TMOD TCGCACCGTGGGTTGAGAT 354 OD F GTCTGATG 262 Rt GAAGTAC 625 RPOCEC_22 TGGTATGCGTGGTCTGA RPOC-EC-232 TGCTAGACCTTTACGTGCA 958 23 2243 F TGGC 263 9 2352 Rt CCGTG_ 626 RPOCEC_23 TGCTCGTAAGGGTCTGG RPOCEC_238 TACTAGACGACGGGTCAGG 960 34 2357 F CGGATAC- 264 0 2403 ft TAACC 627 PC EC_80 CGTCGTGTAATTAACCG RPOCEC_865 ACGTTTTTCGTTTTGAACG 8 833S 2-F TAACAACCG 265 891 ft ATAATGCT 629 CGTCGGGTGATTAACCG RPOCEC_865 GTTTTTCGTTGCGTACGAT 54 8 833 F TAACAACCG 266 889 R GATGTC 628 P00EC_91 TATTGGACAACGGTCGT PCC C100 TTACCGAGCAGGTTCTGAC 961 7 9389 F- CGCGG 267 9 10354 R GGAAACG 607 PCC-C91 TCTGGATAACGGTCGTC ftPOC EtC_100 TCCAGCAGGTTCTGACGGA 959 8 938 F GCGG 268 9 1031 R AACG 606 RPCC-C99 CAAAGGTAAGCAAGGAC P00EC-1O cGCGCAGTGC 57 3 1019 2 F GTTTCCGTCA 269 6 1059 2 ft ACACG 608 RPOC EtC_99 CAAAGGTAAGCAAGGTC PCC C103 CGAACGGCCTGAGTAGTCA 56 3 1019 F GTTTCCGTCA 270 6 1059 ft ACACG 609 SP101_SPET AACCTTAATTGGAAAGA SPl0lSPET1 CCTACCCAACGTTCACCAA 11 1 29 F AACCCAAGAAGT 271 1 92 116 ft GGGCAG 676 SP101_SPET SPl01_SPET1 11_1_29_TM TAACCTTAATTGGAAAG 1_92_116 TM TCCTACCCAACGTTCACCA 446 00 F AAACCCAAGAAGT 272 CD ft AGGGCAG 677 SPlOlSPET SPlolSPETI 11_1154_11 CAATACCGCAACAGCGG 1_1251_1277 GACCCCAACCTGGCCTTTT 797F TGGCTTGGG 273 ft GTCGTTGA 630 SPIOISPET SP101 SPETi 11_1154-11 TcAATACCGCAACAGCG 1_1251_1277 TGACCCCAACCTGGCCTTI' 424 79 TMOD F GTGGCTTGGG 274 TMOD ft TGTCGTTGA 631 SP101_SPET 11_118_147 GCTGGTGAAAATAACCC SP101lSPET1 TGTGGCCGATTTCACCACC 76 F AGATGTCGTCTTC 275 1 213 238 ft TGCTCCT 644 SPI01 SPET SPl01_SPET1 :11_118_147 TGCTGGTGAAAATAACC 1_213_238_T TTGTGGCCGATTTCACCAC 425 TMOD F CAGATGTCGTCTTC 276 M40D ft CTGCTCCT 645 86 SPl01 SPET CGCAAAAAAATCCAGCT 277 SP101 SPETI AAACTATTTTTTTAGCTAT 632 11 1314_13 ATTAGC 1_1403_1431 ACTCGAACAC 6FR SP101_SPET SPl0lSPET1 11_1314_13 TCGCAAAAAAATCCAGC 1_1403_1431 TAAACTATTTTTTTAGCTA 426 367TMOC F TATTAGC 278 TMOD -R TACTCGAACAC 633 SP101_SPET SP101_SPET1 11_1408_14 CGAGTATAGCTAAAAAA 1_-1486_-1515 GGATAATTGGTCGTAACAA 87 37 F ATAGTTTATGACA 279 R. GGGATAGTGAG 634 SP161_SPET SPI01_SPETI 11_1408_14 TCGAGTATAGCTAAAAA 1_1486_ 1515 TGGATAATTGGTCGTAAC-A 427 3-7 TMOD F AATAGTTTATG.ACA 280 TiMOD, P. AGGGATAGTGAG 635 SP101_SPET SP101_SPETI 11_16817 CCTATATTAATCGTTTA 1_1783_1808 ATATGATTATCATTGAACT 88 16 F CAGAAACTGGCT 281 R. GCGGCCG 636 SP101_SPET SP101_SPETI 11_1688 17 TCCTATATTAATCGTTT 1_1783 1808 TATATGATTATCATTGAAC 428 16 TMODF ACAGAAACTGGCT 282 TMOD R. TGCGGCCG 637 SP101 SPET SP101_SPETi 11_1711_17 CTGGCTAAAACTTTGGC 1_1881835 GCGTGACGACCTTCTTGAA 89 33-F AACGGT 283 R. TTGTAATCA 638 SP101 SPET SPI01_SPET1 11_171_17 TCTGGCTAAAACTTTGG 1_1808_ 1835 TGCGTGACGACCTTcTTG.A 429 33-TMOC F CAACGGT 284 TEOD ii ATTGTAATCA 639 SP101_SPET SP101._SPETI 11_18067_18 ATGATTACAATTCAAGA 11901_1927 TTGGACCTGTAATCAGCTG 35-F AGGTCGTCACGC 285 R. AATACTGG 640 SF101_SE] SP101_SPETi 11_1807_18 TATGATTACAATTCAAG 1_1901_1927 TTTGGACCTGTAATCAGCT 430 35 TMOD7 F AAGGTCGTCACGC 286 TMOD -R GAATACTGG 641 SF101_SPET SP101 SPET1 11_1967_19 TAACGGTTATCATGGCC 1 2062 2083 ATTGCCCAGAAATCAAATC 91 91 F CAGATGGG 2B7 P. ATC 642 SF101 SPET SF101 SPETI 11_1967_19 TTAACGGTTATCATGGC 1_2062_2083 TATTGCCCAGAAATCAAAT 431 91 TMOD F CCAGATGGG 288 TMOD R. CATC 643 SP101-SPET 11_216_243 AGCAGGTGGTGAAATCG SP101_SPETI TGCCACTTTGACAACTCCT 77 F GCCACATGATT 289 1 308 333 R. GTTGCTG 654 SF101 SPET SP101_-SPETi 11_216_243 TAGCAGGTGGTGAAATC 1_308_333_T TTGCCACTTTGACAACTCC 432 7M0D Fi GGCCACATGATT 290 MOD R. TGTTGCTG 655 SF101 SPET SP101_SPETi 11 2260_22 CAGAGACCGTTTTATCC 1_2375_2397 TCTGGG;TGACCTG.GTGTTT 92 83-F tATCAGC 291 R. TAGA 646 SF101 SPET SP101_SPETi 112260_22 TCAGAGACCGTTTTATC 1_2375_2397 TTCTGG.GTGACCTG.GTGTT 433 83 TMOD F CTATCAGC 292 TMOD R. TTAGA 647 SF101_SPET SF101_SPETi 11_2375_23 TCTAAAACACCAGGTCA 1 2470_2497 AGCTGCTAGATGAGCTTCT 93 99-F CCCAGAAG 293 P.GCCATGGCC 648 SF101 SPET SP101_SPETi 11_2375_23 TTCTAAAACACCAGGTC 1_2470_2497 TAGCTGCTAGATGAGCTTC 434 99 TI400 F ACCCAGAAG 294 TMOD R. TGCCATGGCC 649 SP101 SPET SP101_SPETi 11_-2468_ -24 ATGGCCATGG.CAGAAGC 1_2543 2570 CCATAAGGTCACCGTCACC 94 87 F TCA 295 R. ATTCAAAGC 650 SPl01_SPET SF101_SPETi 11_2468_24 TATGGCCATGG.CAGAAG 1_2543 2570 TCCATAAGGTCACCGTCAC 435 87 TMOD F CTCA 296 TMOD R CATTCAAAGC 651 SP101-SPET 11_266_295 CTTGTACTTGTGGCTCA SP101_-SPETi GCTG.CTTTGATGGCTGAAT 78 F CACGGCTGTTTGG 297 1 355 380 R. CCCCTTC 661 SF101 sPET SF101_SPETi 11_266_295 TCTTGTACTTGTGGCTC 1_355_380_T TGCTGCTTTG.ATGGCTGAA 436 TMOD F ACACGGCTGTTTGG 298 MOD R. TCCCCTTC 662 SPlOlSPET SPlOl SPET1 11_2961_29 ACCATGACAGAAGGCAT 1_3023_ 3045 GGAATTTACCAGCGATAGA 84 F TTTGACA 299 8.CACC 652 Sp101_SPET SP101 SPET1 11_2961_29 TACCATGACAGAAGGCA 1_3023_3045 TGGAATTTACCAGCGATAG 437 84 TMOD F TTTTGACA 300 TMOD R ACACC 653 SP101_SPET SP101_SPETi 11_3075_31 GATGACTTTTTAGCTAA 1_3168 3196 AATCGACGACCALTCTTGGA 96 03 F TGGTCAGGCAGC 301 ii AAGATTTCTC 656 438 SF101 SPET TGATGAC:TTTTTAGCTA ,302 SP101 SPET1I TAATCGACGACCATCTTGG 657 00 00 -36- 11_3075 31 ATGGTCAGGCAGC 1_3168_3196 AAAGAT'rTCTC 03 TMOD F Th100 R SPl01_SPET SP101lSPETI 11 3085_31 TAGCTAATGGTCAGGCA 1_31-70_3194 TCGACGACCATCTTGGAAA 448 04-F GCC 303 aGATTTC 658 SPl0lSPETI 11_322_344 GTCAAAGTGGCACGTTT SP1OI_SPET1 79 F ACTGGC 304 1 423 441 R ATCCCCTGCTTCTGCTGCC 665 SP1O3. SPET SPl01lSPET1 11_322 344 TGTCAAAGTGGCACGTT 1_423_441_T TATCCCCTGCTTCTGCTGC 439 TMOD F TACTGGC 305 MOD R c 666 SP101_SPET SP101-SPET1 11_336_34 AGCGTAAAGGTGAACCT 1_3480_3506 CCAGCAGTTACTGTCCCCT 97 03 F T 306 R CATCTTTG 659 SP101_SPET SP101_SPETi 11_3386_34 TAGCGTAAAGGTGAACC 1_*34806 3506 TCCAGCAGTTACTGTCCCC 440 03-TM4OD F TT 307 TiMOD i TCATCTTTG 660 S0101_SPET SPl0lSPET1 11_3511_35 GCTTCAGGAATCAATGA 1_3605_3629 GGGTCTACACCTGCACTTG 96 35 F TGGAGCAG 308 -R CATAAC 663 SP101_SPET SP101_SPETi 11_3511_35 TGCTTCAGGAATCAATG 1_3605_ -3629 TGGGTCTACACCTGCACTT 441 35 TMOD F ATGGAGCAG 309 TMOD a GCATAAC 664 SP101_SPET 11_358_387 GGGGATTC-AGCCATcAA SPl01_SPETi CCAACCTTTTCCACAACAG F AGCAGCTATTGAC 310 1 448 473 a AATCAGC 668 SPl0l SPST SPl0lSPEr1 11_356_387 TGGGGATTCAGCCATCA 1_448_473_T TCCAACCTTTTCCACAACA 442 TMOD F AAGCAGCTATTGAC 311 MOD a GAATCAGC 669 SPl0l_SPET 11_364_385 TCAGCCATCAAAGCAGC SPI01lSPETI TACCTTTTCCACAACAGAA 4 4E7 F TATTG 312 1 448 411 a TCAGC 667 SP10l_SPET 11_600_629 CCTTACTTCGAACTATG SP101_SPETi CCCATTTTTTCACGCATGC 81 F AATCTTTTGGAAG 313 1 686 714 R TGAAAATATC 670 SPil lSPET SPl0lSPETi 11_600_629 TCCTTACTTCGAACTAT 1_--686_-714_-T TCCCATTTTTTCACGCATG 443 TMOD F GAATCTTTTGGAAG 314 140D R CTGAAAATATC 671 SPil lSPET 11_658_ 684 GGGGATTGATATCACCG SPl0lSPETi GATTGGCGATAAAGTGATA 82 ATAAGAAGAA 315 1 756 784 a TTTTCTAAAA 672 SPlOlSPET SPl0lSPETi 11_658_684 TGGGGATTGATATCACC 1_756_784_T TGATTGGCGATAAAGTGAT 444 TMOD F GATAAGAAGAA 316 MOD R ATTTTCTAAAA 673 SplOl SPET 11_776_801 TCGCCAATCAAAACTAA SP101_SPETI GCCCACCAGAAAGACTAGC 83 F; GGGAATGGC. 317 1 871 896 a AGGATAA 674 SPlOlSPET SPl0lSPETi 11_776_801 TTCGCCAATCAAAACTA 1_871_896_T TGCCCACCAGAAAGACTAG 445 TMOD F AGGGAATGGC 318 MOD a -CAGGATAA 675 SPil lSPET SP101_SPETi 11_893_921 GGGCAACAGCAGCGGAT 1_988 1012 CATGACAGCCAAGACCTCA 84 F; TGCGATTGCGCG 319 a7 CCCACC 578 SPil lSPET SPl0lSPETi 11_893921 TGGGCAACAGCAGCGGA 1_988 1012 TCATGACAGCC.AAGACCTC 423 TMOD Fi TTGCGATTGCGCG 320 TOD a ACCCACC 679 SSPE-BA_11 TCAAGCAAACGCACAAT SSPEBA_196 TTGCACGTCTGTTTCAGTT 706 4 137 F CAGAAGC 321 222 a GCAAATTC 683 SSPEBA 11 TCAAGCAAACGCA-CAAC SSPE-BA-196 TtGCACGTUT 4

