CA2354234A1 - Method and kit for the characterization of antibiotic-resistance mutations in mycobacterium tuberculosis - Google Patents
Method and kit for the characterization of antibiotic-resistance mutations in mycobacterium tuberculosis Download PDFInfo
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- CA2354234A1 CA2354234A1 CA002354234A CA2354234A CA2354234A1 CA 2354234 A1 CA2354234 A1 CA 2354234A1 CA 002354234 A CA002354234 A CA 002354234A CA 2354234 A CA2354234 A CA 2354234A CA 2354234 A1 CA2354234 A1 CA 2354234A1
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- 241000187479 Mycobacterium tuberculosis Species 0.000 title claims abstract description 40
- 238000000034 method Methods 0.000 title claims abstract description 37
- 230000035772 mutation Effects 0.000 title claims abstract description 33
- 238000012512 characterization method Methods 0.000 title claims abstract description 9
- 238000012163 sequencing technique Methods 0.000 claims abstract description 76
- 230000003321 amplification Effects 0.000 claims abstract description 58
- 238000003199 nucleic acid amplification method Methods 0.000 claims abstract description 58
- 201000008827 tuberculosis Diseases 0.000 claims abstract description 33
- 108090000623 proteins and genes Proteins 0.000 claims abstract description 30
- 101150090202 rpoB gene Proteins 0.000 claims abstract description 19
- 101100038261 Methanococcus vannielii (strain ATCC 35089 / DSM 1224 / JCM 13029 / OCM 148 / SB) rpo2C gene Proteins 0.000 claims abstract description 17
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- 238000001514 detection method Methods 0.000 claims abstract description 16
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- 101150013110 katG gene Proteins 0.000 claims abstract description 10
- 101150022921 pncA gene Proteins 0.000 claims abstract description 9
- 101100509674 Mycolicibacterium smegmatis (strain ATCC 700084 / mc(2)155) katG3 gene Proteins 0.000 claims abstract description 8
- 101150068528 mabA gene Proteins 0.000 claims abstract description 6
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- 229960001225 rifampicin Drugs 0.000 abstract description 8
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- 229960003405 ciprofloxacin Drugs 0.000 abstract description 2
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- 241000186359 Mycobacterium Species 0.000 description 19
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- 238000012360 testing method Methods 0.000 description 14
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 description 9
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
- C12Q1/689—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for bacteria
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/156—Polymorphic or mutational markers
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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 Mycobacterium tuberculosis. These primers can be used in a method for detection and characterization of Mycobacterium tuberculosis 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 M. tuberculosis, and if present to evaluate the rpoB, katG, rpsL/s12 and 23S 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.
(isoniazid), mabA (isoniazid), rpsL/s12 (streptomycin), 16S/rrs (streptomycin), embB (ethambutol), pncA (pyrazinamide), gyrA (ciprofloxacin) and 23S (azithromycin) genes of Mycobacterium tuberculosis. These primers can be used in a method for detection and characterization of Mycobacterium tuberculosis 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 M. tuberculosis, and if present to evaluate the rpoB, katG, rpsL/s12 and 23S 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.
Description
_. WO 00/36142 PCT/CA99/01177 METHOD AND KIT FOR THE CHARACTERIZATION OF
ANTIBI()'TIC-RESISTANCE MUTATIONS IN
M k'(.'c7BA CTERI UM T UBERCUI OSIS
DESCRIPTION
This applicatic>n relates to a method and kit for the characterization of antibiotic resistance mutations :gin Mycobacterium tuberculosis, and particularly to the evaluation of such mutations in clinical samples.
M. tubercr~lo.,~is c;an 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 ofM tuhercuio~;r.s. 'These point mutations render the organism insensitive to the action of the antil7iotic b~~ l.~reventing it's uptake or activation, or by altering the antibiotic target. The observed antibiotic resistance in tL~ tuberculu.sis 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-le;v~~l resistance Rapid and acc;u rote detection oi.' antibiotic resistance in Mycobacterium tuberculosis in sputum sample°:s would greatly improve bath patient treatment and outcome.
Presently, analysis of'M. tubc~r~:ulosis is carried out on DNA recovered from sputum samples handled according to Standard Infectious Disease/Public Health Laboratory practices. T'he sputum sample is decontamirrate;d and a cell sediment isolated. This cell sediment is the sample source for all routines procedures used nn, the detection and isolation ofM
tuberculosis. Portions of this, sample; are used in Bac'l'ec cultures for selective growth ofM
tuberculosis, agar platelagar slant cultures for M tuberculosis, acid-fast bacilli (AFB) smears for mycobacteria detection and molecular biological methods far the detection of M.
tuberculosis and atypical mycc:abacteria. (See 1~ig. 1) Mycobacterial I~NA 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 ofM tuberculosis arty either PC;R-based or probe-based. These tests are used _2,.
grirziarily an rIFB s~e.$t po~tlvC samples. Since thaprc~cuc:c uIM. ruLcrcwhs~
has ah~c~dy been e8tabtisbcd by the ~ smear, these tests are used pyintarily jn a confinnamry capaziry a$ opposed tn a disgr:ostic capacity. Ftuth~:rmore, these tests provide no inforn~ation ou the ppteutia.I antibiotic ttisistanco oP tbrre fit: Ixrberctrlosats sarxlples.
Below is a list of ant~'~iotics usal to t~ut~41!' 7"uh~ra~L~.ric infer:ti~ns.'1'hc gene target of the specific antibiat'ic std re~iaB:~ ~sseciatEd with ~ntibiooc re~szanee are listed, if huo:wn. Tlve rr.~'crences on which the coda~n as,~~nn~encs ~ bases are listed ac the end eF the appl.i catiou.
I. nifampin rpoB ge~G codan SO?-533°
ANTIBI()'TIC-RESISTANCE MUTATIONS IN
M k'(.'c7BA CTERI UM T UBERCUI OSIS
DESCRIPTION
This applicatic>n relates to a method and kit for the characterization of antibiotic resistance mutations :gin Mycobacterium tuberculosis, and particularly to the evaluation of such mutations in clinical samples.
M. tubercr~lo.,~is c;an 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 ofM tuhercuio~;r.s. 'These point mutations render the organism insensitive to the action of the antil7iotic b~~ l.~reventing it's uptake or activation, or by altering the antibiotic target. The observed antibiotic resistance in tL~ tuberculu.sis 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-le;v~~l resistance Rapid and acc;u rote detection oi.' antibiotic resistance in Mycobacterium tuberculosis in sputum sample°:s would greatly improve bath patient treatment and outcome.
Presently, analysis of'M. tubc~r~:ulosis is carried out on DNA recovered from sputum samples handled according to Standard Infectious Disease/Public Health Laboratory practices. T'he sputum sample is decontamirrate;d and a cell sediment isolated. This cell sediment is the sample source for all routines procedures used nn, the detection and isolation ofM
tuberculosis. Portions of this, sample; are used in Bac'l'ec cultures for selective growth ofM
tuberculosis, agar platelagar slant cultures for M tuberculosis, acid-fast bacilli (AFB) smears for mycobacteria detection and molecular biological methods far the detection of M.
tuberculosis and atypical mycc:abacteria. (See 1~ig. 1) Mycobacterial I~NA 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 ofM tuberculosis arty either PC;R-based or probe-based. These tests are used _2,.
grirziarily an rIFB s~e.$t po~tlvC samples. Since thaprc~cuc:c uIM. ruLcrcwhs~
has ah~c~dy been e8tabtisbcd by the ~ smear, these tests are used pyintarily jn a confinnamry capaziry a$ opposed tn a disgr:ostic capacity. Ftuth~:rmore, these tests provide no inforn~ation ou the ppteutia.I antibiotic ttisistanco oP tbrre fit: Ixrberctrlosats sarxlples.
Below is a list of ant~'~iotics usal to t~ut~41!' 7"uh~ra~L~.ric infer:ti~ns.'1'hc gene target of the specific antibiat'ic std re~iaB:~ ~sseciatEd with ~ntibiooc re~szanee are listed, if huo:wn. Tlve rr.~'crences on which the coda~n as,~~nn~encs ~ bases are listed ac the end eF the appl.i catiou.
I. nifampin rpoB ge~G codan SO?-533°
2. Isonia2id katG gene c:odori 2751315f328~
3. Trnrtia~ad mabA gene uttlutown °
4. rsonia~id oxylz-ahp~ intet-aenic region (~'R) nucleotides -48 To ~I-33 - ' 5. A~iduanlycin X36 rRNA. ~wquanoe p~cie4cide356$A' 6. ~ P}~1327tiar!'lldC prlcA g~~e codQn 47185 t 7. Ethambutol ezrth8 gene cadcm 306 s 8. ~tr'eptatnycill rpsUsl2 gene codon a3rss h 9. Scr~plamycin lbSlrrs sednence nuclaotidcs d91, St2, 516, SI3, 903, 9Q4 10. CiProfloxacan gyxA ~eue codon 88-~5' Isrobe--based trsts do ~isr fr~r the ~ste~rimir~~tt7ail of rifampin Tesast~tnce in J1L
tuberculc~srfs {dine probe assay-Tuna?elc~, heat these probes, xely oa prior knowledge of antibiotic resistartco-sssocSated mutations ~n the rpoF3 gears. lViucatians autsida the region opZr~ vy ~.z ~tvl,ra ui new L11t7t3kio~s riot; included xn iha prose roelCtail cau~d still coufet resistance, but would not be delectet~ usirr~ this pra$ttci is it's pc'cscrst ~arm-Alastracts of the lnterscieua~e Conference ~:a Antimicral~isl Agen~a $oa Chemotherapy", vol. 34, page R63 (1994r) describes lice applieatzon of automeced DNA
seduonae xnclyaic of hap65 to specia~cm c~fisolau'~ previously-identified as being~9'.
rut~era~lasis.
