METHOD AND KIT FOR THE CHARACTERIZATION OF ANTIBIOTIC-RESISTANCE MUTATIONS IN MYCOBACTERIUM TUBERCULOSIS

Abstract: Amplification and cycle sequencing primer sets have been developed for the detection and analysis of antibiotic resistance-associated mutations in defined regions of the rpoB (rifampin), katG (isoniazid), oxyR-ahpC PR (isoniazid), mabA (isoniazid), rpsL/s12 (streptomycin), 16S/rrs (streptomycin), embB (ethambutol), pncA (pyrazinamide), gyrA (ciprofloxacin) and 23S (azithromycin) genes of <i>Mycobacterium tuberulosis</i>. These primers can be used in a method for detection and characterization of <i>Mycobacterium tuberculosis</i> present in a sample. The method includes the steps of obtaining a sputum sample suspected of containing M. tuberculosis, performing a first sequencing procedure, with or without prior amplification, on the sample to detect the presence of <i>M. tuberculosis</i>, and if present to evaluate the rpoB, katG, rpsL/s12 and 23S genes for the presence of antibiotic-resistance inducing mutations; and (c) if <i>M. tuberculosis</i> is detected in step (b), performing a second sequencing procedure, with or without prior amplification, on the sample to evaluate the additional genes for the presence of antibiotic-resistance inducing mutations.

Description: METHOD AND KIT FOR THE CHARACTERIZATION OF ANTIBIOTIC-RESISTANCE MUTATIONS IN MYCOBACTERIUM TUBERCULOSIS DESCRIPTION Field of the Invention This application relates to a method and kit for the characterization of antibiotic resistance mutations in Mycobacterium tuberculosis, and particularly to the evaluation of such mutations in clinical samples. Background of the Invention M. tuberculosis can be resistant to all antibiotics that are currently used to treat tuberculosis patients. Antibiotic resistance is due to acquired point mutations in target genes in the genome of M. tuberculosis. These point mutations render the organism insensitive to the action of the antibiotic by preventing it's uptake or activation, or by altering the antibiotic target. The observed antibiotic resistance in M. tuberculosis is not due to an episome-encoded resistance gene transferred from one strain to another and, like other bacteria, is single-step (one point mutation), high-level resistance. Rapid and accurate detection of antibiotic resistance in Mycobacterium tuberculosis in sputum samples would greatly improve both patient treatment and outcome . Presently, analysis of M. tuberculosis is carried out on DNA recovered from sputum samples handled according to Standard Infectious Disease/Public Health Laboratory practices. The sputum sample is decontaminated and a cell sediment isolated. This cell sediment is the sample source for all routine procedures used in the detection and isolation of M. tuberculosis. Portions of this sample are used in BacTec cultures for selective growth of tuberculosis, agar plate/agar slant cultures for . tuberculosis, acid-fast bacilli (AFB) smears for mycobacteria detection and molecular biological methods for the detection of M. tuberculosis and atypical mycobacteria. (See Fig. 1) Mycobacterial DNA is prepared directly from the decontaminated sputum cell sediments according to standard procedures and this mycobacterial DNA is used in the various molecular biological detection procedures. The methods presently in use for the detection of M. tuberculosis are either PCR-based or probe-based. These tests are used primarily on AFB smear-positive samples. Since the presence of M. tuberculosis has already been established by the AFB smear, these tests are used primarily in a confirmatory capacity as opposed to a diagnostic capacity. Furthermore, these tests provide no information on the potential antibiotic resistance of these M. tuberculosis samples. Below is a list of antibiotics used to treat M. Tuberculosis infections. The gene target of the specific antibiotic and regions associated with antibiotic resistance are listed, if known. The references on which the codon assignments are based are listed at the end of the application. 