C'GTTTCAGT

612 4 137P T 'UAGAAGC 321 222P' R TGCAAATTC 684 SSPEBA_11 CAAGCAAACGCACAATC SSPEBA_197 TGCACGTCTGTTTCAGTTG 58 5 137 F AGAAGC 322 222-R CAAATTC 686 SSPE-BA-11 5_137_TMOD TCAAGCAAACGCACAAT SSPEBA_197 TTGCACGTCTGTTTrCAGTT 355 F CAGAAGC 321 222 THOD a GCAAATTC 687 SSPEBA_12 SSPEBA_197 TCTGTTTCAGTTGCAAATT 215 1 137 F- AAcGCAcAATcAGAAGC 323 216 a c 685 551'S BA_12 TGCACAATCAGAAGCTA SSPEBA_202 TTTCACAGCATGCACGTCT 699 3 153 F- AGAAAGCGCAAGCT 324 231 R GTTTCAGTTGC 688 SSPEBA 14 TGCAAGCTTCTGGTGCT BSPEBA_242 TTGTGATTGTTTTGCAGCT 704 6 168 AGC.ATT 325 267 a GATTGTG 689 TGCTTCTGGTGCTAGCA SSPEBA_243 TGATTGTTTTGCAGCTGAT 702 0 168 F- TT 326 264 a TGT 691 TGCTTCTGGCGUCAG SSPE BA_243 TGATTGTTTTGD*AGUrTGA 610 0 18P F -UATT 1326 2641i R- C'CGT69 00 00 -37- SSPEBA_15 SSPEBA_243 _700 6 1.68 F TGGTGCTAGCATT 327 _255 R. TGCAGCTGATTGT 690 SSPE_BA.15 SSPE_BA_243 608 6 168P F TGGCGU'CAGU*ATT 327 255P p. TGUAGUTGACCGT 690 SSPEBA_63 TGCTAGTTATGGTACAG SSPE_-BA_-163 TCATAACTAGCATTTGTGC 705 89 F AGTTTGCGAC 328 191 R TTTGAATGCT 682 SSPEBA_72 TGGTACAGAGTTTGCGA SSPEBA_163 TCATTTGTGCTTTGAATGC 703 89 F C 329 182 R T 681 E SSPEA72 TGGTAUAGAGCCCG SSPEBA163

TC.ATTTGTGCCC'CGAAC

SSPEBA_75 SSPE-BA_163 701 89 F TACAGAGTTTGCGAC 330 177 p. TGTGCTTTGAATGCT 680 TAUAGAGCaC'C'CGU'G SSPEBA_163 6 09 892 F AC 330 1772 R. TGTGCCC*CGAAC'GUt 680 TOXRVBC_1 TCGATTAGGCAGCAACG TOXR .VBC_22 TTCAAAACCTTGCTCTCGC 1099 35 158 F- AAAGCCG 331 1 246 R CAAACAA 692 TRPEAY094 TRPEAY0943 355 1 064_1 TCGACCTTTGGCAGCAA 55_1171 119 TACATCGTTTCGCCC?.AGA 905 086 F CTAGAC 332 6 p. TCAATCA 693 TRPEAY094 TRPE AY0 943 355_1278_1 TCAAATGTACAAGGTGA 55_1392_141 TCCTCTTTTCACAGGCTCT 904 303 F AGTGCGTGA 333 8 p. ACTTCATC 694 TRPEAY094 4TRPE AY0943 355 1445_1 TGGATGGCATGGTGAAA 55_1551_158 TATTTGGGTTTCATTCCAC 903 471 F TGGATATGTC 334 0 R. TCAGATTCTGG 695 TRPEAY094 TRPE-AY0943 355_1467_1 ATGTCGATTGCAATCCG 55_1569_159 TGCGCGAGCTTTTATTTGG 902 491 F TACTTGTG F335 2 p. GTTTC 696 TPPAY094 TRPE AY0943 355_666_68 GTGC-ATGCGGATACAGA 55_769 791 TTCAAAATGCGGAGGCGTA 906 8 f- GCAGAG 336 p. TGTG 697 TRPEAY094 TRPEAY0943 355_-757_-77 TGCAAGCGCGACCACAT 55_864 883 TGCCC-AGGTACAACCTGCA 907 6 F ACG 337 p. T 698 TUFB_BC_22 GCACTATGCACACGTAG TUFE_-EC_-284 TATAGCACCATCCATCTGA 114 5 251 F ATTGTCCTGG 338 30 p. GCGGCAC '706 TUFBEC_23 TTGACTGCCCAGGTCAC TUFB-EC_283 GCCGTCCATTTGAGCAGCA 9 259 2 F GCTG 339 303 2 R cc 704 TUFB_-EC -23 TAGACTGCCCAGGACAC TUEB_EC_283 GCCGTCCATCTGAGCAGCA 59 9 259 F GCTG 340 303 R. cc 705 TUFB_-EC 25 TGCACGCCGACTATGTT TUIFBEC_337 TATGTGCTCACGAGTTTGC 942 1 278 F AAGAACATGAT 341 360 R. GGCAT 707 TUFBB C_-27 TGATCACTGGTGCTGCT TUFBEC_337 TGGATGTGCTCACGAGTCT 941 5 299 F CAGATGGA 342 362 R. GTGGCAT 706 AAGACGACCTGCACGGG TUFBEC_849 117 7 774 F C 343 867 p. GCGCTCCACGTCTTCACGC 709 CCACACGCCGTTCTTCA TUFEEC_103 GGCATCACCATTTCCTTGT 293 7 979 F ACAACT 344 4 1058 R CCTTCG 700 TUFBRC_103 7_979_ THOU TCCACACGCCGTTCTTC 4_1058_TMOD TGGCATCACCATTTCCTTG 367 Pi AACAACT 345 p. TCCTTCG 701 TUFEEC_97 AACTACCGTCCTCAGTT TUFBEC_104 GTTGTCACCAGGCATTACC 62 6 1000 2i F CTACTTCC 346 5 1068 2 R. ATTTC 702 TUFEB C_-97 AACTACCGTCCGCAGTT TUFB-EC_104 GTTGTCGCCAGGCATAACC 61 6 1000 F CTACTTCC 347 5 1068 p. ATTTC 703 TUFBBC_98 CCACAGTTCTACTTCCG TUFB EC 103 TCCAGGCATTACCATTTCT 63 5 1012 F; TACTACTGACG 348 3 162 W. ACTCCTTCTGG 699 VALSEC -11 CGTGGCGGCGTGGTTAT VM.SBC_119 ACGAACTGGATGTCGCCGT 225 05 1124 F CGA 349 5 1214 R. T 710 VALSBC-11 CGTGGCGGCGTGGTTAT VALSBC_119 CGGTACGAACTGGATGTCG 71 05 1124 F CGA 349 5 1218 P. CCGTT 711 05_1124_TM TCGTGGCGGCGTGG;TTA 5_1218THO TCGGTACGAACTGGATGTC 358 00 F TCGA 350 R. GCCGTT 712 VALS_BC_11 TATGCTGACCGACCAGT VALSBC_123 TTCGCGCATCCAGGAGAAG 965 28 1151 F GGTACGT 351 1 12597 p. TACATGTT 713 VAiLS_BC_18 CGACGCGCTGCGCTTCA VALSEC_192 GCGTTCCACAGCTTGTTGC 112 33 1850 F C 352 0 1943 R. AGAAG 714 VALSEC-19 CTTCTGCAACAAGCTGT VALSEC_194 TCGCAGTTCATCAGCACGA 116 20 1943 F GGAACGC 353 8 1970 R AGCG 715 VALS-BC-61 ACCGAGCAAGGAGACCA VALSEC_705 TATAACGCACATCGTCAGG 295 0649_ GC 354 727 R. GTGA 716 WAAA_29692 TCTTGCTCTTTCGTGAG WAAAZ96925 CAAGCGGTTTGCCTCAAAT 931 5 2 29 F TTCAGTAAATG 355 115 138 p. AGTCA 7 17 932 WAAA Z9692 I TCGATCTGGTTTCATGC 356 WAAA Z96925 TGGCACGAGCCTGACCTGT 716I 00