~~MENDED SHEET
WO 00/36142 PCT/CA99/Ol 177 y ._ Thus, there remains a need for a method for detecting antibiotic-resistance mutations in clinical M. tuberrz~leosis 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 n'ethod. It is a further object of this invention to provide amplification and cycle sequencing primer acts, and kits containing such primer sets, for use in the characterization of antibiotic resistance mutations in M. tuberculosis.
~11>1~3I~C9~Y~tlt~tl Amplification and cycle sequencing primer sets have been developed for the detection and analysis of antibicetic resistance-associated mutations in defined regions of the rpoB (rifampin), katG (isaniarid), oxyR-ahpC P'F; (isoniazid), mabA
(isoniazid), rpsL/sl2 (streptomycin), 16S/rrs (streptonuycin), embB (ethambutol), pncA
(pyrazinamide), gyrA
(ciprofioxacin) and 23 S (azithrc°4mycin) genes. Cling these primer sets and the OPENGENETM automated DNA sequencing system, a protocol has been developed which permits both the rapid detection c>f M tuber~culn.~~is and the identification of antibiotic resistance-associated mutations in a series of geese 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' thc~ treatment of tuberculosis. The tests are employed in a hierarchical manner orr both ~,1~'l~ smear-positive or smear-negative samples to determine both the presence and antibiot:ic;-rwesistance of M: tuberculosis in a given sample. This method permits the simultaneous determination ofIVI. tubercnlosi,s presence in a sample and the antibiotic-resistance profile to a.ri entire panel of antibiotics. Standard methods require from 2-6 weeks to culture ll.~ tubercr,rl~~sis and additional time to establish antibiotic resistance.
Although DNA sequence-based (genotypicj tests are not intended to replace the traditional culture-based (phenotypic] mcetlrc>ds, 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 knowan testing protocols for M. tuberculosis; and _. -I _ Fig. 2 shows a hierarchical assay scheme for evaluating M. tuberculosis type in accordance with the invention.
In accordance with the invention, regions of the genome ofM. tuberculosis associated with antibiotic resistance are amplified and sequenced using specifically designed amplification and sequencing primer-s~. Various techniques for amplification are known, including the basic PCR amplif icdatian 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 incorparated 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 cwGle sequencing steps.
In theory, thc. selectic>n of primers to amplify and sequence a known region of interest should be straightfor'~~ard. In fact, however, because of the possibility of primer binding to other sites, campiic ations arising from secandary structure, and other factors which are not fully understood, sa~n~: primers perform better than others for amplification and sequencing of the same regiorn .of interest The present invention provides primers which have been optimized for the arriplific:ation and sequencing of regions associated with each c>f the ten known types of antibi~rtic resistance 'fhese primer sets are shown below, along with the sequence of the genes tluar: 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-rner, bp2201-2220 5' TAC GGT C'GG CG.A tiC.°-C" CiA'r CC 3° SEQ. ID NO. 1 rpoB-R amplification primer, ~'.0-mer, bp2611-2592 5' TAC GGC GTT TCG A'Ti'~i AA(, CC ~' SEQ ID NO. 2 rpoB-Ss sequencing primer, '?0-rner, bp220 k-2"'2 0 5' TAC G(.iT CGG CGA GC'T c:JAT CC 3' SEQ ID NO. 3 rpoB-3 s sequencing primer, <:Ci-mer, bp261 I -~2'S92 5' TAC GGC GTT TCG ATG AAC CC 3" SEQ ID NO. 4 SEQ.ID. NO
caacc gccgcctgcgrar-ggr~gg~ aaarraarr~
tc:
t 2161aaaccgacgac:atcgaccai:gg :
2221aaaaccagatccgggtc~gc~c:ar_gtcgc-ggatggagcgggtggtccgggag cggatgacca 2281cccaggacgtc;gaggccaar.c:ac'<~ccgcac~acgttgatcaacatccggccg gtggtcgccg 2341cgatcaaggagttcr_tc:g~l:ac:cagcc:agctgagccaattcatggaccag aacaacccgc 2401tgtcggggttc~acccac:a;~;cgccga<agtcggcg<-tggggcccggcggt ctgtcacgtg 2461agcgtgccggc3ctggac:;gcq,~gacgtgcacccgt~rgcactacggccgg atgtgcccga :~-2521tcgaaacccct:gaggg~=tc,~vaacatcggtctgatc<3c~c:cgctgtcggtg tacgcgcggg 2581tcaacccgt<:.ggqtt:a~~gaaacgc:cgtac:cgcaaggtggtcgacggc gtc~gttagcg t katG (isoniazid resistance]
katG-F amplification primer, :.~0~-mer, bp'722-741 5' ATG CrGG CTG ATC.' Tt'4<:' G'TCi AA :3' SEQ ID NO. 6 katG-R amplification primer-, ::()-mer, by I 250-~ I 231 5' GGT GTT CCA CJCC' AI:iC' CiAC CrC'. :B SEQ ID NO. 7 katG-Ss sequencing primer 2t)-.nner, bp722-741 5' ATG CrGG CTG .AT(', T,t~(:: GTG AA 3' SEQ ID NO. 8 katG-3s sequencing primer, 2t).-mer, by125()-1~?31 5' GGT C1TT CCA CiCC; ACiC', GAC'. G(: :i' SEQ ID NO. 9 SEQ
ID
NO.
661 gctcggcgatgagcgt.t~~,.:aqo:ggtG3agcgggat.ct.ggagaacccgctggccgcggtgca 721 gatggggctgatctac~gt:.~aa.cccg~;eiggggccgaacggc:aacccggaccccat:ggccgc 7B1 ggcggtcgacattcgcg<i~_~ac~gtt:~c~c~c~cgcatggccatc~aacgacgtcgaaacagcggc - -841 gctgatcgtcggcggt:.c:~i-:a~:r.v~ttcc~gtaagacccatggcgccggcccggccgatctggt -901 cggccccgaar_ccgaggc:c:g:::tccgragqagcagatgggcttgggctggaactagctcgta -961 tggcaccggaaccggt:a,~<~gacgcgatcaccagcggcatc:gaggtcgtatggacgaacac 1021cccgacgaaatgggacva.ac,ac~tttc~tcc~agatcca.gtacggctacgagtgdgagctgac -_ (, .~
1081 gaagagccct gctggcgc: t = ggc:aat ac:ac: cgccaac~gac ggcgccggtg ccgc~caccat 1141 cccggacccg .tt:cgc~cgcxc~~ cacj~ggcgctc ccc:~gacgatg <:aggccactg acct:ctcgct 1201 gcgggtggat ccgat:ct<=t 3 agc:ggatc:ac ~,~r~-~-r~y <~rq~aac-acc-ccgaggaatt 1261 ggccgacgag tt:cgc:caa:~c~~ cct:ggta~::aa grctgatc_rac c::gagacatgg gtcc:cgttgc oxyR-aphC intergenic region ( PR) PR-F amplification primer, 2(i-r/ ~er, bp451-470 5' ACC AC;T GCT T'fG C;CCI (~(~".A C:C 3' SEQ >D NO. 11 PR-R amplification primer, 2t)-rner, bp687-668 5' CCG ATG AGA GCG GT'G tIGC'. TG 3' SEQ ID NO. 12 PR-Ss sequencing primer, 20-~mer, b1r451-470 5' ACC ACT GCT TTG CC(:l ( "~~''"A C~C 3' SEQ 1D NO. 13 PR-3s sequencing primer, 20-rr~er, bp687-E>68 5' CCG ATG ACiA GCG GTG ,~-ICiC' TG 3' SEQ ID NO. 14 SEQ ID NO. 15 361 atgccctggg ggtgcacc~ygac::cggca.t:c ::c~a=~cacc.gc tcgc:cgcaac gtcgactggc 421 tcat<~tcgag aatgcttgc~g~~aca_actga ;3- a, grrr ~a,~gr~ar~r gcggcgaacg 481 cgcgaagccc ggccacgg~~:- ggc:t:agcac:c: ::~:~tt;g_qr_ggc gatgccgata aatatggtgt 541 gatatatcac ctttgcct:g;: c:ac~c:gactt:c ~~:ggcacgat ggaatgtcgc aaccaaatgc 601. at:tgtccgct ttgatgat:g<:. ggac~agt:oat: _x~~cactgcr_a accattggcg atcaat:tccc 661 cgcctac.~aa~-c a cvac,~;;~~ w -.caacgg t_.gaccr_gtcc aaggtcgacg ccaagr.agcc 721 cggcgactac ttc~accact~u tr_<~c:cagtga :~~:3aacacc;:a ggeaagtggc gggtggtgtt mabA (isoniazid resistance) mabA-F amplification primer, fit)-mer, bp56-7's 5' CCT CGC TGC CCA GA.A :~'lCiCx GA :>' SEQ ID NO. 16 mabA-R amplification primer, 20-mer, bp:303-284 5' ATC'. CCG CGG T'T'T CC'f C'CG tJT 3' SEQ ID NO 17 mabA-Ss sequencing primer, 20.