1. Rifampin rpoB gene codon 507-533 a 2. Isoniazid katG gene codon 275/315/328 b 3. Isoniazid mabA gene unknown c 4. Isoniazid oxyR-ahpC intergenic region (PR) nucleotides -48 to +33 5. Azithromycin 23 S rRNA sequence nucleotide 2568A e 6. Pyrazinamide pncA gene codon 47/85 f 7. Ethambutol embB gene codon 306 s 8. Streptomycin rpsL/sl2 gene codon 43/88 h 9. Streptomycin 16S/rrs sequence nucleotides 491, 512, 516, 513, 903, 904 10. Ciprofloxacin gyrA gene codon 88-95 j Probe-based tests do exist for the determination of rifampin resistance inM tuberculosis (line probe assay-InnoTek), but these probes rely on prior knowledge of antibiotic resistance-associated mutations in the rpoB gene. Mutations outside the region covered by the probe or new mutations not included in the probe cocktail could still confer resistance, but would not be detected using this product in it's present form. Thus, there remains a need for a method for detecting antibiotic-resistance mutations in clinical M. tuberculosis sputum samples which is capable of detecting mutations in all of the gene targets which confer antibiotic resistance. It is an object of the present invention to provide such a method. It is a further object of this invention to provide amplification and cycle sequencing primer sets, and kits containing such primer sets, for use in the characterization of antibiotic resistance mutations in M. tuberculosis. Summary of Invention Amplification and cycle sequencing primer sets have been developed for the detection and analysis of antibiotic resistance-associated mutations in defined regions of the rpoB (rifampin), katG (isoniazid), oxyR-ahpC PR (isoniazid), mabA (isoniazid), rpsL/sl2 (streptomycin), 16S/rrs (streptomycin), embB (ethambutol), pncA (pyrazinamide), gyrA (ciprofloxacin) and 23 S (azithromycin) genes. Using these primer sets and the OPENGENE™ automated DNA sequencing system, a protocol has been developed which permits both the rapid detection ofM tuberculosis and the identification of antibiotic resistance— associated mutations in a series of gene targets. The present invention uses a series of tests designed to detect antibiotic resistance-associated mutation in all gene targets for all antibiotics presently in use for the treatment of tuberculosis. The tests are employed in a hierarchical manner on both AFB smear-positive or smear-negative samples to determine both the presence and antibiotic-resistance of M. tuberculosis in a given sample. This method permits the simultaneous determination of M. tuberculosis presence in a sample and the antibiotic-resistance profile to an entire panel of antibiotics. Standard methods require from 2-6 weeks to culture M. tuberculosis and additional time to establish antibiotic resistance. Although DNA sequence-based (genotypic) tests are not intended to replace the traditional culture-based (phenotypic) methods, these tests do represent a rapid, sensitive and accurate protocol which provides clinicians with valuable information regarding antibiotic treatment options within days as opposed to weeks. Brief Description to the Figures Fig. 1 shows known testing protocols for M. tuberculosis; and Fig. 2 shows a hierarchical assay scheme for evaluating M. tuberculosis type in accordance with the invention. Detailed Description of the Invention In accordance with the invention, regions of the genome of tuberculosis associated with antibiotic resistance are amplified and sequenced using specifically designed amplification and sequencing primers. Various techniques for amplification are known, including the basic PCR amplification techniques described in US Patent No. 4,683,202, which is incorporated herein by reference. Similarly, various techniques for sequencing are know, some of which require prior amplification and some of which do not. Included among known sequencing techniques are those disclosed in US Patents Nos. 5,834,189 and 5,789,168, which are incorporated herein by reference. The primers of the invention can be used in any of these sequencing formats, although the invention is exemplified below using separate amplification and cycle-sequencing steps. In theory, the selection of primers to amplify and sequence a known region of interest should be straightforward. In fact, however, because of the possibility of primer binding to other sites, complications arising from secondary structure, and other factors which are not fully understood, some primers perform better than others for amplification and sequencing of the same region of interest. The present invention provides primers which have been optimized for the amplification and sequencing of regions associated with each of the ten known types of antibiotic resistance. These primer sets are shown below, along with the sequence of the genes that they are used to analyze. In the gene sequences, the locations of the primers are underlined. Primers rpoB (rifampin resistance) rpoB-F amplification primer, 20-mer, bp2201-2220 5' TAC GGT CGG CGA GCT GAT CC 3' SEQ. ID NO. 1 rpoB-R amplification primer, 20-mer, bp2611-2592 5' TAC GGC GTT TCG ATG AAC CC 3' SEQ ID NO. 2 rpoB-5s sequencing primer, 20-mer, bp2201-2220 5' TAC GGT CGG CGA GCT GAT CC 3' SEQ IDNO.3 rpoB-3s sequencing primer, 20-mer, bp2611-2592 5' TAC GGC GTT TCG ATG AAC CC 3' SEQ ID NO. 4 SEQ. ID. NO. 5 2161 aaaccgacga catcgaccac ttcggcaacc gccgcctgcg t--.i-ggt-.