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S5_286 311_ TGTTTCAGT I I 394 412R I I I

L

r [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 colil 16S EC gene) 16127994 4033120..4034661 23S rRNA (23S Escherlchia 720 ribosomal RNA colil 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 coll Complement HFLB EC peptidase ftsH) 16127994 (3322645..3324576) infB (protein Escherichia 726 chain initiation colil Complement INFB EC factor infB gene) 16127994 (3310983..3313655) lef (lethal Bacillus Complement 727 LEF BA factor) anthracis 21392688 (149357..151786) pag (protective Bacillus 728 PAG BA antigen) anthracis 21392688 143779..146073 rplB (50S Escherlchia 729 ribosomal protein coll 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 colil RPOC EC beta' chain) 16127994 4182928..4187151 SP101ET Concatenation SPET_1 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) nmur (glutamate 312732..313169 racemase) m utS (DNA mismatch Complement -39repair protein) (1787602..1788007) xpt (xanthine 930977..931425 phosphoribosyl transferase) yqiL (acetyl-CoA- 129471..129903 acetyl transferase) tkt 1391844..1391386 (transketolase) sapE (small acid- 733 soluble spore Bacillus SSPE BA protein) anthracls 30253828 226496..226783 734 tufE (Elongation Escherlchia 734 TUFB EC factor Tu) coli 16127994 4173523..4174707 valS (Valyl-tRNA Escherlchla Complement 735 VALS EC synthetase) coil 16127994 (4461405..447B550) apS (Aspartyl- Escherlchla 16127994 complement(19467 77 736 ASPS EC tRNA synthetase) coli 1948546) 2996286 No extraction CAF1 AF cal (capsular Yerslnia GenBank coordinates 053947 protein cafi) pestis used INVU22 Yerslnia 1256565 74..3772 737 457 inv (invasin) pestis Y. pestls specific 16120353 No extraction chromosomal genes GenBank coordinates LLNCOO difference Yerslnla used 3143 region pestis BONTA X BoNTIA (neurotoxin Clostridium 40381 77..3967 738 52066 type A) 2791983 No extraction 739 MECAY1 meoA methicillin Staphylococcus GenBank- coordinates 4051 resistance gene aureus used trpE (anthranilate 20853695 No extraction TRPE AY synthase (large Acinetobacter GenBank coordinates 094355 component)) baumanil used 740 9965210 No extraction RECAAF recA (recombinase Acinetobacter Genaank coordinates 251469 A) baumanli used 741 4240540 No extraction GYRAAF gyrA (DNA gyrase Acinetobacter GenBank coordinates 100557 subunit A) baumanll used 742 4514436 No extraction GYREAB gyrB (DNA gyrase Acinetobacter GenBank coordinates 008700 subunit B) baumanil used 743 waaA (3-deoxy-D- 2765828 No extraction WAAAZ9 manno-octulosonic- Acinetobacter GenBank coordinates 6925 acid transferase) baumanil used 744 Concatenation comprising: Artificial Sequence* partial gene tkt sequences of 1569415..1569873 (transketolase) Campylobacter CJSTCJ jejunl glyA (serine 367573..368079 hydroxymethyltrans ferase) 15791399 gitA (citrate complement synthase) (1604529..1604930) aspA (aspartate 96692..97168 ammonia lyase) 745 ginA (glutamine complement synthase) (657609 658085) pgm (phosphoglycerate 327773..328270 mutase) uncA (ATP 112163..112651 synthetase alpha chain) RNASEP MNase P Bordetella 33591275 Complement BDP (ribonuclease P) pertussls (3226720..3227933) 746 RNASEP_ Nase P Burkholderia 53723370 Complement BKM (ribonuclease P) mallel (2527296..2528220) 747 RNASEP_ P.ase P BacIllus 16077068 Complement BS (rihonuclease P) subtllls (2330250..2330962) 748 RNASEP RNase P Clostrdwum 18308982 Complement CLB (ribonuclease P) perfrlngens (2291757..2292584) 749 BNASEP MRase P Escherichia 16127994 Complement EC (ribonuclease P) coll (3267457..3268233 750 RNASEP MNase P Rickettsia 15603881 complement(605276..6 RFP (ribonuclease P) prowazekll 06109) 751 RNASEP_ RNase P Staphylococcus 15922990 complement(1559869..

SA (ribonuclease P) aureus 1560651) 752 RNASEP_ Mase P Vibrio 15640032 complement(2580367..

VBC (ribonuclease P) cholerae 2581452) 753 icd (isocitrate CoxIella 29732244 complement(1143867..

ICD CXB dehydrogenase) burnetil 1144235) 754 multi-locus Aclnetobactez 29732244 IS111A insertion baumannii IS1111A element No extraction (mpA (outer Rickettsia 40287451 OMPA AY membrane protein prowazekil 485227 A) No extraction 755 ompB (outer Rickettsla 15603881 OMPBRK membrane protein prowazekil complement(881264..8 p B) 86195) 756 GLTARK gItA (citrate Vlbrlo 15603881 complement(1062547..

P synthase) cholerae 1063857) 757 toxR Francisella 15640032 TOXRVB (transcription tularensls complement(1047143..

C regulator toxR) 1048024) 758 aed (Aspartate Francsella 56707187 semialdehyde tularensls complement(438608..4 ASD FRT dehydrogenase) 39702) 759 GALEFR galE (UDP-glucose Shlgella 56707187 T 4-epimerase) flexnerl 809039..810058 760 IPAHSG ipaH (invasion Campylobacter 30061571 F plasmid antigen) jejuni 2210775..2211614 761 Coxlella complement(849317..8 hupB (DNA-binding burnetil 15791399 49819) HOPE CJ protein Hu-beta) 762 Concatenation comprising: Artificial 763 Sequance* partial gene sequences of Aclnetobacter baumannll trpE (anthranilate synthase component

I))

adk (adenylate Sequenced in-house AB MLST kinase) mutY (adenine glycosylase) fumC (fumarate hydratase) efp (elongation factor p) ppa (pyrophosphate phosphohydratase 00 o -41-

O

S [0089] Note: These artificial reference sequences represent concatenations of partial gene 0 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 S partial gene extractions (100N for SP101_SPET11 (SEQ ID NO: 732); 50N for CJST_CJ (SEQ ID NO: 745); and 40N for AB_MLST (SEQ ID NO: 763)).

(N

Ci [0090] Example 2: DNA isolation and Amplification [0091] Genomic materials from culture samples or swabs were prepared using the DNeasy® 96 C to Tissue Kit (Qiagen, Valencia, CA). All PCR reactions are assembled in 50 il reactions in the 96 well microtiter plate format using a Packard MPII 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 MgCI 2 0.4 M betaine, 800 pM 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 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 to 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, as 25 pl of a 2.5 mg/mL suspension of BioClon amine terminated supraparamagnetic beads were added to 25 to 50 pl 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 3o 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, imidazole, 35% MeOH, plus peptide calibration standards.

0 0 -42- S [0095] Example 4: Mass Spectrometry and Base Composition Analysis [0096] 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 D volume. Thus, components that might be adversely affected by stray magnetic fields, such as n CRT monitors, robotic components, and other electronics, can operate in close proximity to the C FTICR spectrometer. All aspects of pulse sequence control and data acquisition were performed 00 S on a 600 MHz Pentium II data station running Bruker's Xmass software under Windows NT C- 3 operating system. Sample aliquots, typically 15 Rl, 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 pl sample loop integrated with a fluidics handling system that supplies the 100 pl /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 countercurrent flow of dry N 2 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 x0 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 32 scans were co-added for a total data acquisition time of 74 s.

[0097] The ESI-TOF mass spectrometer is based on a Bruker Daltonics MicroTOFM. 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 MicroTOF T M 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 ps.

0 -43-

C

[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 N 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 0 and one injector rinse were required to minimize sample carryover. During a routine screening So 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 as [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 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 A (-15.994) combined with C T (+15.000). For example, one 99-mer nucleic acid strand having a base composition of A 27

G

3 0

C

2 1

T

2 1 has a theoretical molecular mass of 30779.058 while another 99-mer nucleic acid strand having a base composition of A 26

G

31 C22T 2 0 has a theoretical molecular mass of 30780.052. A 1 Da difference in molecular mass may be within the experimental error of a 00 o -44- S molecular mass measurement and thus, the relatively narrow molecular mass range of the four natural nucleobases imposes an uncertainty factor.

[0102] The present invention provides for a means for removing this theoretical 1 Da uncertainty s factor through amplification of a nucleic acid with one mass-tagged nucleobase and three natural N 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 or deoxynucleotide triphosphate (dNTP).

00 C o [0103] 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 Thus, the same the G A (-15.994) event combined with 5-Iodo-C T (-110.900) event would result in a molecular mass difference of 126.894. If the molecular mass of the base composition A 2 7

G

3 0 5-Iodo-C 2 1

T

2 1 (33422.958) is compared with A 26

G

31 5-Iodo-C 22

T

2 o, (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 A 27

G

30 0 Iodo-C 21

T

2 1 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 Iodo-C and Molecular Mass Differences Resulting from Transitions Nucleobase Molecular Mass Transition A Molecular Mass A 313.058 -9.012 A 313.058 -24.012 A 313.058 A-->5-Iodo-C 101.888 A 313.058 15.994 T 304.046 9.012 T 304.046 -15.000 T 304.046 T-->5-IOdo-C 110.900 T 304.046 25.006 C 289.046 24.012 C 289.046 15.000 C 289.046 40.006 414.946 5-IOdo-C-->A -101.888 414.946 5-Iodo-C-->T -110.900 414.946 5-Iodo-C-->G -85.894 G 329.052 -15.994 G 329.052 -25.006 G 329.052 -40.006 G 329.052 G-->5-Iodo-C 85.894 [0104] Example 6: Data Processing S [01051 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 g 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-ofdetection versus probability-of-false-alarm plots for conditions involving complex backgrounds So 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 is 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 ao 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 as 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 00 00

QD

^-i -46corresponding to the appropriate primer set is reported as well as the quantities of primers remaining upon completion of the amplification reaction.