-mer., bp56-75 5' CCT CGC TGC CCA GAA AGCi GA 3k SEQ ID NO. 18 _ WO 00/36142 PCTICA99/Oi 177 mabA-3s sequencing primer, ''>Cf-mer, bp303-284 5' ATC CCC CGG TTT C;C'1" C'.t:.G GT 3' SEQ ID NO. 19 SEQ ID NO. 20 1 agcgcgacat acc:agcag,::g r_vaattcgtag c;qcgtcaata cacccgcagc caggg 61 ~t-,gcccagayQaaatcc::~t: cat~3gtcgaa c~t:gtgct:~ag tcacaccgac aaacgtcacg 121 agcgtaaccc~ cagtgcga-aa gttcccc3ccg c.;aaatcg~~ag ccacgttacg ctcgtggaca 181 taccgatttc gg~::cc<jgc::c. c:ggcgaga~g a. t.aggttgtc ggggtgactg ccac<~gccac 241 tgaaggggcc aa'r3CC_'(_'CCcit.. tcgtatcc,~g t:tcagt-_c:ctg gttgc~raaaa aaaac-~aaaa 301 gatcgggctg gcgat~c:gc<ic:: agcggctggc t:c~c<:gacggc cacaaggtgg ccgtcaccca rpsL/s12 (streptomycin resistance) sl2-F amplification primer, 2~)-~rver, bpl-20 5' CGG TAG ATG CCA A(:'~' /akI"C' CA 3' SEQ ID NO. 21 s12-R amplification primer, 20-rrner, bp384-36 5' GCA TCA GCC C'TT CTt'. t:"1"i' C'.T 3' SEQ ID NO 22 sl2-Ss sequencing primer, 20-rric~r, bpl-20 5' CGG TAG ATG CCA AC'.C', tj~'fC: CA 3' SEQ ID NO. 23 sl2-3s sequencing primer, 20-nnc~r, bp384-36S
5' GCA TCA GC,C CTT CTt:' t~'TT C'T 3' SEQ ID NO. 24 SEQ LD NO. 25 1 ~~Qr cad~cat- ~~~,--~ g~cac~ctggtc: ::gcaagggt:c gtcgggacaa gatcagtaag 61 gtca<~gaccg cggct_ctg,~~=. qggc:agc:<:cca ;:ag:~gtcgtg gtgtatgcac ccgcgtgtac 121 accaccactc cgaagaag~:vcv gaacacgc~rg ;..vac:ggaagg ttgc:ccgcgt gaagttgacg 181 agtcaggtcg aggtracgg~_ gta<:att:c~cc ~gcgagggcc acaacctgca ggagcactcg 241 atggtgctgg tg<:gcggc~3~.~t acc~ggtc~aac~ .:~acr~tgcct_g gtgtgcgcta caagat:catc 301 cgcggttcgc tggatacc.w gggt:gtc:aag aaccgc:aaac aggr_acgcag ccgttacggc 361 gctaagaagg agaagggctct atgr_ca<:gca ~ggggcc°cgc gcccaagcgt ccgttggtca 16S/rrs (streptomycin resistance) 16S-F ampliftcation primer, :; l -rt~er. bp5-25 5' GGT GAT CTG CCC'. TGC.'. ACT 'rCG 3' SEQ ID NO. 26 16S-R amplification primer, '? 1-mer, by 14'7-127 _g_ 5' CGT CAC CCC ACC AAC' ~'~~G (:TG 3" SEQ ID NO. 27 16S-Ss sequencing primer, 21 ~-rrur, bp5-25 5' GGT GA.T CTG CC',C TGC'. ACT '1'CG 3" SEQ ID NO. 28 I 6 S-3 s sequencing primer, 2 I ~-nrke;r, by I 47- I 27 5' CGT CAC CCC AC'.C AAC' ~,A(x C'TG 3' SEQ ID NO. 29 SEQ ID NO. 30 1 cgtgggtgat ctgccctgcva cttc:ggg,sra agcctgggaa actgggtcta ataccggata 61 ggaccacggg atgcatgtcvt r:gtc.~gtggaa agcgctta.ag c_qgtgtggga tgagcccgcg 121 gcctatca~~ rr~.~raa~ ;JQrs'Jara embB (ethambutol resistanceJA
embB-F amplification primer, 2 l--mer, bp'7761-'~'.~B I
5' CGG CAA GC'.T GGC GC;~ C'C'C TCA 3' SEQ ID NO. 31 embB-R amplification primer, 2 I -mer., bp8040-8020 S' AGC CAG CAC ACT .AGt, C°CG (JC',G 3 SEQ >D NO. 32 embB-Ss sequencing primer, ~? I -uner, bp77fi I -7781 5' CGG CAA GC'T GGC (iCA C'C'.T 'CCA 3' SEQ ID NO. 33 embB-3s sequencing primer, 2:V-tner, bp8040-8020 5' AGC CAG CAC ACT' AGC: C'CG GCG 3 SEQ 1D NO. 34 SEQ ID NO. 35 7741 cggcatgcgc cggctgatt:c: rac~caaarra,~~cT~~rrc acc:ctgaccg acgccgtggt 7801 gatattcggc r_rcct:gctc:t ggcatgtc:at, cggcgcgaat tcgtcggacg acggctacat 7861 cctgggcatg gcccr~agnccccgacc:ac gc c,ggc_tacatg tcc:aactatt tccgctggtt 7921 cggcagcccg _gaggatc~~ct ~cggctqgta ttacaacc:tg ctggcgctga tgacccatgt 7981 cagc_gacgcc agtcrgt~~~c;a. t.ctc:gcctgcc agacctggcc gc .,gggc'ra~r_gr~crg pncA (pyrazinamide resistance) pncA-F amplification primer, 2 U-mer; by I -20 5' ATG CGG GCG TT(i ATC ATC GT 3' SEQ 1D NO. 36 ., c,. ..
pncA-F amplification primer, 2(~a-mer, bp561-542 5' TCA GGA GCT GCA AAt'. n:AA ('T 3' S:EQ ID NO. 37 pncA-S s sequencing primer, 20~ mer, by l -20 5' ATG CGG GCG T'TG .AT(:: ,~'TC' CiT 3' S:EQ ID NO. 38 pncA-3s sequencing primer, 2.0-.mer, bp561-54~:
5' TCA GGA GCT GC;A AA(~' ~C"AA CT 3' SEQ 1D NO. 39 SEQ ID. NO. 40 1 atgcgggcgt.tgatcatccat:cgacgtgcag~acgacttctgcgagggtggctcg<aggcg 61gtaaccggtggcgcc:gcg~::a:3gcc:cgrg~:ca:tcagcgactacctggccgaagcgqcggac _ 121taccatcacgtcc:3tggca<:u:caaggactr_coacatcgac~ccgggtgaccacttct=ccggc 181acaccggactat t:cct:cg!::c::gt:ggcc<~c:,gcatt:gcg~cagcggtactcccggcgcggac .
241ttccatcccagtc-tg<3ac.~cgtcc3gcaatcc3~,ggcggtgttctacaagggtgcct:acacc:
301ggagcgtacagcggc:ttc::~aaggagtc:gaccaagaacggcacgccactgctgaatt:ggctg 361cggcaacgccXgcgtcgat::~aggtcgat=gtgcttcqgtattgc~~ac:cgatcattgtgtgcgc .
421cagacggccgaggacc3cg~.3t..acgcaat:g<~ct:tggc;cacc:agggtgctggtggacctgaca 481gcgggtgtgtcggccgat~ac:vcaccgtcgcc~cg<:aggar~gagatgcgcaccgccagcgtc 541g3~:$ r~ 9'~a~crc~G-.a__i3 rt-gyrA (fluoroquinitone/ciproflo:xacin resistance) gyrA-F amplification primer, 2(~~ mer., bp2383-x:402 5' CAG CTA CAT C'.GA C'.T:'~ 'fGC tJA 3' SEQ ID NO. 41 gyrA-R amplification primer, 2O.-mer, bp2702-2(i8'.3 5' GGG CTT CGG TGT AC(~' 7"CA TC 3" SEQ ID NO. 42 gyrA-Ss sequencing primer, ~~0~-riser, bp2383-2102 5' CAG CTA CAT CGA CTA 7 GC (JA 3" SEQ m NO. 43 gyrA-3s sequencing primer, ::',0-riser, bp2'7()2-2cp83 S' GGG C'rT CCiG TGT ACC'. TCA TC :3' SEQ ID NO. 44 SEQ >D NO. 45 2341 cgaccggatc gaaccgct.t~:a acatcgagc:a ggagatgcag cgcagctaca tcgactatgc 2401 ~3tgagcgtg atcgtcggcw: grc~cgctgcc ggaggtgcgc gac:gggctca agccc:gtgca 2461 tcgccgggtg ctcr_atgc~~~~.~ tgt:tcgatt;c cggcttrcgc ccggaccgca gccacgccaa - 1 (?
2521 gtcggcccgg tcggttgevc.~ agac:catggg c,aactaccac: ccgcacggcg acgcgtcgat 2581 ctacgacagc ctggtgcgca~ tggc:cca<~ .rc-.:tggtr_gctg c:gctar_ccgc tggtggacgg 2641 ccagggcaac ttcggctcgc: caggcaatga c=cacrggcg gcyarga~yr a~a~c~Qaaac-2701 ~ggctgacc ccgttggc:ge~ tggagatctr~r_ gagggaaatc: gacgaggaga cagtcgattt 23S (macrolide/azithromycin ~rc~sistance) 23S-F amplification primer, 2i:>-mer~, hp2444-241:3 5' CGA AAT TCC TTG T'C(_i (:=it:.:rT AA SEQ ID NO. 46 3' 23S-R amplification primer, 20-mer, bp2683-2664 5' GTA TTT CAA CAA C'.GA (:'T(~ (A 3' SEQ ID NO. 47 23S-5s sequencing primer, 20-mf,r, bp2444-2463 5' CGA AAT TCC TTG TCCi (i(JT' A.A 3' S:EQ ID NO. 48 23S-3s sequencing primer, 2C!-mer, bp2683-2664 5' GTA TTT CAA CAA (:GA ~(~TC (T;A 3' SEQ ID NO. ~49 SEQ B7 NO. 50 2401 gccccagtaa arggrggt::g~ taactataac catcctaagg tag~gaaa r c-c-rt-Qr 2461 ~gttccga cc~tgcac~::~aa t:gc~cgtaacg a~ct:tcc~aac tgtc:tcaacc atagactcgg 2521. cgaaattgca ct:.acgagc::aa agatgc:t-gt tac:gc~gr~ggc aggacgaaaa gacc:ccggga 2581 cctt.cactac aactt;ggt;at t:ggtgttc~gg t.ac:ggt-.tgt gt:aggatagg tggc;agactt 2641. tgaagcacag .ac::gc~cvagv: t t c~tcrgg_~~rryrt-~rrgaaa rar~cactctg atcgtattgg To facilitate detection of the sequencing products using real-time fluorescence-based electrophoresis apparat.u:~ (for example, a~ 'Visible Genetics OPENGENETM sequencer), at least one of the sequencing l7rirners 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 cyanune dye such as (;'~'S.0 OR CY5.5.