-gg.- g-.gri-gat-.-T- 2221 aaaaccagat ccgggtcggc atgtcgcgga tggagcgggt ggtccgggag cggatgacca 2281 cccaggacgt ggaggcgatc acaccgcaga cgttgatcaa catccggccg gtggtcgccg 2341 cgatcaagga gttcttcggc accagccagc tgagccaatt catggaccag aacaacccgc 2401 tgtcggggtt gacccacaag cgccgactgt cggcgctggg gcccggcggt ctgtcacgtg 2461 agcgtgccgg gctggaggtc cgcgacgtgc acccgtcgca ctacggccgg atgtgcccga 2521 tcgaaacccc tgaggggccc aacatcggtc tgatcggctc gctgtcggtg tacgcgcggg 2581 tcaacccgtt cgggttcatc gaaacgccgt accgcaaggt ggtcgacggc gtggttagcg katG (isoniazid resistance) katG-F amplification primer, 20-mer, bp722-741 5ΑTG GGG CTG ATC TAC GTG AA 3' SEQ ID NO. 6 katG-R amplification primer, 20-mer, bpl250-1231 5' GGT GTT CCA GCC AGC GAC GC 3' SEQ ID NO. 7 katG-5s sequencing primer, 20-mer, bp722-741 5ΑTG GGG CTG ATC TAC GTG AA 3' SEQ ID NO. 8 katG-3s sequencing primer, 20-mer, bpl250-1231 5' GGT GTT CCA GCC AGC GAC GC 3 ' SEQ ID NO. 9 SEQ ID NO. 10 661 gctcggcgat gagcgttaca gcggtaagcg ggatctggag aacccgctgg ccgcggtgca 721 gatggggctg atctacgtga acccggaggg gccgaacggc aacccggacc ccatggccgc 781 ggcggtcgac attcgcgaga cgtttcggcg catggccatg aacgacgtcg aaacagcggc 841 gctgatcgtc ggcggtcaca ctttcggtaa gacccatggc gccggcccgg ccgatctggt 901 cggccccgaa cccgaggctg ctccgctgga gcagatgggc ttgggctgga agagctcgta 961 tggcaccgga accggtaagg acgcgatcac cagcggcatc gaggtcgtat ggacgaacac 1021 cccgacgaaa tgggacaaca gtttcctcga gatcctgtac ggctacgagt gggagctgac 1081 gaagagccct gctggcgctt ggcaatacac cgccaaggac ggcgccggtg ccggcaccat 1141 cccggacccg ttcggcgggc cagggcgctc cccgacgatg ctggccactg acctctcgct 1201 gcgggtggat ccgatctatg agcggatcac gr.gr r.gr.t.gg c ggaacac ccgaggaatt 1261 ggccgacgag ttcgccaagg cctggtacaa gctgatccac cgagacatgg gtcccgttgc oxyR-aphC intergenic region (PR) PR-F amplification primer, 20-mer, bp451-470 5' ACC ACT GCT TTG CCG CCA CC 3' SEQ ID NO.11 PR-R amplification primer, 20-mer, bp687-668 5' CCG ATG AGA GCG GTG AGC TG 3' SEQ ID NO.12 PR-5s sequencing primer, 20-mer, bp451-470 5' ACC ACT GCT TTG CCG CCA CC 3' SEQ ID NO.13 PR-3s sequencing primer, 20-mer, bp687-668 5' CCG ATG AGA GCG GTG AGC TG 3' SEQ ID NO. 14 SEQ ID NO. 15 361 atgccctggg ggtgcaccga gaccggcttc cgaccaccgc tcgccgcaac gtcgactggc 421 tcatatcgag aatgcttgcg gcactgctga accaπf.σctt tσccσc acc gcggcgaacg 481 cgcgaagccc ggccacggcc ggctagcacc tcttggcggc gatgccgata aatatggtgt 541 gatatatcac ctttgcctga cagcgacttc acggcacgat ggaatgtcgc aaccaaatgc 601 attgtccgct ttgatgatga ggagagtcat gccactgcta accattggcg atcaattccc 661 cgcctaccaσ ctcaccαctc tcatcσσcασ tgacctgtcc aaggtcgacg ccaagcagcc 721 cggcgactac ttcaccacta tcaccagtga cgaacaccca ggcaagtggc gggtggtgtt mabA (isoniazid resistance) mabA-F amplification primer, 20-mer, bp56-75 5' CCT CGC TGC CCA GAA AGG GA 3' SEQ ID NO. 16 mabA-R amplification primer, 20-mer, bp303-284 5' ATC CCC CGG TTT CCT CCG GT 3' SEQ LD NO. 17 mabA-5s sequencing primer, 20-mer, bp56-75 5' CCT CGC TGC CCA GAA AGG GA 3' SEQ LD NO. 18 mabA-3s sequencing primer, 20-mer, bp303-284 5' ATC CCC CGG TTT CCT CCG GT 3' SEQ ID NO.19 SEQ ID NO.20 1 agcgcgacat acctgctgcg caattcgtag ggcgtcaata cacccgcagc c.aagar.r. r.g 61 rtαrc agaa aααgat.ccα catggtcgaa gtgtgctgag tcacaccgac aaacgtcacg 121 agcgtaaccc cagtgcgaaa gttcccgccg gaaatcgcag ccacgttacg ctcgtggaca 181 taccgatttc ggcccggccg cggcgagacg ataggttgtc ggggtgactg ccacagccac 241 tgaaggggcc aaacccccat tcgtatcccg ttcagtcctg g accggag gaaarrgggg 301 gatcααqctq gcgatcgcac agcggctggc tgccgacggc cacaaggtgg ccgtcaccca rpsL/sl2 (streptomycin resistance) sl2-F amplification primer, 20-mer, bpl-20 5' CGG TAG ATG CCA ACC ATC CA 3' SEQ ID NO.21 sl2-R amplification primer, 20-mer, bp384-365 5' GCA TCA GCC CTT CTC CTT CT 3' SEQ ID NO 22 sl2-5s sequencing primer, 20-mer, bpl-20 5' CGG TAG ATG CCA ACC ATC CA 3' SEQ ID NO.23 sl2-3s sequencing primer, 20-mer, bp384-365 5' GCA TCA GCC CTT CTC CTT CT 3' SEQ LD NO.24 SEQ ID NO.25 1 rggtagatgc caaccar.cca gcagctggtc cgcaagggtc gtcgggacaa gatcagtaag 61 gtcaagaccg cggctctgaa gggcagcccg cagcgtcgtg gtgtatgcac ccgcgtgtac 121 accaccactc cgaagaagcc gaactcggcg cttcggaagg ttgcccgcgt gaagttgacg 181 agtcaggtcg aggtcacggc gtacattccc ggcgagggcc acaacctgca ggagcactcg 241 atggtgctgg tgcgcggcgg ccgggtgaag gacctgcctg gtgtgcgcta caagatcatc 301 cgcggttcgc tggatacgca gggtgtcaag aaccgcaaac aggcacgcag ccgttacggc 361 gctaagaagg agaagggctg atgccacgca aggggcccgc gcccaagcgt