[0108] Example 7: Use of Broad Range Survey and Division Wide Primer Pairs for 5 Identification of Bacteria in an Epidemic Surveillance Investigation [0109] 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 clade 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 o 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 is 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 TMODR 389 16S rRNA 168 EC 713 732 F 26 16S EC 789 809 388 16S rRNA 347 16S EC_785 806_TMODF 30 168 EC 880 897_TMODR 392 16S rRNA 11 168 EC 785 806 F 29 16S EC 880 897 R 391 16S rRNA 348 16S_EC_960 981 TMODF 38 16S_EC 1054_1073_TMODR 363 16S rRNA 14 16S EC 960 981 F 37 16S EC 1054 1073 R 362 16S rRNA 349 23S EC 1826 1843_THOD_F 49 23S_EC_1906_1924_TMOD_R 405 23S rRNA 16 233 EC 1826 1843 F 48 23S EC 1906 1924 R 404 23S rRNA 352 INFB EC 1365_1393_TMODF 161 INFB EC_1439_1467_TMODR 516 infB 34 INFB EC 1365 1393 F 160 INFB EC 1439 1467 R 515 infB 354 RPOCEC 2218 2241 TMOD F 262 RPOCEC_2313_2337 TMODR 625 rpoC 52 RPOC EC 2218 2241 F 261 RPOC EC 2313 2337 R 624 rpoC 355 SSPEBA 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 VALSEC_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 23SEC 2646_2667 TMOD_F 60 23SEC_2745_2765_TMOD_R 416 23S rRNA 118 23S EC_2646 2667 F 59 23SEC 2745 2765_R 415 23S rRNA 17 23S EC 2645 2669 F 58 23S EC 2744 2761 R 414 23S rRNA 00

O

00

O

O

O

^0

(-N

-47- 361 16S_EC_1090_ 1_2_TMOD_F 5 16S EC 1175_1196_TMODR 370 16S rRNA 3 16S EC 1090 1111 2 F 6 16S EC 1175 1196 R 369 16S rRNA 362 RPOB EC_3799_3821_TMOD_F 245 RPOB EC 3862_3888 TMODR 603 rpoB 289 RPOB EC 3799 3821 F 246 RPOB EC 3862 3888 R 602 rpoB 363 RPOC EC_2146_2174 TMOD_F 257 RPOCEC_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 TMODF 345 TUFB_EC_1034_1058_TMOD_R 701 tufB 293 TUFB EC 957 979 F 344 TUFB EC 1034 1058 R 700 tufB 449 RPLB EC 690 710 F 237 RPLBEC_737_758 R 589 rplB 357 RPLB EC 688 710_TMODF 236 RPLB_EC_736_757_TMOD_R 588 rplB 67 RPLB EC 688 710 F 235 RPLB EC 736 757 R 587 rplB [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 to 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 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 ao 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 drilldown 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 -48constitutes 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 CAPCBA_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 CYA BA 1353 1379 F 127 CYA BA 1448 1467 R 482 cyA 353 LEFBA_756_781 TMODF 175 LEF BA 843 872 TMODR 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 0 amplified to obtain bioagent identifying amplicons using the primer pair numbers listed in the upper right hand comer 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 2t 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 as 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.

-49- 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 [AG C T] A G C T] [AG C T] Klebsiella [29 32 25 13] [23 38 28 26] [26 32 28 pneumonfliae MGH78578 [29 31 25 131* [23 37 28 26]* [26 31 28 CO-92 Biovar [29 30 28 29] Yersinia pestis Orientalis [29 32 25 131 [22 39 28 26] [30 30 27 291* 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 [2B 31 23 17] [24 37 25 27] [29 30 28 29] Pseudomonas [26 36 29 24] aeruginosa PAO1 [30 31 23 15] [27 36 29 231* [26 32 29 29] Pseudomonas fluorescens PfO-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 15] [33 33 23 27] [29 26 28 31] Francisella tularensis schu 4 [32 29 22 16] [28 38 26 26] [25 32 28 31] Bordetella pertussis Tohama 1 [30 29 24 16] [23 37 30 24] [30 32 30 261 Burkholderia C27 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 26 281 [24 36 29 27] Neisseria meningitides MC58 (serogroup B) [29 28 26 16] [27 34 27 27] (25 35 30 26] Neisseria meningitides serogroup C, FAM18 [29 28 26 16] (27 34 27 27] [25 35 30 26] Neisseria meningitides 32491 (serogroup A) [29 28 26 16] [27 34 27 27] [25 35 30 26] Chlamydophila pneumoniae TW-183 [31 27 22 19] NO DATA [32 27 27 29] Chlamydophile pneumoniae AR39 [31 27 22 19] NO DATA [32 27 27 29] Chlamydophila pneumoniae CWLO29 [31 27 22 19] NO DATA [32 27 27 29] Chlamydophile 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 avlum k10 [27 36 21 15] [22 37 30 28] [21 36 27 Mycobacterium avium 104 (27 36 21 15] [22 37 30 28] [21 36 27 Mycobacterium tuberculosis 0CS0#93 [27 36 21 15] [22 37 30 28] [21 36 27 Mycobacterlum tuberculosis CDC 1551 (27 36 21 15] [22 37 30 281 [21 36 27 Mycobacterium tuberculosis B37Rv (lab strain) [27 36 21 15] (22 37 30 28] (21 36 27 Mycoplasma pneumonlae 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 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 21 211 [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 Staphylococcus (25 35 30 26] [30 29 30 29] aureus NCTC 8325 [27 30 21 21] [25 35 31 261* [30 29 29 Streptococcus [24 36 31 agalactlae NEM316 [26 32 23 18] [24 36 30 261* [25 32 29 Streptococcus equi NC 002955 [26 32 23 18] [23 37 31 25] [29 30 25 32] Streptococcus pyogenes MGAS8232 [26 32 23 18] (24 37 30 25] [25 31 29 31] Streptococcus yogenes 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 (M1) [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 Streptococcus pneumoniae R6 [26 32 23 181 [25 35 28 28] [25 32 29 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 125 32 29 mitis NCTC 12261 [26 32 23 181 [25 35 30 26] [24 31 35 29]* 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 [AG C T] [AG C T] [AG C T] Klebsiella pneumonlae MGH78578 [25 31 25 22] (33 37 25 27] NO DATA CO-92 Biovar [25 31 27 Yersinia pestis Orientalis [25 32 26 20]* [34 35 25 28] NO DATA P12 (Biovar [25 31 27 Yersinia pestis Mediaevalis) [25 32 26 20]* [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 25 271 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 PfO-1 NO DATA [30 37 27 281 NO DATA Pseudomonas putida KT2440 [24 31 26 20] [30 37 27 281 NO DATA Legionella pneumophila Philadelphia-1 [23 30 25 23] [30 39 29 24] NO DATA Francisella tularensls 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 Burkholderla cepacia J2315 [23 27 22 20] [31 37 28 261 NO DATA Burkholderia pseudomalIlei 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 MCS8 (serogroup B) [25 27 22 18] [34 37 25 261 NO DATA Nelsseria 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 -51eiingitidis Ciamydophil pneumonliae TW-183 [30 28 27 18] NO DATA NO DATA hlamydophila pneumoniae AR39 [30 28 27 18] NO DATA NO DATA Chlamydophila pneumoniao CWLO29 [(30 28 27 18] NO DATA NO DATA Chlamydophila pneumonisoe J138 [30 28 27 18] NO DATA NO DATA Corynebacterium diphtheriae NCTC13129 NO DATA [29 40 28 25] NO DATA ycobacterium avium k10 NO DATA [33 35 32 22] NO DATA ycobacterium 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 Mycoba c teri um tuberculosis H37Rv (lab strain) NO DATA [30 36 34 22] NO DATA Mycoplasma pneumonfliae 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 31 271 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 31271 Staphylococcus aureus N315 [26 30 25 20] [31 38 24 29] [33 30 3127] Staphylococcus aureus NCTC 8325 [26 30 25 20] [31 38 24 29] [33 30 31 271 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 Streptococcus pyogenes MHGAS8232 [28 31 23 19] [33 37 24 28] [38 31 29 231 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 (Ml) [28 31 22 201* [33 37 24 28] [38 31 29 23] Streptococcus pneumonlae 670 [28 31 22 20] [34 36 24 28] [37 30 29 Streptococcus pneumoniae R6 [28 31 22 20] [34 36 24 29] [37 30 29 Streptococcus pneumoniae TIGR4 [28 31 22 20] [34 36 24 28] [37 30 29 Streptococcus gordonii NCTC7868 [28 32 23 20] [34 36 24 28] [36 31 29 Streptococcus [28 31 22 mitis NCTC 12261 (29 30 22 20]* [34 36 24 28] [37 30 29 StreptococcusI mutans UA159 [26 32 23 22] [34 37 24 271 NO DATA -52- 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] [AG C T] [AG 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 26 20 P12 (Biovar Yersinia pestis Mediaevalia) NO DATA (29 31 33 29] [32 28 20 Yersinia pestis 91001 NO DATA [29 31 33 291 NO DATA Haemophilus influenzae KW20 NO DATA (30 29 31 32] NO DATA Pseudomonas aeruginosa PAO1 NO DATA [26 33 39 24] NO DATA Pseudomonas fluorescens PfO-l 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 Francisella 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 596243 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 MCSB (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 22491 (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 Chlamydophile 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 19 271 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 35] (36 24 19 26] -53- Staphylococcus aureus N315 [17 20 21 17] [30 27 30 35] [36 24 19 261 Staphylococcus aureus NCTC 8325 [17 20 21 17] [30 27 30 35] [35 24 19 271 Streptococcus a alactiae 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 36] 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 36] NO DATA Streptococcus pyogenes Manfredo (M5) [23 21 19 12] [24 32 30 36] NO DATA Streptococcus pyogenes SF370 (Ml) [23 21 19 12] [24 32 30 36] NO DATA Streptococcus pneumoniae 670 [22 20 19 14] [25 33 29 35] [30 29 21 Streptococcus pneumonlae R6 [22 20 19 14] [25 33 29 35] (30 29 21 Streptococcus neumoniae TIGR4 [22 20 19 14] [25 33 29 35] [30 29 21 Streptococcus gordoni 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 [AG C T] [AG C T] [AG C T] Klebsiella pneumonlae 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] KIMS 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 19 221 Haemophilus influenzae KW20 NO DATA NO DATA NO DATA Pseudomonas aeruginosa PAO1 NO DATA NO DATA NO DATA Pseudomones fluorescens Pf0-1 NO DATA NO DATA NO DATA Pseudomonas putida KT2440 NO DATA [21 37 37 21] NO DATA Legionelle pneumophila Philadelphia-1 NO DATA NO DATA NO DATA Francsella 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 pseudomellei 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 meningtidis serogroup C, FAM418 NO DATA NO DATA NO DATA -54- Neisseria meningitidis Z2491 (serogroup A) NO DATA NO DATA NO DATA Chlamydophila pneumonlae 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 kl0 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 I' 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 (Ml) 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 [AG C T] AG C T] [AG 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 P12 (Biovar yersinia pestis Mediaevalis) [20 34 18 20] NO DATA NO DATA.

yersinia pestis 91001 [20 34 18 20] NO DATA NO DATA Haemophilus influenzae KW20 NO DATA NO DATA NO DATA Pseudomonas aeruginosa PAO1 [19 35 21 171 [16 36 28 22] NO DATA Pseudomonas fluorescens PfO-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 Burkholderia pseudomallel K96243 [19 34 19 20] [15 37 26 22] [25 27 32 Neisseria gonorrhoeae FA 1090, ATCC 700625 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 22491 (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 CWLO29 NO DATA NO DATA NO DATA Chlamydophila pneumoniae 3138 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] Mycobacteriumn avium 104 [19 34 23 16] NO DATA [24 26 35 19] Mycobacterium tuberculosis CSU#93 [19 31 25 17) NO DATA [25 25 34 Mycobacterium tuberculosis CDC 1551 [19 31 24 18] NO DATA [25 25 34 Mycobacterium tuberculosis H37Rv (lab strain) [19 31 24 18] NO DATA [25 25 34 Mycoplasma pneumonihe 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 00

IND

IND

N

k 00 -56aureus Staphylococcus aureus NCTC 8325 NO DATA NO DATA NO DATA Streptococcus agalactiae NEM316 NO DATA NO DATA NO DATA Streptococcus eqlUi 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-I 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 (Ml) 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 gordonll 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 to 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 Is 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 00 -57- S 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.