The primers of the invention are suitably packaged in a kit. This kit will contain individually packagec:l 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), anti preferably includes the primer sets for a plurality of resistance genes, most preferably the primer sets for all ten resistance genes.
_ WO 00136142 PCT/CA99101177 -ll.-The suitable pr~ol.c:~co1 for the utilization of these primer sets in the evaluation ofM. tuberculosis in clinical ;samples utilizes P(.".R amplification, followed by cycle sequencing. DNA for use in the test is obtained. i=rorn a .sample of sputum (IOOuI-lOml). The sputum sample is processed according to Standard Infectious Disease/Public Health Laboratory practices (Mycobac~e~riology Bench Manual, Laboratory Services Branch, December 1997, Ontario Ministrwy of 1-Iealti~). The sputum sample is homogenized, decontaminated and concentr,~t~~d. Mycobacterial DNA is prepared directly from a portion of the concentrated cell sediment ( 100-:?OOuI) using standard DNA extraction methods or commercially available kits.
Amplification of the DNA is performed using the amplification primer sets described above. PCR reagent:. can be prepared for individual reactions, or may be prepared as a master mix which can be u,e;d fcsr multiple t<~sts e.g., 10 PC;R
reactions. Exemplary combinations of reagents are su mmarized in the 1E'ollowing table.
PCR mix 1 PCR 10 PCRs final conc. / PCR
genomic DNA (20ng~'u1) 1 Oul 20ng (~0. 5 fM) lOX PCR buffer I 2.5ui 25.Ou1 IX
2.5mM dNTP mix (l : l :l :1 y 2.5u~ 25 0ul 250uM
DMSO 1 3u1 l3.Oul 5%
Taq DNA polymerase (11J) 0.2u1 2.Oul 1 unit molecular grade water I e:i 5ul 165.Ou1 MTB gene primers ( I OuM) 1.0u1 1 O.OuI l Opmol per primer total volume per PCR 25.Ou1 If the master mix as shown in the column labeled I 0 PCRs is utilized, the mastermix contains all the necessary PCR reagerkt~ other than the gc.nomic DNA. In this example, 24.Ou1 of the mastermix is added to a PCR tube, that already contains I .Oul of genomic DNA, prior to the addition of the mineral oil overlay and placement in the thermocycler.
The genomic CI~NA preparation utilized must be of sufl'rcient quality and integrity for robust and reproducible PCR Suitable DNA preparation can be obtained using WO 00/36142 PCT/CA99/0117"
c! ._ the Gentra PuregeneTM DNA. i=.~alation kit. The kit components are appropriate for the isolation of genomic DNA from blood, fresh or ~TOZen tissue, archival material and paraf~m-embedded tissue.
Each primer pair is used to amplify a single gene region under the following conditions:
1. Denaturation 't4C 5 minutes 1 cycle 2. Denaturation '~4C 30 seconds Annealing 6t)C 3f> seconds 35 cycles Extension '72C 60 seconds 3. Extension 72C s minutes 1 cycle 4. Hold !~"C
The temperature change durin;the cycles of the step 2 is desirably set to ramp at a rate of 1 °C/sec.
After amplifican~ion, :>..Oul from the 25.Ou1 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 sequerACing reaction. (comparable verification of sequencing purity is performed on each of the other amplification products Sequence anaty sis is e;arried 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 ~;en~e.
For initial sequence analysis of rpoB, the rpoB-Ss primer should be used. For confrrmatory sequence analysis the rpoB-3s primer should be used. For each template to be sequenced, aliquot 3.Ou1 of each of the nucleotide ternunation mixes into four separate tubes marked <A>, <C>, v~G> anci ~~"lrv=~ and store on ice until the sequencing mastermix is prepared.
rpoB template 2 Oul lOX VGI Sequenace TM butler 2 Sul _. WO 00/36142 PCT/CA99/0117T
-13v DMSO 3.Sul 2.5uM dye-sequencing primer 2 Ou1 PCR grade water 9 Oul X10 diluted The moc . 1 n c 0 5 ul total volume ~. ~? Oul Mix the DMSO and other corriponents in the mastermix well by repeated pipetting (5 times) with a micropipette. Store t'~~e mastermix on ic;e until ready to add to the nucleotide termination mixes.
Add 5.Ou1 of the mastermix to c,ach of the four marked tubes containing the nucleotide termination m~xe~;, Add ~.Oul lightweight mineral c>il to each of the four marked tubes containing the mastermix and nucleotide termination mixc~.s.
Store on ice u.mtil ready to load into the thermocycler.
paramgterc fir .nculg cycl c .d ~
I . Denaturation 94C 5 minutes I X
2. Denaturation 94C 30 seconds Annealing 60C 30 seconds 35X
Extension 7~C.' 60 seconds 3. Extension 7~C 5 minutes 1X
4. Hold ~''C.
The temperature change during; the cycles of the step 2 is desirably set to ramp at a rate of 1 °C/sec.
At the end of the cycle sequencing reaction add 6.Ou1 of the Stop Loading Dye directly to each of the four to b es to stop the :sequencing reaction. The sequencing samples are heated at 95°C for '~ minutes and then placed on ice before loading 2.Oul (from a total volume of l4ul) on the CLIPPERT"~' ~~equencer. 'The remainder of the sequencing reaction can be stored at -20°C for subsequent use.
._ WO 00/36142 _ 1.1 -The CL.IPPERT~'~ sequences is sex-up as described in the OPENGENE
Automated DNA Sequencin~;l .yystem User Manual. Run parameters for the CLIPPERT'M
sequences are 54°C/ 1300voltsl t).Ssec sampling/35min run/50% laser power. The samples loaded included 2 ul each of t.hc, Forward and reverse sequencing reaction products for the target gene, differentially labaled, for example with C1~'S.0 and CY5.5 cyanine dye labels.
Once the run is completed, tire base-called data is analysed by comparison of the test sequence to the rpoB sequence database in GEl\fELIBRAR.IANTM. This sequence alignment.
compares the test sequence to the standard control sequence and allows sequence ambiguities to be assessed. Once edited the°'test sequence ran be screened far antibiotic resistance-associated mutations using Cil=:1'vfELIBRARIA1'~'rM
3'estircg far multiple types oi~ 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 M. tubercz~Jnci.c' are only performed on AFB smear-positive sputum samples. 'These methods serve; as canfirrnatary tests for the presence ofM.
tuberculosis. In addition to these molecular hiological method;,, the culture-based procedures for M.
tuberculosis detection (Bac Tec liquid culture, agar plate and slant cultures) are performed in parallel. AFB smear-negative sputum samples 2~re processed with only the culture-based detection procedures (Figure 1 ).
In the present irmention bath AFB smear-positive and smear-negative sputum samples can be processed using; bath culture-!eased 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 X000 bacteria/ul the AFB stain will be negative. In addition to this is the observ~ltion that the decontamination procedure used to prepare the sputum sample usually kills l0'-20°io 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 a.re culture-positive for M.
tuberculosis (Ontario Public Health Laboratory). 'This level of myc;abacteria is easily detected by molecular biological methods and is t:h~~refore 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 Ivl.tuberculosis infections (rpoB, katG, rpsL/s12, PK, embB, pncA) WO 00/36142 PC'T/CA99/01177 lc,-and detect other species of nryc;cibacteria (23 S) in the absence of M.
tuberculosis (Figure 2).
Group I analyses are performed before both Group Il and Group III. Group I
analysis will provide information on the antibiotic resistance; status to rifampin (rpoB), isoniazid (katG), streptomycin (rpsL/sl2) and a.~:iithronrycin (23~>). In addition the rpoB
amplification indicates the presence ofM. tuberculcrsn,s and in the absf;nce of rpoB amplification the 23S sequence allows identification of most oi.' the clinically relevant mycobacterial species. Group II
analysis provides information care antibiotic resistance mutations in the "second-line"
antibiotics used to treat M. tr~b~erculosis infections namely, isoniazid (PR), ethambutol (embB), pyrazinamide (pncA) and civprofloxacin (gyrA). Group III contains gene targets in which mutations associated vvitt~ 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. tuherculo..w.s infections, and thus the associated antibiotic resistance mutation patterns. ~~ary geographically. As shown in Figure 2 the culture-based methods are performed in parallel. The molecular biological methods would permit the idenstification ofM: tuberculosis from both AFB smear-positive and smear-negative sln.~tum samples a.nd further provide information on the antibiotic resistance profile ~;~f t hese samples yell in advance of current culture-based methods. This information wcmuld be crucial tc~ the initiation of appropriate and effective antibiotic treatment regimens fiar t~~! tubercr~lrr.~is infections.
Examples A pool of DI~,~ samples from antibiotic-sensitive M. tuberculosis isolates was obtained from the L,C>7C; I--Ieauth and Wc;lfares Canada. Ottawa, Ontario.
Wild-type sequence traces, for all gene targets hn~3~wn to harbor mutations in antibiotic-resistant M. tuberculosis, were generated.
A panel of 1_:)rd:'~ samples from five phenotypic streptomycin-resistant M.
tuberculosis isolates was obt;~ined lronr the Public Health Laboratory, Ontario Ministry of Health, 'Coronto, Ontario. ~Cr~ese Ir)NA samples were examined for antibiotic resistance-associated mutations in all l~~ antibiotic gene targets listed above.