ccgttggtca 16S/rrs (streptomycin resistance) 16S-F amplification primer, 21-mer, bp5-25 5' GGT GAT CTG CCC TGC ACT TCG 3' SEQ LD NO.26 16S-R amplification primer, 21-mer, bpl47-127 5' CGT CAC CCC ACC AAC AAG CTG 3' SEQ ID NO.27 16S-5s sequencing primer, 21-mer, bp5-25 5' GGT GAT CTG CCC TGC ACT TCG 3' SEQ ID NO.28 16S-3s sequencing primer, 21-mer, bpl47-127 5' CGT CAC CCC ACC AAC AAG CTG 3' SEQ ID NO. 29 SEQ ID NO. 30 1 cgtgggtgat ctgccctgca cttcgggata agcctgggaa actgggtcta ataccggata 61 ggaccacggg atgcatgtct tgtggtggaa agcgctttag cggtgtggga tgagcccgcg 121 grrtat-f-π r ttgttggtgg ggt.ga r.g embB (ethambutol resistance) embB-F amplification primer, 21-mer, bp7761-7781 5' CGG CAA GCT GGC GCA CCT TCA 3' SEQ ID NO.31 embB -R amplification primer, 21-mer, bp8040-8020 5' AGC CAG CAC ACT AGC CCG GCG 3 SEQ LD NO.32 embB-5s sequencing primer, 21-mer, bp7761-7781 5' CGG CAA GCT GGC GCA CCT TCA 3' SEQ ID NO.33 embB-3s sequencing primer, 21-mer, bp8040-8020 5' AGC CAG CAC ACT AGC CCG GCG 3 SEQ LD NO. 34 SEQ LD NO. 35 7741 cggcatgcgc cggctgattc rggraagrtg grgr.ar.r.t r ar.r.r.r.gar.r.g acgccgtggt 7801 gatattcggc ttcctgctct ggcatgtcat cggcgcgaat tcgtcggacg acggctacat 7861 cctgggcatg gcccgagtcg ccgaccacgc cggctacatg tccaactatt tccgctggtt 7921 cggcagcccg gaggatccct tcggctggta ttacaacctg ctggcgctga tgacccatgt 7981 cagcgacgcc agtctgtgga tgcgcctgcc agar.-tgg.-r- grrgggrtag tgt-grt-ggrf- pncA (pyrazinamide resistance) pncA-F amplification primer, 20-mer, bpl-20 5' ATG CGG GCG TTG ATC ATC GT 3' SEQ ID NO.36 pncA-F amplification primer, 20-mer, bp561-542 5' TCA GGA GCT GCA AAC CAA CT 3' SEQ ID NO.37 pncA-5s sequencing primer, 20-mer, bpl-20 5' ATG CGG GCG TTG ATC ATC GT 3' SEQ ID NO.38 pncA-3s sequencing primer, 20-mer, bp561-542 5' TCA GGA GCT GCA AAC CAA CT 3' SEQ ID NO. 39 SEQ ID. NO. 40 1 atgcgggcgt tgatcatcgt cgacgtgcag aacgacttct gcgagggtgg ctcgctggcg 61 gtaaccggtg gcgccgcgct ggcccgcgcc atcagcgact acctggccga agcggcggac 121 taccatcacg tcgtggcaac caaggacttc cacatcgacc cgggtgacca cttctccggc 181 acaccggact attcctcgtc gtggccaccg cattgcgtca gcggtactcc cggcgcggac 241 ttccatccca gtctggacac gtcggcaatc gaggcggtgt tctacaaggg tgcctacacc 301 ggagcgtaca gcggcttcga aggagtcgac gagaacggca cgccactgct gaattggctg 361 cggcaacgcg gcgtcgatga ggtcgatgtg gtcggtattg ccaccgatca ttgtgtgcgc 421 cagacggccg aggacgcggt acgcaatggc ttggccacca gggtgctggt ggacctgaca 481 gcgggtgtgt cggccgatac caccgtcgcc gcgctggagg agatgcgcac cgccagcgtc 541 gagM-ggtt-t- gragrtrrtg a gyrA (fluoroquinilone/ciprofloxacin resistance) gyrA-F amplification primer, 20-mer, bp2383-2402 5' CAG CTA CAT CGA CTA TGC GA 3' SEQ ID NO.41 gyrA-R amplification primer, 20-mer, bp2702-2683 5' GGG CTT CGG TGT ACC TCA TC 3' SEQ LD NO. 42 gyrA-5s sequencing primer, 20-mer, bp2383-2402 5' CAG CTA CAT CGA CTA TGC GA 3' SEQ LD NO. 43 gyrA-3s sequencing primer, 20-mer, bp2702-2683 5' GGG CTT CGG TGT ACC TCA TC 3' SEQ ID NO. 44 SEQ ID NO. 45 2341 cgaccggatc gaaccggttg acatcgagca ggagatgcag cgcagctaca tcgactatgc 2401 .gatgagcgtg atcgtcggcc gcgcgctgcc ggaggtgcgc gacgggctca agcccgtgca 2461 tcgccgggtg ctctatgcaa tgttcgattc cggcttccgc ccggaccgca gccacgccaa 2521 gtcggcccgg tcggttgccg agaccatggg caactaccac ccgcacggcg acgcgtcgat 2581 ctacgacagc ctggtgcgca tggcccagcc ctggtcgctg cgctacccgc tggtggacgg 2641 ccagggcaac ttcggctcgc caggcaatga cccaccggcg gr.ga gaggt- acarrgaagc 2701 ccgαctαacc ccgttggcga tggagatgct gagggaaatc gacgaggaga cagtcgattt 23S (macrolide/azithromycin resistance) 23S-F amplification primer, 20-mer, bp2444-2463 5' CGA AAT TCC TTG TCG GGT AA 3' SEQ ID NO. 46 23S-R amplification primer, 20-mer, bp2683-2664 5' GTA TTT CAA CAA CGA CTC CA 3' SEQ ID NO. 47 23S-5s sequencing primer, 20-mer, bp2444-2463 5' CGAAAT TCC TTG TCG GGT AA 3' SEQ ID NO.48 23S-3s sequencing primer, 20-mer, bp2683-2664 5' GTA TTT CAA CAA CGA CTC CA 3' SEQ ID NO. 49 SEQ ID NO. 50 2401 gccccagtaa acggcggtgg taactataac catcctaagg t agrgaaatt r.rt r.gt r.ggg 2461 taaαttccαa cctgcacgaa tggcgtaacg acttcccaac tgtctcaacc atagactcgg 2521 cgaaattgca ctacgagtaa agatgctcgt tacgcgcggc aggacgaaaa gaccccggga 2581 ccttcactac aacttggtat tggtgttcgg tacggtttgt gtaggatagg tgggagactt 2641 tgaagcacag acgccagttt gt-gt-ggagt-c gt tgt r.gaaa tarcac r g atcgtattgg To facilitate detection of the sequencing products using real-time fluorescence- based electrophoresis apparatus (for example, a Nisible Genetics OPEΝGEΝE™ sequencer), at least one of the sequencing primers is preferably labeled with a flourescent label. The label is selected for compatibility with the sequencing apparatus employed, and may be, for example, fluorescein or a cyanine dye such as CY5.0 OR CY5.5. The