S[0116] In addition to the identification of Streptococcus pyogenes, other potentially pathogenic S organisms were identified concurrently. Mass spectral analysis of a sample whose nucleic acid c was amplified by primer pair number 349 (SEQ ID NOs: 49 and 405) exhibited signals of 0 bioagent identifying amplicons with molecular masses that were found to correspond to c o 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 which 3 is incorporated herein by reference in its entirety.

[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 S 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 Haemophilus 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 as 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.

[01181 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 00 -58-

O

N flora dominated by multiple, commensal non-beta-hemolytic Streptococcal species, including the a viridans group streptococci 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 S military recruits in the midst of a respiratory disease outbreak had a dramatically different N microbial population than that experienced by the general population in the absence of epidemic disease.

0 0 [0119] Example 8: Drill-down Analysis for Determination of emm-Type of Streptococcus o 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 ao 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), as 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 emm-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.

-59- 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_SPET11 358 387 SP101 SPET11 448 442 TMOD_F 311 473_TMOD_R 669 gki SP101_SPET11 358 387 310. SP101 SPET11 448 668 gki F 473 TMOD R SP101 SPETI1 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 F 714 R SP101 SPET11_1314 133 SP101 SPET11 1403 426 6 TMOD F 278 _1431_TMOD_R 633 murI 86 SP101 SPET11 1314_133 277 SP101_SPET11_1403 632 murl 6 F1431

R

SP101 SPET11 1807 183 SP101_SPET11 1901 430 5_TMODF 286 _1927_TMOD_R 641 mutS SP101 SPET11 1807 183 285 SP101 SPET11_1901 640 mutS F 1927 R SP101 SPET11_3075 310 SP101 SPET11_3168 438 3 TMOD F 302 _3196_TMOD_R 657 xpt 96 SP101 SPET11_3075_310 301 SP101_SPET11_3168 656 xpt 3 F 3196 R SP101_SPET11_3511_353 SP101 SPET11_3605 441 5_TMODF 309 _3629_TMOD_R 664 yqiL 98 SP101 SPET11 3511 353 308 SP101_SPET11_3605 663 yqiL F 3629 R [0122] 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.

[0123] Of the 51 samples taken during the peak of the November/December 2002 epidemic (Table 8A-C rows 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 emf -type by emm lnGene Location murI auts instof e Mass Sqecn (sml) Year (Primer Pair (Primer Pair Insanc Sspectrometry SNo.in smpe 426) No. 430) 48 3 3 MCR San A39 G25 C20 T34 A38 G27 C23 T33 2 6 6 Diego 2002 A440 G24 C20 T34 A38 G27 c23 T33 1 28 28 A439 G25 C20 T34 A38 G27 C23 T33 3 ND ~~~~~~~(Cultured)A3G2 2T3 A8G7C3T3 3 3D A39 G25 C20 T34 A38 G27 C23 T33 3 5,58 5 1440 G24 C20 734 A438 G27 C23 733 6 6 6 NHRC San A440 G24 C20 T34 A438 G27 C23 733 1 11 11 Diego- A439 G25 C2D T34 A438 G27 C23 T33 3 12 12 Archive 2003 A440 G24 C20 734 A438 G26 C24 733 1 22 22 (Cultured) A39 G25 C20 734 A438 G27 C23 T33 3 25,75 75 A439 G25 C20 T34 1438 G27 C23 T33 4 44/61,82,9 44/61 A440 G24 C20 734 A438 G26 C24 T33 2 53,91 91 G25 C20 T34 A438 G27 C23 733 1 2 2 A439 G25 C20 734 A438 G27 C24 732 2 3 3 A439 G25 C20 T34 A38 G27 C23 T33 1 4 4 A439 G25 C20 T34 A38 G27 C23 T33 1 6 6 Ft. A40 G24 C20 T34 A438 G27 C23 T33 11 25 or 75 Weod 2003 A39 G25 C20 734 1438 G27 C23 T33 25,75, 33, 1 34,4,52,84 75 (Cultured) A439 025 C20 T34 1438 G27 C23 733 44/61 or 82 1 or 9 44/61 1440 G24 C20 T34 A438 G26 C24 T33 2 5 or 58 5 G24 C20 T34 A38 G27 C23 T33 3 1 1 1440 G24 C20 T34 1438 G27 C23 T33 2 3Ft Sll 2003 A39 G25 C20 T34 A438 G27 C23 T33 1 4 4 (Cultured) A439 G25 C20 T34 A438 G27 C23 T33 1 28 28 G25 C20 734 1438 G27 C23 T33 1 3 3 A439 G25 C20 T34 A438 G27 C23 733 1 4 4 A39 025 C20 734 A438 G27 C23 T33 3 6 6 A440 G24 C20 734 A438 G27 C23 733 1 11 11 Ft. 1439 G25 C20 T34 1438 G27 C23 733 1 13 94** Benning 2003 1440 G24 C20 T34 "A38 G27 C23 T33 44/61 or 82 (Cultured) 1 or 9 82 1440 G24 C20 T34 1438 G26 C24 T33 1 5 or 58 58 1A40 G24 C20 734 1438 G27 C23 T33 1 78 or 89 89 G25 C20 T34 A436 G27 C23 733 2 5 or 58 14kan 40 G24 C20 T34 A438 G27 C23 733 1 2 AFB 1439 G25 C20 T34 1438 027 C24 T32 1 81 or 90 ND (hot 2003 A440 G24 C20 T34 A438 G27 C23 733 1. 78 (Twat A38 G26 C20 734 1438 G27 C23 T33 No detection No detection No detection 7 3 ND 1439 025 C20 734 A438 G27 C23 T33 1 3- ND MCRD San No detection 1438 G27 C23 733 1 R ieo2002 No detection No detection 1 3 ND (Throat No detection No detection 2 3 ND Swabs) No detection A438 G27 C23 T33 3 No detection IND rNo detection No detection 61 Table 8B1: 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 -m-type by amGene Location xtYL intne ass eunig (ape Year (Primer Pair (Primer Pair Insanc S pectrometry Ieunig (ape No. 438) No. 441) 48 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 (Clue)A30 G36 C20 T36 A41 G28 P1.8 T32 is _ND in G1 C20 36 AC G2tu19ed) 3 3D A30 G36 C20 T36 A40 G29 C19 T31 6 3,5 3 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 C19T'31 2 53,91 91 A30 G36 C19 T37 A40 G29 C19 T131 1 2 2 A30 G36 C20 T136 A40 G29 C19 T131 2 3 3 A30 G36 C20 T36 A40 G29 C19 T131 1 4 4 A30 G36 C19 T37 A41 G28 C19 T131 1 6 6 Ft. A30 G36 C20 T36 A40 G29 C19 T31 Weoad 20 A30 G36 C20 T36 A40 G29 C19 T131 25,75, 33, 1 34,4,52,84 75 (Cultured) A30 G36 C19 T137 A40 G29 C19 T131 1 44/61 or 82 I or 9 44/61 A30 G36 C20 T36 A41 G28 C19 T131 2 5 or 58 5 A30 G36 C20 T36 A40 G29 C19 T31 3 1 1 .A30 G36 C19 T137 A40 G29 C19 T31 2 3 3 Ft. Sill_ 2003 30G36 C20 T36 A40 G29 C19 T31 1 4 4 (Cultured) A30 G36 C19 T137 A41 G28 C19 T31 1 28 28 G36 C20 T136 _A41 G28 C18 T132 1 3 3 A30 G36 C20 T36 A40 G29 C19 T31_ 1 4 4 A30 G36 C19 T37 A41 G28 C19 '31 3 6- 6 A30 G36 C20 T36 A40 G29 C19 T131 1 11 11 Ft. A30 G36 C20 T36 A40 G29 C19 T131 1 13 94** Benning 2003 A30 G36 C20 T36- Al1 G28 C19 T31 44/61 or 82 (Cultured) 1 or 9 82 A30 G36 C20 T36 A41 G28 C19 '31 1 5 or 56 58 A30 G36 C20 '136 A40 G29 C19 T131 1 78 or 89 89 A30 G36 C20 T36 A41 G28 C19 '131 2 5 or 58 Lklad A30 G36 C20 T36 A40 G29 C19 T31 1 2 AFB A30 G36 C20 T36 A40 G29 C19 T131 1 81 or 90 ND 2003 A30 G36 C20 T36 A40 G29 C19 T31 1 78 (Throat A30 G36 C20 T136 A41 G28 C19 T31 Swabs) No detection detection No detection 7 3 ND A30 G36 C20 T36_ A40 G29 C19 T31 1 3 ND NCRD San A30 G36 C20 T36 A40 G29 C19 T31 1 3 D Dego2002 A30 G36 C20 T36 No detection 1 3 N4D (Throat No detection A40 G29 C19 T31 2 3 ND Swabs) A30 G36 C20 T36 A40 G29 C19 '31 3 No detection ND detection No detection -62- 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 of em-tp by earn-Gene Location kgt Isacs mass Seunig (ape ear (Primer Pair ((Primer Pair Insanc spectometry uecig (mpe No. 442) No. 443) 48 3 CRD San A32 G35 C17 T32 A.39 G28 C16 T32 2 6 6 Diego 202 31 G35 C17 T33 A.39 G28 C15 T33 1 28 28 (Clurd 30 G36 C17 T33 A.39 G28 C16 T32 3ND (CAue)P32 G35 C17 T32 A.39 G28 C16 T32 6 3 3 A.32 G35 C17 T32 A.39 G28 C16 T32 3 5,58 5 A.30 G36 C20 T30 A.39 G28 C15 T33 6 6 6 NHCSa 31 G35 C17 T33 A.39 G28 C15 T33 1 11 11 Diego- A.30 G36 C20 T30 A.39 G28 C16 T32 3 12 12 Archive 2003 A.31 G35 C17 T33 A.39 G28 C15 T33 1 22 22 (Cultured) A.31 G35 C17 T33 A.38 G29 C15 T33 3 25,75 75 A.30 G36 C17 T33 A39 G28 C15 T33 4 44/61,82,9 44/61 A.30 G36 C18 T32 A39 G28 C15 T33 2 53,91 91 A32 G35 C17 T32 A.39 G28 C16 T32 1 2 2 A.30 G36 C17 T33 A.39 G28 C15 T33 2 3 3 A.32 G35 C17 T32 A.39 G28 C16 T32 1 4 4 A31 G35 C17 T33 A.39 G28 C15 T33 1 6 6 Ft..31 G35 C17 T33 A.39 G28 C15 733 11 25 or 75 75 Weoad 203 A30 G36 C17 T33 A.39 G28 C15 733 25,75, 33, 1 34,4,52,84 75 (Cultured) A.30 G36 C17 T33 A.39 G28 C15 T33 44/61 or 82 1 or 9 44/61 A.30 G36 C18 T32 A.39 G28 C15 733 2 5 or 58 5 G36 C20 730 A39 G28 C15 T33 3 1 1 A30 G36 C18 T32 A39 G28 C15 T33 2 3 3Ft. Sill 203 32 G35 C17 T32 A.39 G28 C16 T32 1 4 4 (Cultured) A.31 G35 C17 T33 A.39 G28 C15 T33 1 28 28 G36 C17 T33 A.39 G28 C16 T32 1 3 3 A.32 G35 C17 732 A.39 G28 C16 T32 1 4 4 -A31 G35 C17 T33 A.39 G28 C15 T33 3 6 6 A.31 G35 C17 T33 A.39 G28 C15 T33 1 1 11 Ft. A.30 G36 C20 T30 A.39 G28 C16 T32 1 13 94** Benning 2003 A.30 G36 C19 T31 A.39 G28 C15 T33 44/61 or 82 (Cultured) I or 9 62 A.30 G36 C18 T32 A.39 G28 C15 T33 1 5 or 58 58 A.30 G36 C20 T30 A.39 G28 C15 T33 1 78 or 89 89 A.30 G36 C18 T32 A.39 G28 C15 T33 2 5 or 58 Lakiad A30 G36 C20 T30 A.39 G28 C15 T33 1 2 P.FB A.30 G36 C17 T33 A.39 G28 C15 T33 1 81 or 90 N 2003 A.30 G36 C17 T33 A.39 G28 C15 T33 1 78 (Throat P.30 G36 C18 T32 A.39 G28 C15 T33 Swabs) No detection detection No detection 7 3 ND A.32 G35 C17 T32 A.39 G28 C16 T32 1 3 ND MCRl San No detection No detection 1_3____Diego 2002 A.32 G35 C17 T32 A.39 G28 C16 T32 1 3 ND (Throat A.32 G35 C17 T32 No detection 2 3 ND Swabs) P.2 G35 C17 T32 No detection 3 No detection ND rNo detection No detection 00 -63- C 1 [0124] 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 s bioagent identifying amplicons corresponding to the primers of the broad surveillance set (Table I 4) and the Bacillus anthracis drill-down set (Table [0126] Calibration sequences were designed to simulate bacterial bioagent identifying amplicons 0 produced by the T modified primer pairs shown in Table 4 (primer names have the designation S "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 or reverse CR) primer name indicates the coordinates of an extraction representing a gene of a standard reference bacterial genome to which the primer hybridizes the forward primer name 16SEC_713 732 TMODF 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 !o sequence ofE. 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 as from molecular mass data (vide supra).