Streptomycin resistance-associated mutations were dc;tected in the rpsL/s12 gene in four isolates.
Parallel antibiotic resistance-associated mutatic~rws in the rpoB ~:rifampin), kat(.i (isoniazid), PR (isoniazid), _. 1 ~ ..
embB (ethambutol), pncA (pyr~~inarnide) and g~rrA (ciprofloxacin) genes were also identified which underscores the impor~:a~~ce of examining all the gene targets for first-line antibiotics used in the treatment of A~ trrber«culosi.s. A sunnmary of the results is shown in 'Cable 1.
The following references are cited herein and are incorporated herein by reference for all states which aliow such incorporation.
DL Wiliiams et al. (1f94). Characterisation of rifampin resistance in pathogenic mycobacteria. Antimicrob Agents (:hemother 38: 2380-2386.
WH Haas et al. (19971. Molecular analysis of katG gene mutations in strains of Mycobacterium tuberculosis complex from Africa« Antimicrob Agents Chemother 41: 1b01-1603.
S Sreevatsan et al. (1!197). Analysis of the oxyR-ahpC region in isoniazid-resistant and -susceptible Mycobacterium tuberculosis complex organisms recovered from disea:;ed humans and animals in diverse localities. Antimicrob Agents Chemother 41i : 600-606.
A Teienti et al. (19941. C:enotypic assessment of isoniazid and rifampin resistance in Mycobacterium tutrerculosis: a blind study at the reference laboratory level.
Antimicrob Agents C'hemother 35: ''19-723.
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: 1134-640.
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-ti40.
MA Lety et al. (1997).. A single point mutation in the embB gene is responsible for resistance to etharnbutol in Mycobacterium smegmatis. Antimicrob Agents C',hemother 41: 2629-2633.
A Scorpio et al. (1997). Characaterisation of pncA mutations in pyrazinamide-resistant Mycobacterium tuberculosis. Antimicrob Agents Chemather 41: 540-543.
_ [7 _ C Xu et al. (1996). Fluoroquinilone resistance associated with specific gyrase mutations in clinical isolates of muitidrug-resistant Mycobacterium tuberculosis.
J Infect Disease 174- 1127-1130.
ICA Nash et al. ( 1990. 4Jenetic basis of macrolide resistance in Mycobacterium avium isolated from p;~tients with disseminated disease. Antimicrob Agents Chemother 39: 2625-2(i30.
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<110> Visible Genetics Inc.
Shipman, Robert <120> Method and Kit for the ~'~.°haracterization of Antibiotic-Resistance Mutaauons in Mycobacterium tuberculosis <130> VGEN.P-055-WO
<140>
<141>
<150> 60/111,794 <151> 1998-12-11 <160> 50 <170> PatentIn Ver. 2.1 <210> 1 <211 > 20 <212> DNA
<213> Mycobacterium tuberculosis <220>
<223> rpoB-F amplification primer <400> 1 tacggtcggc gagctgatcc 20 <210> 2 <211> 20 <212> DNA
<213> Mycobacterium tuberculosis <220>
<223> rpoB-R amplification primer <400> 2 tacggcgttt cgatgaaccc 20 2_ <210> 3 <211> 20 <212> DNA
<213> Mycobacterium tuberculosis <220>
<223> rpoB-Ss sequencing primer <400> 3 tacggtcggc gagctgatcc 20 <210> 4 <211> 20 <212> DNA
<213> Mycobacterium tubc:rnulosis <220>
<223> rpoB-3s sequencing primer <400> 4 tacggcgttt cgatgaaccc 20 <210>
<211 > 480 <212> DNA
<213> Mycobacterium tuberculosis <220>
<223> rpoB (rifarnpin resistance) PCTICA99/Ol 177 <400> 5 aaaccgacga catcgaccac ttcg~~c.aacc gccgcctgcl; tacggtcggc gagctgatcc 60 aaaaccagat ccgggtcggc atgtcgcgga tggagcggg;t ggtccgggag cggatgacca 120 cccaggacgt ggaggcgatc acac:cgcaga cgttgatcaa catccggccg gtggtcgccg 180 cgatcaagga gttcttcggc accagccagc tgagccaatt catggaccag aacaacccgc 240 tgtcggggtt gacccacaag cgcc~yactgt cggcgctgl;g gcccggcggt ctgtcacgtg 300 agcgtgccgg gctggaggtc cgc~;~acgtgc acccgtcgc:a ctacggccgg atgtgcccga 360 tcgaaacccc tgaggggccc aaca~tcggtc tgatcggctc gctgtcggtg tacgcgcggg 420 tcaacccgtt cgggttcatc ,gaasrc;ccgt accgcaagg~t ggtcgacggc gtggttagcg 480 <210> 6 <211> 20 WO 00/36142 PCT'/CA99/01177 <212> DNA
<213> Mycobacterium tuberculosis <220>
<223> katG-F amplification primer <400> 6 atggggctga tctacgtgaa 2«
<210> 7 <211> 20 <212> DNA
<213> Mycobacterium tuberculosis <220>
<223> katG-R amplification primer <400> 7 ggtgttccag ccagcgacgc 20 <210> 8 <211> 20 <212> DNA
<213> Mycobacterium tuberculosis <220>
<223> katG-Ss sequencing pranler <400> 8 atggggctga tctacgtgaa 20 <210> 9 <211> 20 <212> DNA
<213> Mycobacterium tubercv.losis <220>
<223> katG-3s sequencing primer <400> 9 ggtgttccag ccagcgacgc 20 WO 00/36142 PC'T/CA99/01177 _ 4 <210> 10 <211 > 660 <212> DNA
<213> Mycobacterium tuberculosis <220>
<223> katG (isoniazid resistance) <400> 10 gctcggcgat gagcgttaca gcggtaagcg ggatctggag aacccgctgg ccgcggtgca 60 gatggggctg atctacgtga acccggaggg gccgaacggc aacccggacc ccatggccgc 120 ggcggtcgac attcgcgaga cgtttcl;~;cg c,atggccatg aacgacgtcg aaacagcggc 180 gctgatcgtc ggcggtcaca ctttcf;gtaa gacccatggc ~c;cggcccgg ccgatctggt 240 cggccccgaa cccgaggctg ctccg~rigga gcagatggg,c ttgggctgga agagctcgta 300 tggcaccgga accggtaagg acgcgatcac cagcggcatc gaggtcgtat ggacgaacac 360 cccgacgaaa tgggacaaca gtttcc~rc:ga gatcctgtac p;gctacgagt gggagctgac 420 gaagagccct gctggcgctt ggcaat~~cac c:gccaaggac; ggcgccggtg ccggcaccat 480 cccggacccg ttcggcgggc caggg,cgctc cccgacgatg ctggccactg acctctcgct 540 gcgggtggat ccgatcta.tg agcggat4:ac gcgtcgctgg caggaacacc ccgaggaatt 600 ggccgacgag ttcgccaagg cctgg'.a~Gaa gctgatccac cgagacatgg gtcccgttgc 660 <210> 11 <211> 20 <212> DNA
<213> Mycobacterium tubi:rc~ulosis <220>
<223> PR-F amplification pricier <400> 11 accactgctt tgccgccacc 2t1 <210> 12 <211> 20 <212> DNA
<213> Mycobacterium tuberculosis <220>
<223> PR-R amplification primer <400> 12 ccgatgagag cggtgagctg 2t~
WO 00/36142 ~ PCT/CA99/01177 <210> 13 <21 I > 20 <212> DNA
<213> Mycobacterium tuber~;ulosis _ <220>
<223> PR-Ss sequencing p~°inaer <400> 13 accactgctt tgccgccacc 20 <210> 14 <211>20 <212> DNA
<213> Mycobacterium tuber<;ulosis <220>
<223> PR-3s sequencing prinu~,r <400> 14 ccgatgagag cggtgagctg 20 <210> 15 <211 > 420 <212> DNA
<213> Mycobacterium tuberculosis <220>
<223> oxyR-ahpC intergen~c :~e;gion (PRj <400> 15 atgccctggg ggtgcaccga gaccggcttc cgaccaccgc tcgccgcaac gtcgactggc 60 tcatatcgag aatgcttgcg gcactgct~~a accactgctt tgccgccacc gcggcgaacg I20 cgcgaagccc ggccacggcc ggctal;c°acc tcttggcggc; gatgccgata aatatggtgt gatatatcac ctttgcctga cagcgacttc acggcacgat ggaatgtcgc aaccaaatgc 240 attgtccgct ttgatgatga ggagagtcat gccactgcta accattggcg atcaattccc 300 cgcctaccag ctcaccgctc tcatcggr.~;g tgacctgtcc aaggtcgacg ccaagcagcc 360 cggcgactac ttcaccacta tcaccagt~;a cgaacacc;ca ggcaagtggc gggtggtgtt 420 <2I0> 16 <211> 20 <212> DNA
<213> Mycobacterium tube.rctrlosis <220>
<223> fabG-H amplification primer <400> 16 cctcgctgcc cagaaaggga 20 <210> 1~
<211> 20 <212> DNA
<213> Mycobacterium tuber~:wlosis <220>
<223> fabG-R amplification primer <400> 17 atcccccggt ttcctccggt 20 <210> 18 <211> 20 <212> DNA
<213> Mycobacterium tubt~rr,ulosis <220>
<223> fabG-Ss sequencing primer <400> 1 g cctcgctgcc cagaaaggga 20 <210> 19 <211> 20 <212> DNA
<213> Mycobacterium tuberculosis <220>
<223> fabG-3s sequencing primer <400> 19 atcccccggt ttcctccggt 20 WO 00/36142 PCT/CA99/011??