primers of the invention are suitably packaged in a kit. This kit will contain individually packaged amplification and sequencing primers sets for each resistance gene to be evaluated by the kit. Thus, the kit of the invention includes at least 4 primers (two amplification and two sequencing primers), and preferably includes the primer sets for a plurality of resistance genes, most preferably the primer sets for all ten resistance genes. The suitable protocol for the utilization of these primer sets in the evaluation of tuberculosis in clinical samples utilizes PCR amplification, followed by cycle sequencing. DNA for use in the test is obtained from a sample of sputum (lOOul-lOml). The sputum sample is processed according to Standard Infectious Disease/Public Health Laboratory practices (Mycobacteriology Bench Manual, Laboratory Services Branch, December 1997, Ontario Ministry of Health). The sputum sample is homogenized, decontaminated and concentrated. Mycobacterial DNA is prepared directly from a portion of the concentrated cell sediment (100-200ul) using standard DNA extraction methods or commercially available kits. Amplification of the DNA is performed using the amplification primer sets described above. PCR reagents can be prepared for individual reactions, or may be prepared as a master mix which can be used for multiple tests e.g., 10 PCR reactions. Exemplary combinations of reagents are summarized in the following table. PCR mix 1 PCR 10 PCRs final cone. / PCR genomic DNA (20ng/ul) l.Oul 20ng (~0.5fM) 10X PCR buffer I 2.5ul 25.0ul IX 2.5mM dNTP mix (1 : 1 : 1 : 1) 2.5ul 25.0ul 250uM DMSO 1.3ul 13.0ul 5% Taq DNA polymerase (1U) 0.2ul 2.0ul 1 unit molecular grade watei 16.5u 165.0ul MTB gene primers (lOuM) l.Oul lO.Oul 1 Opmol per primer total volume per PCR 25.0ul If the master mix as shown in the column labeled 10 PCRs is utilized, the mastermix contains all the necessary PCR reagents other than the genomic DNA. In this example, 24.0ul of the mastermix is added to a PCR tube, that already contains 1.Oul of genomic DNA, prior to the addition of the mineral oil overlay and placement in the thermocycler. The genomic DNA preparation utilized must be of sufficient quality and integrity for robust and reproducible PCR. Suitable DNA preparation can be obtained using the Gentra Puregene™ DNA isolation kit. The kit components are appropriate for the isolation of genomic DNA from blood, fresh or frozen tissue, archival material and paraffin- embedded tissue. Each primer pair is used to amplify a single gene region under the following conditions: 1. Denaturation 94°C 5 minutes 1 cycle 2. Denaturation 94°C 30 seconds Annealing 60°C 30 seconds 35 cycles Extension 72°C 60 seconds 3. Extension 72°C 5 minutes 1 cycle 4. Hold 6°C The temperature change during the cycles of the step 2 is desirably set to ramp at a rate of C/sec. After amplification, 2.0ul from the 25.0ul PCR is analysed for purity on a 0.8% agarose gel. Samples displaying single PCR product bands can be used directly for sequence analysis. The yield and purity of the PCR product determines the amount to be used in the subsequent cycle sequencing reaction. Comparable verification of sequencing purity is performed on each of the other amplification products. Sequence analysis is carried out on the amplified product. The basic procedures and conditions are the same for each region. Accordingly, the invention will be exemplified using the rpoB gene. For initial sequence analysis of rpoB, the rpoB-5s primer should be used. For confirmatory sequence analysis the rpoB-3s primer should be used. For each template to be sequenced, aliquot 3.0ul of each of the nucleotide termination mixes into four separate tubes marked <A>, <C> <G> and <T> and store on ice until the sequencing mastermix is prepared. Cycle sequencing mastermix rpoB template 2.0ul 10X VGI Sequenace ™ buffer 2.5ul DMSO 3.5ul 2.5uM dye-sequencing primer 2.0ul PCR grade water 9.0ul 1 10 diluted Thermosequenase ( ul total volume 22.0ul Mix the DMSO and other components in the mastermix well by repeated pipetting (5 times) with a micropipette. Store the mastermix on ice until ready to add to the nucleotide termination mixes. Add 5.0ul of the mastermix to each of the four marked tubes containing the nucleotide termination mixes. Add 8.0ul lightweight mineral oil to each of the four marked tubes containing the mastermix and nucleotide termination mixes. Store on ice until ready to load into the thermocycler. 