[0127] The 419 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 -64reaction 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 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 16SEC_1090 1111_2_T 5 16SEC_1175 1196 TMOD R 370 Bacillus 764 MOD F anthracls 346 16S EC 713732THOD_ 27 169_EC_789_809_TODR 389 BacIllus 765 F anthracis 347 16SEC_785_806TMDD_ 30 16SEC_880_897TODP 392 Bacillus 766 F anthracls 348 16SEC_960_981THOD_ 38 16S CC 1054_1073_THOO R 363 Bacillus 767 F anthracis 349 23S EC 1826 1843 TMO 49 23S BC 1906_1924_TMOD R 405 Bacillus 768 D C authzacis 360 23SEC_2646_2667TMO 60 239_EC_2745_2765TMOD R 416 Bacillus 769 I D F anthracis 350 CAECBA_274_303_TMOD 98 CAPC BA 349 376 TUOfR 452 Bacillus 770 F anthracls 351 CYABA 1353_1379 TMO 128 CYABA_1448_1467 TMODR 483 Bacillus 771 oF anthracis 352 INFB EC_1365_1393_TM 161 INFB BC 1439 1467 THOD 516 Bacillus 772 OD F R anthracls 353 LEFBA_756_781_TM0D_ 175 LEF BA 843_872_TMODR 531 Bacillus 773 I anthracis 356 RPLOEC_650_679_TMOD 232 RPLBBC_739_762_TMODB 592 Clostrldi um 774 F botulinum 449 RPLB EC_690_710_F 237 RPLBEC737_758_R 589 Clostrldlum 775 bocullnum 359 P0OBEC_1845_1866 TM 241 RPOBEC_1909_1929 71D 597 Yersinla 776 OD 0 F a Pea tin 362 RPOB-C 3799_3821 TN 245 RPOB BC 3862 3888 71OD 603 Burkholderla 777 OD F R mallei 363 RPOCEC_2146 2114_TM 257 RPOCEC_2227 2245 7140 621 Burkholderla 778 00 F mallei 354 RPOCEC_2218_2241_TM 262 RPOCEC_2313_2337TMOD 625 Bacillus 779 OD F R anthracla 355 SSPE BA 115 137 TMOD 321 SSPEBA_197 222 TMOD R 687 Bacillus 780 F r anthracis 367 TUFB EC957_979_THOD 345 TUFEqC1034 1058 TMOD 701 Burkholderia 781 F R mallel 358 VALSEC_1105 1124 TM 350 VALS BC 1195_1218_TMOD 712 Yerainia 782 O0 F R Fasts Table 10: Primer Pair Gene Coordinate References and'Calibration Polynucleotide Sequence Coordinates within the Combination Calibration Polynucleotide Bacterial Gene Gene Eseraction Coordinates Reference Geflank GI No. of Primer Pair Coordionles of Calibration and Species of Genomic or Plasmid Sequence Gnomic or Plaamid No. Sequence In Combination Sequence Calibration Polynucleodde (SEQ EDNO:783) 16S B. coil 4033120..4034661 16127994 346 16. .109 16S E. coll 4033120..4034661 16127994 GI 347 83. .190 16S E. coll 4033120..4034661 16127994 348 246..353 16S E. coil 4033120..4034661 16127994 361 368..469 23S B. coll 4166220..4169123 16127994 349 743. .837 23S E. Coil 4166220..4169123 16127994 360 865..981 rpoB Z. 4178823..4182851 16127994 359 1591..1672 Coll. (complemen strand) rpoB E. coll 4176823..4182851 16127994 362 2081..2167 (complement strand) rpoC coll 4182928..4187151 16127994 354 1810..1926 rpoC E. col 4182928..4187151 16127994 363 2183..2279 infB S. coll 3313655..3310983 16127994 352 1692..1791 (complement strand) tufB B. coll 4173523..4174707 16127994 367 2400..2498 rplB Z. Coil 3449001..3448180 16127994 (0 356 1945..2060 rplB E. coll 3449001. .3448180 16127994 449 1986..2055 valS S. coll 4481405..4478550 16127994 358 1462..1572 I_ (complement strand) I I

OC

C

C

(N

(N

OC

C

(N

i capC 56074..55628 (complement 6470151 350 2517..2616 B. anthracai strand) cya 156626..154288 4894216 351 1338..1449 B, anthracis (complement strand) lef 127442..129921 4894216 353 1121..1234 8. anthracis sspE 226496..226783 30253828 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 s 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 o 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) s 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 ;o 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 "CJSTCJ." Housekeeping genes to which the primers hybridize o3 and produce bioagent identifying amplicons include: tkt (transketolase), glyA (serine -66hydroxymethyltransferase), gitA (citrate synthase), aspA (aspartate ammonia lyase), ginA (glutamine synthase), pgm (phosphoglycerate mutase), and uncA (ATP synthetase alpha chain).