<210> 20 <211> 360 <212> DNA
<213> Mycobacterium tuberculosis <220>
<223> fabG (isoniazid resistance;) <400> 20 agcgcgacat acctgctgcg caattc~,tag ggcgtcaata cacccgcagc cagggcctcg 60 ctgcccagaa agggatccgt catggtc;gaa gtgtgctgag tcacaccgac aaacgtcacg 120 agcgtaaccc cagtgcgaaa gttcc:c~;c~cg gaaatcgcag, c;cacgttacg ctcgtggaca 1$0 taccgatttc ggcccggccg cggcgagacg ataggttgtc ggggtgactg ccacagccac 240 tgaaggggcc aaacccccat tcgtatcc.cg ttcagtcctg gtt:accggag gaaaccgggg 300 gatcgggctg gcgatcgcac agc~;g~a~;gc tgccgacggc cacaaggtgg ccgtcaccca 360 <210> 21 <211> 20 <212> DNA
<213> Mycobacterium tuberculosis <220>
<223> s12-F amplification primer <400> 21 cggtagatgc caaccatcca 20 <210> 22 <211> 20 <212> DNA
<213> Mycobacterium tubc:rcaulosis <220>
<223> s12-R ampliLicatior~ primer <400> 22 gcatcagccc ttctccttct 20 <210> 23 <211> 20 <212> DNA
<213> Mycobacterium tuberculosis <220>
<223> s12-Ss sequencing primer <400> 23 cggtagatgc caaccatcca 20 <210> 24 <211> 20 <212> DNA
<213> Mycobacterium tubc;rc~;ulosis <220>
<223> sl2-3s sequencing pricrcer <400> 24 gcatcagccc ttctccttct 20 <210> 25 <211 > 420 <212> DNA
<2I3> Mycobacterium tuberculosis <220>
<223> rpsL/sl2 (streptomycin resistance) <400> 25 cggtagatgc caaccatcca gcagctggtc cgcaagggtc gtcgggacaa gatcagtaag 60 gtcaagaccg cggctctgaa gggca~gcccg cagcgtcgt~; gtgtatgcac ccgcgtgtac 120 accaccactc cgaagaagcc gaactcggcg cttcggaagg~ ttgcccgcgt gaagttgacg 180 agtcaggtcg aggtcacggc gtacattccc ggcgagggc:c~ acaacctgca ggagcactcg 240 atggtgctgg tgcgcggcgg ccgg~tgaag gacctgcctg; gtgtgcgcta caagatcatc 300 cgcggttcgc tggatacgca gggtgts;aag aaccgcaaa.c aggcacgcag ccgttacggc 360 gctaagaagg agaagggctg atgcc:acgca. aggggccc~;c gcccaagcgt ccgttggtca 420 <210> 26 <211> 21 <212> DNA
<213> Mycobacterium tube~r~:ulosis <220>
<223> 16S-F amplificatic>n ~:rime;r PCT/CA99/0117?
<400> 26 ,,1 ggtgatctgc cctgcacttc g ' <210> 27 <211> 21 <212> DNA
<213> Mycobacterium tubm-culosis <220>
<223> 16S-R amplification ~~rimer <400> 27 21 cgtcacccca ccaacaagct g <210> 28 <211> 21 <212> DNA
<213> Mycobacterium tuberculosis <220>
<223> 16S-Ss sequencing lorimer <400> 28 21 ggtgatctgc cctgcacttc g <210> 29 <211=~ 21 <212=> DNA
<213:> Mycobacterium cuheerculosis <220:>
<223> 16S-3s sequencing, primer <400> 29 21 cgtcacccca ccaacaagct 1;
<210> 30 <211> 147 <212> DNA
<21'3> Mycobacteriunn. tuberculosis <220>
<223> 16S/rrs (streptomycin resistance;) <400> 30 cgtgggtgat ctgccctgca cttcgggat~ agcctgggaa actgggtcta ataccggata 60 ggaccacggg atgcatgtct tgtggtgg~~a. agcgctttag cggtgtggga tgagcccgcg 120 gcctatcagc ttgttggtgg ggtgacg 14 <210> 31 <211> 21 <212> DNA
<213> Mycobacterium tubercuiosis <220>
<223> embB-F amplification primer <400> 31 cggcaagctg gcgcaccttc a 21 <210> 32 <211> 21 <212> DNA
<213> Mycobacterium tubercurosis <220>
<223> embB-R amplification primer <400> 32 agccagcaca ctagcccggcl; 21 <210> 33 <211> 21 <212> DNA
<213> Mycobacterium tuberc:u~losis <220>
<223> embB-Ss seuqencing primer PCTlCA99/01177 <400> 3 3 cggcaagctg gcgcaccttc a 21 _ 11 <210> 34 <211> 21 <212> DNA
<213> Mycobacterium tuberculosis <220>
<223> embB-3s sequencing pri~rner <400> 34 agccagcaca ctagcccggc g 21 <210> 35 <211> 300 <212> DNA
<213> Mycobacterium tuberculosis <220>
<223> embB (ethambutol resistance) <400> 35 cggcatgcgc cggctgattc cggcaa;~ctg gcgcaccttc accctgaccg acgccgtggt 60 gatattcggc ttcctgctct ggcatgtc~~t cggc.gcgaat tc~;tcggacg acggctacat 120 cctgggcatg gcccgagtcg ccgaccacgc cggctacat~; tccaactatt tccgctggtt 180 cggcagcccg gaggatccct tcggct;ggta ttacaacctg caggcgctga tgacccatgt 240 cagcgacgcc agtctgtgga tgcg~;c~t~;cc agacctggcc gccgggctag tgtgctggct 300 <210> 36 <211> 20 <2I2> DNA
<213> Mycobacterium tuberculosis <220>
<223> pncA-F amplification larimer <400> 36 atgcgggcl,~t tgatcatcgt 20 <210> 37 <211> 20 <212> DNA
<213> Mycobacterium tuberculosis _ 1~
<220>
<223> pncA-F amplification yrimer <400> 37 tcaggagctg caaaccaact 20 <210> 38 <211> 20 <212> DNA
<213> Mycobacterium tuberculosis <220>
<223> pncA-Ss sequencing primer <400> 38 atgcgggcgt tgatcatcgt 20 <210> 39 <211> 20 <212> DNA
<213> Mycobacterium tubcrc;ulasis <220>
<223> pncA-3s sequencing primer <400> 39 tcaggagctg caaaccaact 20 <210> 40 <211> 561 <212> DNA
<213> Ivlycobacterium tub<;rc;ulosis <220>
<223> pncA (pyrazinamide, r~:sistarace) <400> 40 atgcgggegt tgatcatcgt cgacgt~~;cag aacgacttct gcgagggtgg ctcgctggcg 60 gtaaccggtg gcgccgcgct ggccc;~cgcc atcagcgact acctggccga agcggcggac 120 taccatcacg tcgtggcaac caaggael~tc cacatcgacc cl;ggtgacca cttctccggc 180 acaccggact attcctcgtc gtggccaccg cattgcgtca ~;cggtactcc cggcgcggac 240 ttccatccca gtctggacac gtcggcaatc gaggcggtgt ;ctacaaggg tgcctacacc 300 WO 00/36142 PC'TICA99/01177 __ ggagcgtaca gcggcttcga aggagtc:~;ac gagaacggca cgccactgct gaattggctg 360 cggcaacgcg gcgtcgatga ggtcgatgtg gtcggtattg c:caccgatca ttgtgtgcgc 420 cagacggccg aggacgcggt acgcairtggc ttggccacca gggtgctggt ggacctgaca 480 gcgggtgtgt cggccgatac cacc~ytc:gcc gcgctggagg agatgcgcac cgccagcgtc 540 gagttggttt gcagctcctg a 561 <210> 41 <211> 20 <212> DNA
<213> Mycobacterium tuberculosis <220>
<223> gyrA-F amplification prumer <400> 41 cagctacatc gactatgcga 20 <210> 42 <211> 20 <212> DNA
<213> Mycobacterium tubercsrlosis <220>
<223> gyrA-K amplification l~r~imer <400> 42 gggcttcggt gtacctcatc 20 <210> 43 <211> 20 <212> DNA
<213> Mycobacterium tuberculosis <220>
<223> gyrA-Ss sequencing primer <400> 43 cagctacatc gactatgcga 20 <210> 44 <211> 20 <212> DNA
<213> Mwcobacterium tuberc~~losis <220>
<223> gyrA-3s sequencing priuner <400> 44 gggcttcggt gtacctcatc 20 <210> 45 <211 > 420 <212> DNA
<213> Mycobacterium tuberculosis <220>
<223> gyrA (fluoroquiniloneiciprofloxacin resistance) <400> 4~
cgaccggatc gaaccggttg acatcg;a~;ca ggagatgcag cgcagctaca tcgactatgc 60 gatgagcgtg atcgtcggcc gcgcgctgcc ggaggtgcgc gacgggctca agcccgtgca 120 tcgccgggtg ctctatgcaa tgttcgatt~; cggcttccgc ccggaccgca gccacgccaa 180 gtcggcccgg tcggttgccg agaccatggg caactaccac c;cgcacggcg acgcgtcgat 240 ctacgacagc ctggtgcgca tggcc;cagcc ctggt.cgctg cgctacccgc tggtggacgg 300 ccagggcaac ttcggctcgc caggcw~itga cccaccggcg gcgatgaggt acaccgaagc 360 ccggctgacc ccgttggcga tggaga4tgct gagggaaatc l;acgaggaga cagtcgattt 420 <210> 46 <211> 20 <212> DNA
<213> Mycobacterium tuberculosis, <220>
<223> 23S-F amplification primer <400> 46 cgaaattcct tgtcgggtaa 20 <210> 47 <211> 20 <212> DNA
<213> Mycobacterium tuberculosis WO 00/36142 ~ 5 PCT/CA99/01177 <220>
<223> 23S-R amplification primer <400> 47 gtatttcaac aacgactcca 20 <210> 48 <21 I > 20 <212> DNA
<213> Mycobacterium tuberculosis <220>
<223> 23S-Ss sequencing primer <400> 48 cgaaattcct tgtcgggtaa 20 <210> 49 <211> 20 <212> DNA
<213> Mycobacterium tuberculosis <220>
<223> 23S-3s sequencing primer <400> 49 gtatttcaac aacgactcca 20 <210> 50 <211> 300 <212> DNA
<213> Mycobacterium tuberculosis <220>
<223> 23S (macrolidelazithromycin resistance) <400> 5 G
gccccagtaa acggcggtgg taacta~taac catcctaagg tagcgaaatt ccttgtcggg 60 taagttccga cctgcacgaa tggcgtaacg acttcccaac tgtctcaacc atagactcgg 120 cgaaattgca ctacgagtaa agatgctcgt tacgcgcggc aggacgaaaa gaccccggga 180 ccttcactac aacttggtat tggtgttcgg tacggtttgt gtaggatagg tgggagactt 240 tgaagcacag acgccagttt gtgtgg~~~~tc gttgttgaaa taccactctg atcgtattgg 300
tuberculc~srfs {dine probe assay-Tuna?elc~, heat these probes, xely oa prior knowledge of antibiotic resistartco-sssocSated mutations ~n the rpoF3 gears. lViucatians autsida the region opZr~ vy ~.z ~tvl,ra ui new L11t7t3kio~s riot; included xn iha prose roelCtail cau~d still coufet resistance, but would not be delectet~ usirr~ this pra$ttci is it's pc'cscrst ~arm-Alastracts of the lnterscieua~e Conference ~:a Antimicral~isl Agen~a $oa Chemotherapy", vol. 34, page R63 (1994r) describes lice applieatzon of automeced DNA
seduonae xnclyaic of hap65 to specia~cm c~fisolau'~ previously-identified as being~9'.