1. Denaturation 94°C 5 minutes IX 2. Denaturation 94°C 30 seconds Annealing 60°C 30 seconds 35X Extension 72°C 60 seconds 3. Extension 72°C 5 minutes IX 4. Hold 6°C The temperature change during the cycles of the step 2 is desirably set to ramp at a rate of l°C/sec. At the end of the cycle sequencing reaction add 6.0ul of the Stop Loading Dye directly to each of the four tubes to stop the sequencing reaction. The sequencing samples are heated at 95°C for 2 minutes and then placed on ice before loading 2.0ul (from a total volume of 14ul) on the CLIPPER™ sequencer. The remainder of the sequencing reaction can be stored at -20°C for subsequent use. The CLIPPER™ sequencer is set-up as described in the OPENGENE Automated DNA Sequencing System User Manual. Run parameters for the CLIPPER™ sequencer are 54°C/ 1300volts/ 0.5sec sampling/35 min run/50% laser power. The samples loaded included 2 ul each of the forward and reverse sequencing reaction products for the target gene, differentially labeled, for example with CY5.0 and CY5.5 cyanine dye labels. Once the run is completed, the base-called data is analysed by comparison of the test sequence to the rpoB sequence database in GENELLBRARIAN™. This sequence alignment compares the test sequence to the standard control sequence and allows sequence ambiguities to be assessed. Once edited the test sequence can be screened for antibiotic resistance- associated mutations using GENELLBRARIAN™. Testing for multiple types of antibiotic-resistance mutations can be carried out using a hierarchical assay, as summarized in Fig. 2. At present molecular biological methods for the detection of tuberculosis are only performed on AFB smear-positive sputum samples. These methods serve as confirmatory tests for the presence ofM tuberculosis. In addition to these molecular biological methods, the culture-based procedures forM tuberculosis detection (BacTec liquid culture, agar plate and slant cultures) are performed in parallel. AFB smear-negative sputum samples are processed with only the culture-based detection procedures (Figure 1). In the present invention both AFB smear-positive and smear-negative sputum samples can be processed using both culture-based and molecular biological methods. A limitation of the AFB stain methodology is it's limit of detection. If a sputum sample has a mycobacterial concentration of less than 5000 bacteria/ul the AFB stain will be negative. In addition to this is the observation that the decontamination procedure used to prepare the sputum sample usually kills 10-20% of the mycobacteria present. This would suggest that two-thirds of the AFB smear-negative samples potentially contain mycobacteria. In practice 10-20%> of the AFB smear-negative samples are culture-positive forM tuberculosis (Ontario Public Health Laboratory). This level of mycobacteria is easily detected by molecular biological methods and is therefore incorporated in the present invention. The hierarchy proposed incorporates tests that specifically detect M. tuberculosis (rpoB), detect mutations in genes associated with resistance to the "first-line" antibiotics used to treat M. tuberculosis infections (rpoB, katG, rpsL/sl2, PR, embB, pncA) and detect other species of mycobacteria (23 S) in the absence ofM tuberculosis (Figure 2). Group I analyses are performed before both Group II and Group III. Group I analysis will provide information on the antibiotic resistance status to rifampin (rpoB), isoniazid (katG), streptomycin (rpsL/sl2) and azithromycin (23 S). In addition the rpoB amplification indicates the presence ofM tuberculosis and in the absence of rpoB amplification the 23 S sequence allows identification of most of the clinically relevant mycobacterial species. Group II analysis provides information on antibiotic resistance mutations in the "second-line" antibiotics used to treat M tuberculosis infections namely, isoniazid (PR), ethambutol (embB), pyrazinamide (pncA) and ciprofloxacin (gyrA). Group III contains gene targets in which mutations associated with antibiotic resistance are infrequently found. This protocol permits specific gene targets to be examined according to the local treatment procedures since the both antibiotics used to treat M tuberculosis infections, and thus the associated antibiotic resistance mutation patterns, vary geographically. As shown in Figure 2 the culture-based methods are performed in parallel. The molecular biological methods would permit the identification ofM tuberculosis from both AFB smear- positive and smear-negative sputum samples and further provide information on the antibiotic resistance profile of these samples well in advance of current culture-based