Table 11: Campylobacter Drill-down Primer Pairs Primer Fonvard Primer Name Forward Primer Reverse Primer Name Reverse Primer Target Gene Pair (SEQ ED NO:) (SEQ ED NO:) No. 1_ 1053 CJST CJ 10B0 1110 F 102 CJST CJ 1166 1198 R 456 qt 1064 CJST CJ 16B0 1713 F 107 CJST CJ 1795 1822 R 461 gy 1054 CJST CJ 2060 2090 F 109 CJST CJ 2148 2174 R 463 m 1049 CJST CJ 2636 2668 F 113 CJST CJ 2753 2777 R 467 tkt 1048 CJST CJ 360 394 F 119 CJST CJ 442 476 R 472 ap 1047 CJST CJ 584 616 F 121 CJST CJ 663 692 R 474 ln 101331 The primers were used to amplify nucleic acid from 50 food product samples provided by the USDA, 25 of which contained Campylobacterjejuni and 25 of which contained Campylobacter coli. Primers used in this study were developed primarily for the discrimination of Campylobacterjejuni clonal complexes and for distinguishing Campylobacterjejuni from Campylobacter ccli. Finer discrimination between Campylobacter coli types is also possible by using specific primers targeted to loci where closely-related Campylobacter coli isolates to 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 Drilldown MLST Primer Pair Nos: 1048 and 1047 Base Ease MLUST type or MSTyecomposition of Composition of Clnlor C lona Bioagent Biosgont Gop Seis Isolato Complex by o ColbySri Identifying Identifying Grioip Spasie Seqteain Amplicon IAmplicon Comostio aalyisObtained with Obtained with omoinanalysis Primer Pair No: Primer Pair (aaphl No; 1047 (ginA) J-1 C. Goose ST 690ST91 R37 A3G2C1T4 A7G1C6T5 jejuni /692/707/991 S 9 137 .0G5C616 P4 2 1 2 J7-2 C.na Complex ST 356, jejunl ua 206/46/353 complex RM44192 P.30 G25 C16 T46 A.48 G21 C17 T23 353 J~ Hu~man Complex ST 436 RM44194 A30 G25 C15 T47 A48 G21 C18 T22 Jelunl 3541179 ST 257, J-*4 C. Human Complex 257 complex RU44197 A30 G25 C16 T46 A.48 G21 C18 T22 jejunl 257 C. Human Complex 52 ST 52, R144277 A30 G25 C115 T46 A.46 G21 C17 T23 Iejuni complex 52 c.ST 51, RM4275 P.30 G25 C15 T47 A.48 G21 C17 T23 .7-6 C. Human Complex 443 complex M27 P.025CS47 .8G1C713 jejuni R47 3 2 1 4 4 2 1 2 J7 C. Hua ope 2 ST 604, RM41864 A.30 G25 C15 T47 A.48 G21 CIO T22 jejunl oe omlx4 complex 42 Hmn Complex ST 362, jejn 42.4H/ma2 complex 81M3193 A.30 G25 C15 T47 A.48 G21 CIO T22 J9 C. Human Complex ST 147, 8130 P30G5C547 .7G2Ci13 jejuni 45/263 Complex 45 M0 3 2 1 4 4 2 2 2 ejn Human Consistent ST 628 RM44183 A.31 827 C20 739 A46 G21 C16 124 67 with 74 closely related sequence types (none belong to a clonal complex) ST 832 I P11169 A31 G27 C20 T39 A48 G21 C16 T24 Poultry ST 1056 RM1857 A31 G27 C20 T39 A48 G21 C16 T24 ST 889 RM1166 A31 G27 C20 T39 A48 G21 C16 T24 ST 829 RM1182 A31 G27 C20 T39 A48 G21 C16 T24 ST 1050 RM1518 A31 G27 C20 T39 A48 G21 C16 T24 ST 1051 RM1521 A31 G27 C20 T39 A48 G21 C16 T24 ST 1053 RM1523 A31 G27 C20 T39 A48 G21 C16 T24 ST 1055 RM1527 A31 G27 C20 T39 A48 G21 C16 T24 ST 1017 RM1529 A31 G27 C20 T39 A48 G21 C16 T24 ST 860 RM1840 A31 G27 C20 T39 A48 G21 C16 T24 ST 1063 RM2219 A31 G27 C20 T39 A48 G21 C16 T24 ST 1066 RM2241 A31 G27 C20 T39 A48 G21 C16 T24 ST 1067 8M2243 P31 G27 C20 T39 A.48 G21 C16 T24 ST 1068 RM2439 A31 G27 C20 T39 A48 G21 C16 T24 ST 1016 R143230 A31 G27 C20 T39 A48 G21 C16 T24 ST 1069 RM3231 A31 G27 C20 T39 A48 G21 C16 T24 ST 1061 RM1904 A31 G27 C20 T39 A48 G21 C16 T24 ST 825 RH1534 A31 G27 C20 T39 A48 G21 C16 T24 C. coll Swine Unknown ST 901 RM1505 A31 G27 C20 T39 A48 G21 C16 T24 C-2 C. coll 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. coll 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 G2!1 C16 T24 clonal Marmoset complex) ST 891 RM1531 A31 G27 C20 T39 P48 G21 C16 T24 Table 12B -Results of Base Composition Analysis of 50 Campylobacter Samples with Drilldown MLST Primer Pair Nos: 1053 and 1064 Base Base MLST type or TComposition of Composition of or Cln Bioagont Bioagent Isolate Complex by Complexhb Strain Identifying Identifying Group Speies origin Base Cemple Aplicon Amplicon aquence Obtained with Obtained with analysis Primer Pair Primer Pair No: 1053 (gltA) No: 1064 (91yA) A.24 625 C23 T47 P.40 629 C29 J-1 C. Goose ST 690 ST 991 RM3673 jeJuni 1692/707/991 J-2aC Complex ST 356, A24 G25 C23 T47 A40 G29 C29 -2 .uman 206/48/353 complex RH4192 JeJuni 0353 C. Complex ST 436 PM4194 A24 G25 C23 T47 A40 G29 C29 J-3 jejuni Human 354/179 436 RM4194 C. ST 257, A24 G25 C23 T47 A40 G29 C29 Human Complex 257 complex RM4197 257 C. Human Complex 52 ST 52, RM4277 A24 G25 C23 T47 A39 G30 C26 T48 leiunl complex 52 C. ST 51, RM4275 A24 G25 C23 T47 A39 G30 C28 T46 J-6 jjunl Human Complex 443 complex 443 RM4279 A24 G25 C23 T47 A39 G30 C28 T46 A24 G25 C23 T47 R39 G30 C26 T48 C. ST 604, RM1864 ejunl Human Complex 42 complex 42 68 umC. Complex ST 362, A24 G25 C23 T47 IL38 G31 C28 T46 J- JeJul Human 42/49/362 complex RM3193 A24 G25 C23 T47 A38 G31 C28 T46 C. Complex ST 147, J-9 jeJunl Human 45/283 Complex 45 RM3203 C n ST 828 RM4183 A23 G24 C26 T46 A39 G30 C27 T47 Human ST 832 RM41169 A23 24 C26 T46 A39 G30 C27 T47 ST 1056 R41857 A23 G24 C26 T46 A39 G30 C27 T47 ST 889 RHI166 A23 G24 C26 T46 P39 G30 C27 T41 ST 829 R41182 A23 G24 C26 T46 A39 G30 C27 T47 ST 1050 RM1518 23 G24 C26 T46 P39 G30 C27 T47 ST 1051 RI521 PA23 G24 C26 T46 A39 G30 C27 T47 ST 1053 RM1523 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. Coll types (none ST 860 R1140 A.23 624 C26 T46 P.39 630 C27 T47 belong to aI clonal ST 1063 42 2.19 .A23 G24 C26 T46 A39 G30 C27 T47 ST 1066 RM2241 23 G24 C26 T46 .39 630 C27 T47 ST 1067 RM12243 A23 G24 C26 T46 A39 G30 C27 T47 ST 106 RM12439 A23 G24 C26 T46 A39 G30 C27 T47 ST 1016 RH3230 A23 G24 C26 T46 A39 G30 C27 T47 Swine ST 1069 M3231 P23 G24 026 T46 NO DATA ST 1061 R1904 P23 G24 C26 T46 A39 G30 C27 T47 ST 825 91534 A23 G24 C26 T46 A39 G30 C27 T47 ST 901 RM1505 A23 G24 C26 T46 A39 G30 C27 T47 C-2 C. Coll Human ST 895 ST 895 RM1532 A23 G24 C26 T46 P39 G30 C27 T47 Consistent ST 1064 RM2223 A23 G24 C26 T46 39 G30 C27 T47 with 63 closely ST 1082 RM1170 A23 G24 C26 T46 P39 G30 C27 T47 Poultry relatedP.3640547 P96002T7 C-3 C. coil sequence ST 1054 RM1525 A23 G24 C25 T47 A39 G30 C27 T47 types (none belong to a ST 1049 91517 A23 G24 C26 T46 A39 G30 C27 T47 Maoset c81 13 A23 G24 C26 T46 A39 G30 C27 147 Table 12C Results of Base Composition Analysis of 50 Campylobacter Samples with Drilldown MiST Primer Pair Nos: 1054 and 1049 13as Base k4LST type or MLTTp Compoition of Composition of closely T18 Rl7 Islt lnlor Clonal Bioagent flioagent Group Speeltex b3 Copdentifyng Identifying orioip Base oequence T y Sta l on plicon o aseComposition analysis Obtained with Obtained with Co analysis Priar Pair No: Primer Pair (Pgm) No: 1049 (tkt) J- Goose ST 690a jejuni /692/707/991 S 9 137 .6630818 P4 2 3 3 C. Humsn Complex ST 356, T 2 20/8/ complex 944192 A26 G33 C9 T37 PA41 G28 036 T37 353ano 206/8/35 353 na Jo Human Complex ST 436 RMn4194 Jai C. Goe T9_T9_3_ 354/179 A27 G332 C19 T37 A42 G28 C36 T36 00

U

00 69

C.

jejuni Human Complex 257 ST 257, complex 257 RM1117 A27 G32 C19 T37 A41 G29 C35 T37 C. ST 52, RM4277 jejuni Human Complex 52 complex 52 A26 G33 C18 T38 A41 G28 C36 T37 C. ST 51, RM4275 A27 G31 C19 T38 A41 G28 C36 T37 j-6 jejuni Human Complex 443 complex 443 RM4279 443__RM4279 A27 G31 C19 T38 A41 G28 C36 T37 j7 C. Human Complex 42 ST 604, RM1864 A27 G32 C19 T37 A42 G28 C35 T37 jejuni complex 42 ST 362, 8 C. n Complex complex RM3193 A26 G33 C19 T37 A42 G28 C35 T37 J ejun a 42/49/362 362 C. Complex ST 147, RM3203 A28 G31 C19 T37 A43 028 C36 J-9 jejuni Human 45/2B3 Complex 45 C. ST 828 RM4183 jejuni A27 G30 C19 T39 A46 G28 C32 T36 Human ST 832 RM1169 A27 G30 C19 T39 A46 G28 C32 T36 ST 1056 RM1657 A27 G30 C19 T39 A46 G28 C32 T36 ST 889 RM1166 A27 G30 C19 T39 A46 G28 C32 T36 ST 829 9941182 A27 G30 C19 T39 A46 G28 C32 T36 ST 1050 R91518 A27 G30 C19 T39 A46 G28 C32 T36 ST 1051 9941521 A27 G30 C19 T39 A46 G28 C32 T36' ST 1053 RM1523 A27 G30 C19 T39 A46 G28 C32 T36 Conaistent ST15 9452 with 741066 RM A27 G30 C19 T39 A46 G28 C32 T36 Poultry closely related ST 1017 RM41529 ST 108 H23 A27 G30 C19 T39 A46 G28 C32 T36 sequence C-i C. coil types (none ST 860 RM41840 belong to a R 0 I A27 G30 C19 T39 A46 G28 C32 T36 clonal ST 1063 9242219 complex) 11 R 4 A27 G30 C19 T39 A46 G28 C32 T36 ST 1066 RM42241 ST 901 _R_1505 A27 G30 C19 T39 A46 G28 C32 T36 ST 1067 9942243 C-2_ C o H n8S 9 M A27 G30 C19 T39 A46 G28 C32 T36 ST 1068 9942439 Consistent ST 1064 R22 A27 G30 C19 T39 A46 G28 C32 T36 ST 1016 9943230 630 019 T39 A46 G28 032 T36 Swine 6316 R33 Soiney ST 1069 RM1 IA27 G30 C19 T39 A46 G28 C32 T36 ST 1061 RM1904 types (none A27 G30 C19 T39 A46 G29 C32 T36 ST 825 RM1534 Unknown A27 G30 C19 T39 A46 G28 C32 T36 ST 901 9941505 A27 630 019 T39 A46 628 032 T36 0-2 C. coil Human ST 895 ST 895 99M1532 A2 63 01T9 A4 6902T3 comlex RM531 A27 G30 C19 T39 IA4 5 G29 C32 T36 ST 1064 9942223 Consistent method A27 630 019 T39 A45 029 032 T36 with 63 closly S 102 RMI78 A27 G30 019 T39 A45 629 032 T36 Poultry related 0-3 C. coil sequence ST 1054 99M1525 types (none 27 030 019 T39 A45 629 032 T36 belong to a ST 1049 9941517 clone1 27 G30 C19 T39 A45 629 032 T36 complex) Marmoset ST 891 9141531 630 019 T39 A45 629 032 T36 [01341 The base composition analysis method was successful in identification of 12 different strain groups. Campylobacter jejuni and Campylobacter coli are generally differentiated by all 0 0 0- loci. Ten clearly differentiated Campylobacterjejuni isolates and 2 major Campylobacter coli 0) groups were identified even though the primers were designed for strain typing of Campylobacterjejuni. One isolate (RM4183) which was designated as Campylobacterjejuni was found to group with Campylobacter coli and also appears to actually be Campylobacter coli by full MLST sequencing.