rut~era~lasis.
~~MENDED SHEET
WO 00/36142 PCT/CA99/Ol 177 y ._ Thus, there remains a need for a method for detecting antibiotic-resistance mutations in clinical M. tuberrz~leosis 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 n'ethod. It is a further object of this invention to provide amplification and cycle sequencing primer acts, and kits containing such primer sets, for use in the characterization of antibiotic resistance mutations in M. tuberculosis.
~11>1~3I~C9~Y~tlt~tl Amplification and cycle sequencing primer sets have been developed for the detection and analysis of antibicetic resistance-associated mutations in defined regions of the rpoB (rifampin), katG (isaniarid), oxyR-ahpC P'F; (isoniazid), mabA
(isoniazid), rpsL/sl2 (streptomycin), 16S/rrs (streptonuycin), embB (ethambutol), pncA
(pyrazinamide), gyrA
(ciprofioxacin) and 23 S (azithrc°4mycin) genes. Cling these primer sets and the OPENGENETM automated DNA sequencing system, a protocol has been developed which permits both the rapid detection c>f M tuber~culn.~~is and the identification of antibiotic resistance-associated mutations in a series of geese 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' thc~ treatment of tuberculosis. The tests are employed in a hierarchical manner orr both ~,1~'l~ smear-positive or smear-negative samples to determine both the presence and antibiot:ic;-rwesistance of M: tuberculosis in a given sample. This method permits the simultaneous determination ofIVI. tubercnlosi,s presence in a sample and the antibiotic-resistance profile to a.ri entire panel of antibiotics. Standard methods require from 2-6 weeks to culture ll.~ tubercr,rl~~sis and additional time to establish antibiotic resistance.
Although DNA sequence-based (genotypicj tests are not intended to replace the traditional culture-based (phenotypic] mcetlrc>ds, 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 knowan testing protocols for M. tuberculosis; and _. -I _ Fig. 2 shows a hierarchical assay scheme for evaluating M. tuberculosis type in accordance with the invention.
In accordance with the invention, regions of the genome ofM. tuberculosis associated with antibiotic resistance are amplified and sequenced using specifically designed amplification and sequencing primer-s~. Various techniques for amplification are known, including the basic PCR amplif icdatian 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 incorparated 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 cwGle sequencing steps.
In theory, thc. selectic>n of primers to amplify and sequence a known region of interest should be straightfor'~~ard. In fact, however, because of the possibility of primer binding to other sites, campiic ations arising from secandary structure, and other factors which are not fully understood, sa~n~: primers perform better than others for amplification and sequencing of the same regiorn .of interest The present invention provides primers which have been optimized for the arriplific:ation and sequencing of regions associated with each c>f the ten known types of antibi~rtic resistance 'fhese primer sets are shown below, along with the sequence of the genes tluar: 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-rner, bp2201-2220 5' TAC GGT C'GG CG.A tiC.°-C" CiA'r CC 3° SEQ. ID NO. 1 rpoB-R amplification primer, ~'.0-mer, bp2611-2592 5' TAC GGC GTT TCG A'Ti'~i AA(, CC ~' SEQ ID NO. 2 rpoB-Ss sequencing primer, '?0-rner, bp220 k-2"'2 0 5' TAC G(.iT CGG CGA GC'T c:JAT CC 3' SEQ ID NO. 3 rpoB-3 s sequencing primer, <:Ci-mer, bp261 I -~2'S92 5' TAC GGC GTT TCG ATG AAC CC 3" SEQ ID NO. 4 SEQ.ID. NO
caacc gccgcctgcgrar-ggr~gg~ aaarraarr~
tc:
t 2161aaaccgacgac:atcgaccai:gg :
2221aaaaccagatccgggtc~gc~c:ar_gtcgc-ggatggagcgggtggtccgggag cggatgacca 2281cccaggacgtc;gaggccaar.c:ac'<~ccgcac~acgttgatcaacatccggccg gtggtcgccg 2341cgatcaaggagttcr_tc:g~l:ac:cagcc:agctgagccaattcatggaccag aacaacccgc 2401tgtcggggttc~acccac:a;~;cgccga<agtcggcg<-tggggcccggcggt ctgtcacgtg 2461agcgtgccggc3ctggac:;gcq,~gacgtgcacccgt~rgcactacggccgg atgtgcccga :~-2521tcgaaacccct:gaggg~=tc,~vaacatcggtctgatc<3c~c:cgctgtcggtg tacgcgcggg 2581tcaacccgt<:.ggqtt:a~~gaaacgc:cgtac:cgcaaggtggtcgacggc gtc~gttagcg t katG (isoniazid resistance]
katG-F amplification primer, :.~0~-mer, bp'722-741 5' ATG CrGG CTG ATC.' Tt'4<:' G'TCi AA :3' SEQ ID NO. 6 katG-R amplification primer-, ::()-mer, by I 250-~ I 231 5' GGT GTT CCA CJCC' AI:iC' CiAC CrC'. :B SEQ ID NO. 7 katG-Ss sequencing primer 2t)-.nner, bp722-741 5' ATG CrGG CTG .AT(', T,t~(:: GTG AA 3' SEQ ID NO. 8 katG-3s sequencing primer, 2t).-mer, by125()-1~?31 5' GGT C1TT CCA CiCC; ACiC', GAC'. G(: :i' SEQ ID NO. 9 SEQ
ID
NO.