methods. This information would be crucial to the initiation of appropriate and effective antibiotic treatment regimens forM tuberculosis infections. Examples A pool of DNA samples from antibiotic-sensitive M tuberculosis isolates was obtained from the LCDC, Health and Welfare Canada. Ottawa, Ontario. Wild-type sequence traces, for all gene targets known to harbor mutations in antibiotic-resistant M tuberculosis, were generated. A panel of DNA samples from five phenotypic streptomycin-resistant M tuberculosis isolates was obtained from the Public Health Laboratory, Ontario Ministry of Health, Toronto, Ontario. These DNA samples were examined for antibiotic resistance- associated mutations in all 10 antibiotic gene targets listed above. Streptomycin resistance- associated mutations were detected in the rpsL/sl2 gene in four isolates. Parallel antibiotic resistance-associated mutations in the rpoB (rifampin), katG (isoniazid), PR (isoniazid), embB (ethambutol), pncA (pyrazinamide) and gyrA (ciprofloxacin) genes were also identified which underscores the importance of examining all the gene targets for first-line antibiotics used in the treatment ofM tuberculosis. A summary of the results is shown in Table 1. The following references are cited herein and are incorporated herein by reference for all states which allow such incorporation. a DL Williams et al. (1994). Characterisation of rifampin resistance in pathogenic mycobacteria. Antimicrob Agents Chemother 38: 2380-2386. b WH Haas et al. (1997). Molecular analysis of katG gene mutations in strains of Mycobacterium tuberculosis complex from Africa. Antimicrob Agents Chemother 41: 1601-1603. c S Sreevatsan et al. (1997). Analysis of the oxyR-ahpC region in isoniazid- resistant and -susceptible Mycobacterium tuberculosis complex organisms recovered from diseased humans and animals in diverse localities. Antimicrob Agents Chemother 41: 600-606. d A Telenti et al. (1994). Genotypic assessment of isoniazid and rifampin resistance in Mycobacterium tuberculosis: a blind study at the reference laboratory level. Antimicrob Agents Chemother 35: 719-723. e C Katsukawa et al. (1997). Characterisation of the rpsL and rrs genes of streptomycin-resistant clinical isolates of Mycobacterium tuberculosis in Japan. J Appl Microbiol 83: 634-640. C Katsukawa et al. (1997). Characterisation of the rpsL and rrs genes of streptomycin-resistant clinical isolates of Mycobacterium tuberculosis in Japan. J Appl Microbiol 83: 634-640. MA Lety et al. (1997). A single point mutation in the embB gene is responsible for resistance to ethambutol in Mycobacterium smegmatis. Antimicrob Agents Chemother 41: 2629-2633. h A Scorpio et al. (1997). Characaterisation of pncA mutations in pyrazinamide- resistant Mycobacterium tuberculosis. Antimicrob Agents Chemother 41: 540- 543. C Xu et al. (1996). Fluoroquinilone resistance associated with specific gyrase mutations in clinical isolates of multidrug-resistant Mycobacterium tuberculosis. J Infect Disease 174: 1127-1130. KA Nash et al. (1995). Genetic basis of macrolide resistance in Mycobacterium avium isolated from patients with disseminated disease. Antimicrob Agents Chemother 39: 2625-2630. OPH#1 OPH#2 OPHB3 OPH#4 0PH#11 bp/codon/aa bp/codon/aa bptcodon/aa bp/codon/aa bp/codon/aa gene (antibiotic) rpoB (rifampin) cac526tac, His526Tyr tcg553ttg, Ser553Leu cac526gac. His526Asp tcg553Ug, Ser553Leu w t katG.1 (isoniazid) agc513acc, Ser513Thr agc513acc, Ser513Thr agc513acc, Ser513Thr wt wt oxyR-ahpC PR (isoniazid) g541a wt wt wt g541a fabG (isoniazid) wt wt wt wt t rpsL/s12 (streptomycin) t aag43agg, Lys43Arg aag43agg, Lys43Arg aagδδagg, Lys88Arg aag43agg, Lys43Arg 16s/rrs (streptomycin) t wt wt wt wt embB (ethambutol) t gtc292ttc, va!292phe wt wt pncA (pyrazinamide) tcc65tct. Ser65Ser wt att133aat, lle133Asn t tcc65tct, Ser65Ser gyrA (ciprofloxacin) agc95acc, Se.95Thr agc95acc, Ser95Thr agc95acc, Ser95Thr agc95acc. Ser95T r agc95acc, Ser95Thr 23s (azithromycin) t wt t wt wt