V) [0135] Example 12: Identification ofAcinetobacter baumannii Using Broad Range Survey Ci and Division-Wide Primers in Epidemiological Surveillance 0 [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 0o was identified in the remaining two samples. In addition, 14 different strain types (containing single nucleotide polymorphisms relative to a reference strain ofAcinetobacter 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.

as [01381 The epidemiology of strain type 7 ofAcinetobacter 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.

00 -71- C1 [0139] The epidemiology of strain type 3 ofAcinetobacter 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 0 0 methods of analysis of bacterial bioagent identifying amplicons provide the means for So0 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- Ss 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/mlst/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 -o include anthranilate synthase component I (trpE), adenylate kinase (adk), adenine glycosylase (mutY), 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 1155-1157 hybridize to and amplify segments of adk. Primer pair numbers 1158-1164 hybridize to and Ss 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 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.

-72- Table 13: MST 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 NOt) (SEQ ID NO:) 1151 AB MLST-11-OF007 62 91 F 83 AB MLST-11-OIF00 7 169 203 R 426 1152 AB MLST-11-OIF007 185 214 F 76 AB MLST-11-OIF007 291 324 Rt 432 1153 AB HLST-11-OF007 260 269 F 79 AB MLST-11-OIF007 364 393 R 434 1154 AB MLST-11-OIF007 206 239 F 78 AB MLST-11--1F007 318 344 f 433 1155 AR HLST-11-OIF007 522 552 F 80 AB HLST-11-01F007 587 610 f 435 1156 AR MLST-11-OIF007 547 571 F 81 AB MLST-11-OIF007 656 686 ft 436 1157 AB MLST-11-OIF007 601 627 F 82 AB MLST-11-OIF007 710 736 R 437 1158 ARMLST-11- OIFO0I 1202 1225 F AB NLST-11-FOI007 1266 1296 R 420 1159 ARMLS?-11- 01F007 1202 1225 F AB MLST-11-01F00 7 1299 1316 R 421 1160 ARMLST-11- 66 OIF007 1234 1264 F AR MLST-11-OIF007 1335 1362 R 422 1161 ARMLST-11- 67 OIFO07 1327 1356 F AB MLST-11-OIF007 1422 1448 Rt 423 1162 ARMLST-11- 68 OIF007 1345 1369 F AB MLST-11-IFDO 7 1470 1494 R 424 1163 ABRMLST-11- 69 OIF007 1351 1375 F AB HLST-11-01FD07 1470 1494 ft 424 1164 ABMLST-11- OIF007 1387 1412 F AR RLST-11-01F007 1470 1494 Rt 424 1165 ABk4LST-11- 71 01F007 1542 1569 F AB MLST-11-OIF007 1656 1680 R 425 1166 ARMIST-11- 72 01F007 1566 1593 F AR MLST-11-01F007 1656 1680 R 425 1167 AB KLST-11- 73 01F007 1611 1638 F AB HLST-11-01F007 1731 1757 f 427 1168 AB MLST-11- 74 OIF007 1726 1752 F AB HLST-11-OIF007 1790 1821 R 428 1169 ABMLST-11- OIF007 1792 1826 F AB MLST-11-OIF007 1876 1909 f 429 1170 AB HLST-11- O1F007 1792 1826 AB MLST-11-OIF007 1895 1927 f 430 1171 AB MLST-i1- 77 AB MLST-11-OIF007 2097 2118 R 431 oc

C

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-73- 01F007 1970 2002 F [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 28 .5 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 correlationllinkage.

[0144] All of the sample isolates were tested against a broad panel of antibiotics to characterize their antibiotic resistance profiles. As n example of a representative result from antibiotic susceptibility testing, STI 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 penicillins, extended spectrum penicillins, cephalosporins, carbipenem, protein synthesis inhibitors, nucleic acid synthesis inhibitors, anti-metabolites, and anti-cell membrane antibiotics.

S 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 0 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. Each reference (including, but not -6 limited to, journal articles, U.S. and non-U.S. patents, patent application publications, international patent application publications, gene bank accession numbers, internet web sites, and the like) cited in the present application is incorporated herein by reference in its entirety.

Claims (16)

1. An oligonucleotide primer selected from the group consisting of: an oligonucleotide primer 21 to 35 nucleobases in length comprising 70% to 100% sequence identity with SEQ ID NO: 97, an oligonucleotide primer 20 to 35 nucleobases in length comprising 70% to 100% sequence identity with SEQ ID NO: 451, an oligonucleotide primer 19 to 35 nucleobases in length comprising 70% to 100% sequence identity with SEQ ID NO: 127, an oligonucleotide C- primer 14 to 35 nucleobases in length comprising 70% to 100% sequence identity with SEQ ID NO: 482, an oligonucleotide primer 19 to 35 nucleobases in length comprising 70% to 100% 00 sequence identity with SEQ ID NO: 174, an oligonucleotide primer 21 to 35 nucleobases in 0 jo length comprising 70% to 100% sequence identity with SEQ ID NO: 530, an oligonucleotide primer 21 to 35 nucleobases in length comprising 70% to 100% sequence identity with SEQ ID NO: 310, an oligonucleotide primer 19 to 35 nucleobases in length comprising 70% to 100% sequence identity with SEQ ID NO: 668, an oligonucleotide primer 21 to 35 nucleobases in length comprising 70% to 100% sequence identity with SEQ ID NO: 313, an oligonucleotide primer 21 to 35 nucleobases in length comprising 70% to 100% sequence identity with SEQ ID NO: 670, an oligonucleotide primer 17 to 35 nucleobases in length comprising 70% to 100% sequence identity with SEQ ID NO: 277, an oligonucleotide primer 21 to 35 nucleobases in length comprising 70% to 100% sequence identity with SEQ ID NO: 632, an oligonucleotide primer 21 to 35 nucleobases in length comprising 70% to 100% sequence identity with SEQ ID .o NO: 285, an oligonucleotide primer 19 to 35 nucleobases in length comprising 70% to 100% sequence identity with SEQ ID NO: 640, an oligonucleotide primer 21 to 35 nucleobases in length comprising 70% to 100% sequence identity with SEQ ID NO: 301, an oligonucleotide primer 21 to 35 nucleobases in length comprising 70% to 100% sequence identity with SEQ ID NO: 656, an oligonucleotide primer 18 to 35 nucleobases in length comprising 70% to 100% as sequence identity with SEQ ID NO: 308, and an oligonucleotide primer 18 to 35 nucleobases in length comprising 70% to 100% sequence identity with SEQ ID NO: 663.

2. A composition comprising one or more of the oligonucleotide primers of claim 1. 3 0

3. A composition comprising two or more of the oligonucleotide primers of claim 1.

4. The composition of claim 3 wherein either or both of said oligonucleotide primers comprises at least one modified nucleobase. O C

5. The composition of claim 3 wherein either or both of said oligonucleotide primers comprises a non-templated T residue on the

6. The composition of claim 3 wherein either or both of said oligonucleotide primers comprises at least one non-template tag. V

7. The composition of claim 3 wherein either or both of said oligonucleotide primers N- comprises at least one molecular mass modifying tag. 00 3

8. A kit comprising the composition of claim 3.

9. The kit of claim 8 further comprising at least one calibration polynucleotide.

The kit of claim 8 further comprising at least one ion exchange resin linked to magnetic beads.

11. A method for identification of an unknown bacterium comprising: amplifying nucleic acid from said bacterium using the composition of claim 3 to obtain an amplification product; 0 o determining the molecular mass of said amplification product; optionally determining the base composition of said amplification product from said molecular mass; and comparing said molecular mass or base composition of said amplification product with a plurality of molecular masses or base compositions of known bacterial bioagent identifying as amplicons, wherein a match between said molecular mass or base composition of said amplification product and the molecular mass or base composition of a member of said plurality of molecular masses or base compositions identifies said unknown bacterium.

12. The method of claim 11 wherein said molecular mass is determined by mass spectrometry.

13. A method of determining the presence or absence of a bacterium of a particular clade, genus, species, or sub-species in a sample comprising: 00 -76- O amplifying nucleic acid from said sample using the composition of claim 3 to obtain an S amplification product; determining the molecular mass of said amplification product; N, optionally determining the base composition of said amplification product from said molecular mass; and I comparing said molecular mass or base composition of said amplification product with CN the known molecular masses or base compositions of one or more known clade, genus, species, or sub-species bioagent identifying amplicons, wherein a match between said molecular mass or 00 base composition of said amplification product and the molecular mass or base composition of S one or more known clade, genus, species, or sub-species bioagent identifying amplicons indicates the presence of said clade, genus, species, or sub-species in said sample. 1

14. The method of claim 13 wherein said molecular mass is determined by mass spectrometry.

A method for determination of the quantity of an unknown bacterium in a sample comprising: contacting said sample with the composition of claim 3 and a known quantity of a calibration polynucleotide comprising a calibration sequence; concurrently amplifying nucleic acid from said bacterium in said sample with the composition of claim 3 and amplifying nucleic acid from said calibration polynucleotide in said sample with the composition of claim 3 to obtain a first amplification product comprising a bacterial bioagent identifying amplicon and a second amplification product comprising a calibration amplicon; aS determining the molecular mass and abundance for said bacterial bioagent identifying amplicon and said calibration amplicon; and distinguishing said bacterial bioagent identifying amplicon from said calibration amplicon based on molecular mass, wherein comparison of bacterial bioagent identifying amplicon abundance and calibration amplicon abundance indicates the quantity of bacterium in said sample. -77- 00 O

16. The method of claim 15 further comprising determining the base composition 0 of said bacterial bioagent identifying amplicon. Dated 12 December, 2008 Ibis Biosciences, Inc. IN Patent Attorneys for the Applicant/Nominated Person SPRUSON FERGUSON 00