Claims: - 18 - CT.ATMS 1. A method for detection and characterization of Mycobacterium tuberculosis present in a sample, comprising the steps of: (a) obtaining a sputum sample suspected of containing M tuberculosis, (b) performing a first sequencing procedure, with or without prior amplification, on the sample to detect the presence ofM tuberculosis, and if present to evaluate the rpoB, katG, rpsL/sl2 and 23 S genes for the presence of antibiotic-resistance inducing mutations; and (c) if M tuberculosis is detected in step (b), performing a second sequencing procedure, with or without prior amplification, on the sample to evaluate the additional genes for the presence of antibiotic-resistance inducing mutations. 2. The method of claim 1, wherein the second sequencing procedure evaluates PR, embB pncA and gyrA genes for the presence of antibiotic-resistance mutations. 3. The method of claim 3, further comprising the step of performing a third sequencing procedure whenM tuberculosis was detected in step (b) to evaluate 16S/rrs and mabA genes for the presence of antibiotic-resistance mutations. 4. The method of any of claims 1 to 3, wherein the first sequencing procedure for rpoB is performed using amplification primers as set forth in Seq. ID Nos. 1 and 2 and sequencing primers as set forth in Seq. ID. Nos. 3 and 4. 5. The method of any of claims 1 to 4, wherein the first sequencing procedure for katG is performed using amplification primers as set forth in Seq. ID Nos. 6 and 7 and sequencing primers as set forth in Seq. ID. Nos. 8 and 9. 6. The method of any of claims 1 to 5, wherein the first sequencing procedure for rpsL/sl2 is performed using amplification primers as set forth in Seq. ID Nos. 21 and 22 and sequencing primers as set forth in Seq. ID. Nos. 23 and 24. - 19 - 7. The method of any of claims 1 to 6, wherein the second sequencing procedure for 23 S is performed using amplification primers as set forth in Seq. ID Nos. 46 and 47 and sequencing primers as set forth in Seq. ID. Nos. 48 and 49. 8. The method of any of claims 1 to 7, wherein the second sequencing procedure for PR is performed using amplification primers as set forth in Seq. ID Nos. 11 and 12 and sequencing primers as set forth in Seq. ID. Nos. 13 and 14. 9. The method of any of claims 1 to 8, wherein the second sequencing procedure for pncA is performed using amplification primers as set forth in Seq. ID Nos. 36 and 37 and sequencing primers as set forth in Seq. ID. Nos. 38 and 39. 10. The method of any of claims 1 to 9, wherein the second sequencing procedure for embB is performed using amplification primers as set forth in Seq. ID Nos. 31 and 32 and sequencing primers as set forth in Seq. ED. Nos. 33 and 34. 11. The method of any of claims 1 to 10, wherein the second sequencing procedure for gyrA is performed using amplification primers as set forth in Seq. ID Nos. 41 and 42 and sequencing primers as set forth in Seq. ID. Nos. 43 and 44. 12. The method of any of claims 2 to 11, wherein the third sequencing procedure for 16S/rrs is performed using amplification primers as set forth in Seq. ID Nos. 26 and 27 and sequencing primers as set forth in Seq. ED. Nos. 28 and 29. 13. The method of any of claims 2 to 12, wherein the third sequencing procedure for mabA is performed using amplification primers as set forth in Seq. ID Nos. 16 and 17 and sequencing primers as set forth in Seq. ID. Nos. 18 and 19. 14. A kit for evaluation of antibiotic-resistance mutations in a sample of Mycobacterium tuberculosis, comprising one or more pairs of amplification primers and one or more matched pairs of sequencing primers for amplification and sequencing regions within - 20 - the genome ofM tuberculosis, characterized in that the amplification and sequencing primer pairs are selected from among: (a) amplification primers of Seq. ID Nos. 1 and 2 in combination and sequencing primers of Seq. ID Nos. 3 and 4; (b) amplification primers of Seq. ID Nos. 6 and 7 in combination and sequencing primers of Seq. ID Nos. 8 and 9; (c) amplification primers of Seq. ID Nos. 11 and 12 in combination and sequencing primers of Seq. ID Nos. 13 and 14; (d) amplification primers of Seq. ID Nos. 16 and 17 in combination and sequencing primers of Seq. ID Nos. 18 and 19; (e) amplification primers of Seq. ID Nos. 21 and 22 in combination and sequencing primers of Seq. ID Nos. 23 and 24; (f) amplification primers of Seq. ID Nos. 26 and 27 in combination and sequencing primers of Seq. ID Nos. 28 and 29; (g) amplification primers of Seq. ID Nos. 31 and 32 in combination and sequencing primers of Seq. ED Nos. 33 and 34; (h) amplification primers of Seq. ID Nos. 36 and 37 in combination and sequencing primers of Seq. ED Nos. 38 and 39; (i) amplification primers of Seq. ED Nos. 41 and 42 in combination and sequencing primers of Seq. ED Nos. 43 and 44; and (j) amplification primers of Seq. ED Nos. 46 and 47 in combination and sequencing primers of Seq. ID Nos. 48 and 49.

Inventors: SHIPMAN ROBERT, SHIPMAN, ROBERT
Applicants: SHIPMAN ROBERT, VISIBLE GENETICS INC, SHIPMAN, ROBERT, VISIBLE GENETICS INC.
Application year: 1999
Main classification: C12Q1
IPC codes: C12N15, C12Q1