661 gctcggcgatgagcgt.t~~,.:aqo:ggtG3agcgggat.ct.ggagaacccgctggccgcggtgca 721 gatggggctgatctac~gt:.~aa.cccg~;eiggggccgaacggc:aacccggaccccat:ggccgc 7B1 ggcggtcgacattcgcg<i~_~ac~gtt:~c~c~c~cgcatggccatc~aacgacgtcgaaacagcggc - -841 gctgatcgtcggcggt:.c:~i-:a~:r.v~ttcc~gtaagacccatggcgccggcccggccgatctggt -901 cggccccgaar_ccgaggc:c:g:::tccgragqagcagatgggcttgggctggaactagctcgta -961 tggcaccggaaccggt:a,~<~gacgcgatcaccagcggcatc:gaggtcgtatggacgaacac 1021cccgacgaaatgggacva.ac,ac~tttc~tcc~agatcca.gtacggctacgagtgdgagctgac -_ (, .~
1081 gaagagccct gctggcgc: t = ggc:aat ac:ac: cgccaac~gac ggcgccggtg ccgc~caccat 1141 cccggacccg .tt:cgc~cgcxc~~ cacj~ggcgctc ccc:~gacgatg <:aggccactg acct:ctcgct 1201 gcgggtggat ccgat:ct<=t 3 agc:ggatc:ac ~,~r~-~-r~y <~rq~aac-acc-ccgaggaatt 1261 ggccgacgag tt:cgc:caa:~c~~ cct:ggta~::aa grctgatc_rac c::gagacatgg gtcc:cgttgc oxyR-aphC intergenic region ( PR) PR-F amplification primer, 2(i-r/ ~er, bp451-470 5' ACC AC;T GCT T'fG C;CCI (~(~".A C:C 3' SEQ >D NO. 11 PR-R amplification primer, 2t)-rner, bp687-668 5' CCG ATG AGA GCG GT'G tIGC'. TG 3' SEQ ID NO. 12 PR-Ss sequencing primer, 20-~mer, b1r451-470 5' ACC ACT GCT TTG CC(:l ( "~~''"A C~C 3' SEQ 1D NO. 13 PR-3s sequencing primer, 20-rr~er, bp687-E>68 5' CCG ATG ACiA GCG GTG ,~-ICiC' TG 3' SEQ ID NO. 14 SEQ ID NO. 15 361 atgccctggg ggtgcacc~ygac::cggca.t:c ::c~a=~cacc.gc tcgc:cgcaac gtcgactggc 421 tcat<~tcgag aatgcttgc~g~~aca_actga ;3- a, grrr ~a,~gr~ar~r gcggcgaacg 481 cgcgaagccc ggccacgg~~:- ggc:t:agcac:c: ::~:~tt;g_qr_ggc gatgccgata aatatggtgt 541 gatatatcac ctttgcct:g;: c:ac~c:gactt:c ~~:ggcacgat ggaatgtcgc aaccaaatgc 601. at:tgtccgct ttgatgat:g<:. ggac~agt:oat: _x~~cactgcr_a accattggcg atcaat:tccc 661 cgcctac.~aa~-c a cvac,~;;~~ w -.caacgg t_.gaccr_gtcc aaggtcgacg ccaagr.agcc 721 cggcgactac ttc~accact~u tr_<~c:cagtga :~~:3aacacc;:a ggeaagtggc gggtggtgtt mabA (isoniazid resistance) mabA-F amplification primer, fit)-mer, bp56-7's 5' CCT CGC TGC CCA GA.A :~'lCiCx GA :>' SEQ ID NO. 16 mabA-R amplification primer, 20-mer, bp:303-284 5' ATC'. CCG CGG T'T'T CC'f C'CG tJT 3' SEQ ID NO 17 mabA-Ss sequencing primer, 20.-mer., bp56-75 5' CCT CGC TGC CCA GAA AGCi GA 3k SEQ ID NO. 18 _ WO 00/36142 PCTICA99/Oi 177 mabA-3s sequencing primer, ''>Cf-mer, bp303-284 5' ATC CCC CGG TTT C;C'1" C'.t:.G GT 3' SEQ ID NO. 19 SEQ ID NO. 20 1 agcgcgacat acc:agcag,::g r_vaattcgtag c;qcgtcaata cacccgcagc caggg 61 ~t-,gcccagayQaaatcc::~t: cat~3gtcgaa c~t:gtgct:~ag tcacaccgac aaacgtcacg 121 agcgtaaccc~ cagtgcga-aa gttcccc3ccg c.;aaatcg~~ag ccacgttacg ctcgtggaca 181 taccgatttc gg~::cc<jgc::c. c:ggcgaga~g a. t.aggttgtc ggggtgactg ccac<~gccac 241 tgaaggggcc aa'r3CC_'(_'CCcit.. tcgtatcc,~g t:tcagt-_c:ctg gttgc~raaaa aaaac-~aaaa 301 gatcgggctg gcgat~c:gc<ic:: agcggctggc t:c~c<:gacggc cacaaggtgg ccgtcaccca rpsL/s12 (streptomycin resistance) sl2-F amplification primer, 2~)-~rver, bpl-20 5' CGG TAG ATG CCA A(:'~' /akI"C' CA 3' SEQ ID NO. 21 s12-R amplification primer, 20-rrner, bp384-36 5' GCA TCA GCC C'TT CTt'. t:"1"i' C'.T 3' SEQ ID NO 22 sl2-Ss sequencing primer, 20-rric~r, bpl-20 5' CGG TAG ATG CCA AC'.C', tj~'fC: CA 3' SEQ ID NO. 23 sl2-3s sequencing primer, 20-nnc~r, bp384-36S
5' GCA TCA GC,C CTT CTt:' t~'TT C'T 3' SEQ ID NO. 24 SEQ LD NO. 25 1 ~~Qr cad~cat- ~~~,--~ g~cac~ctggtc: ::gcaagggt:c gtcgggacaa gatcagtaag 61 gtca<~gaccg cggct_ctg,~~=. qggc:agc:<:cca ;:ag:~gtcgtg gtgtatgcac ccgcgtgtac 121 accaccactc cgaagaag~:vcv gaacacgc~rg ;..vac:ggaagg ttgc:ccgcgt gaagttgacg 181 agtcaggtcg aggtracgg~_ gta<:att:c~cc ~gcgagggcc acaacctgca ggagcactcg 241 atggtgctgg tg<:gcggc~3~.~t acc~ggtc~aac~ .:~acr~tgcct_g gtgtgcgcta caagat:catc 301 cgcggttcgc tggatacc.w gggt:gtc:aag aaccgc:aaac aggr_acgcag ccgttacggc 361 gctaagaagg agaagggctct atgr_ca<:gca ~ggggcc°cgc gcccaagcgt ccgttggtca 16S/rrs (streptomycin resistance) 16S-F ampliftcation primer, :; l -rt~er. bp5-25 5' GGT GAT CTG CCC'. TGC.'. ACT 'rCG 3' SEQ ID NO. 26 16S-R amplification primer, '? 1-mer, by 14'7-127 _g_ 5' CGT CAC CCC ACC AAC' ~'~~G (:TG 3" SEQ ID NO. 27 16S-Ss sequencing primer, 21 ~-rrur, bp5-25 5' GGT GA.T CTG CC',C TGC'. ACT '1'CG 3" SEQ ID NO. 28 I 6 S-3 s sequencing primer, 2 I ~-nrke;r, by I 47- I 27 5' CGT CAC CCC AC'.C AAC' ~,A(x C'TG 3' SEQ ID NO. 29 SEQ ID NO. 30 1 cgtgggtgat ctgccctgcva cttc:ggg,sra agcctgggaa actgggtcta ataccggata 61 ggaccacggg atgcatgtcvt r:gtc.~gtggaa agcgctta.ag c_qgtgtggga tgagcccgcg 121 gcctatca~~ rr~.~raa~ ;JQrs'Jara embB (ethambutol resistanceJA
embB-F amplification primer, 2 l--mer, bp'7761-'~'.~B I
5' CGG CAA GC'.T GGC GC;~ C'C'C TCA 3' SEQ ID NO. 31 embB-R amplification primer, 2 I -mer., bp8040-8020 S' AGC CAG CAC ACT .AGt, C°CG (JC',G 3 SEQ >D NO. 32 embB-Ss sequencing primer, ~? I -uner, bp77fi I -7781 5' CGG CAA GC'T GGC (iCA C'C'.T 'CCA 3' SEQ ID NO. 33 embB-3s sequencing primer, 2:V-tner, bp8040-8020 5' AGC CAG CAC ACT' AGC: C'CG GCG 3 SEQ 1D NO. 34 SEQ ID NO. 35 7741 cggcatgcgc cggctgatt:c: rac~caaarra,~~cT~~rrc acc:ctgaccg acgccgtggt 7801 gatattcggc r_rcct:gctc:t ggcatgtc:at, cggcgcgaat tcgtcggacg acggctacat 7861 cctgggcatg gcccr~agnccccgacc:ac gc c,ggc_tacatg tcc:aactatt tccgctggtt 7921 cggcagcccg _gaggatc~~ct ~cggctqgta ttacaacc:tg ctggcgctga tgacccatgt 7981 cagc_gacgcc agtcrgt~~~c;a. t.ctc:gcctgcc agacctggcc gc .,gggc'ra~r_gr~crg pncA (pyrazinamide resistance) pncA-F amplification primer, 2 U-mer; by I -20 5' ATG CGG GCG TT(i ATC ATC GT 3' SEQ 1D NO. 36 ., c,. ..
pncA-F amplification primer, 2(~a-mer, bp561-542 5' TCA GGA GCT GCA AAt'. n:AA ('T 3' S:EQ ID NO. 37 pncA-S s sequencing primer, 20~ mer, by l -20 5' ATG CGG GCG T'TG .AT(:: ,~'TC' CiT 3' S:EQ ID NO. 38 pncA-3s sequencing primer, 2.0-.mer, bp561-54~:
5' TCA GGA GCT GC;A AA(~' ~C"AA CT 3' SEQ 1D NO. 39 SEQ ID. NO. 40 1 atgcgggcgt.tgatcatccat:cgacgtgcag~acgacttctgcgagggtggctcg<aggcg 61gtaaccggtggcgcc:gcg~::a:3gcc:cgrg~:ca:tcagcgactacctggccgaagcgqcggac _ 121taccatcacgtcc:3tggca<:u:caaggactr_coacatcgac~ccgggtgaccacttct=ccggc 181acaccggactat t:cct:cg!::c::gt:ggcc<~c:,gcatt:gcg~cagcggtactcccggcgcggac .
241ttccatcccagtc-tg<3ac.~cgtcc3gcaatcc3~,ggcggtgttctacaagggtgcct:acacc:
301ggagcgtacagcggc:ttc::~aaggagtc:gaccaagaacggcacgccactgctgaatt:ggctg 361cggcaacgccXgcgtcgat::~aggtcgat=gtgcttcqgtattgc~~ac:cgatcattgtgtgcgc .
421cagacggccgaggacc3cg~.3t..acgcaat:g<~ct:tggc;cacc:agggtgctggtggacctgaca 481gcgggtgtgtcggccgat~ac:vcaccgtcgcc~cg<:aggar~gagatgcgcaccgccagcgtc 541g3~:$ r~ 9'~a~crc~G-.a__i3 rt-gyrA (fluoroquinitone/ciproflo:xacin resistance) gyrA-F amplification primer, 2(~~ mer., bp2383-x:402 5' CAG CTA CAT C'.GA C'.T:'~ 'fGC tJA 3' SEQ ID NO. 41 gyrA-R amplification primer, 2O.-mer, bp2702-2(i8'.3 5' GGG CTT CGG TGT AC(~' 7"CA TC 3" SEQ ID NO. 42 gyrA-Ss sequencing primer, ~~0~-riser, bp2383-2102 5' CAG CTA CAT CGA CTA 7 GC (JA 3" SEQ m NO. 43 gyrA-3s sequencing primer, ::',0-riser, bp2'7()2-2cp83 S' GGG C'rT CCiG TGT ACC'. TCA TC :3' SEQ ID NO. 44 SEQ >D NO. 45 2341 cgaccggatc gaaccgct.t~:a acatcgagc:a ggagatgcag cgcagctaca tcgactatgc 2401 ~3tgagcgtg atcgtcggcw: grc~cgctgcc ggaggtgcgc gac:gggctca agccc:gtgca 2461 tcgccgggtg ctcr_atgc~~~~.~ tgt:tcgatt;c cggcttrcgc ccggaccgca gccacgccaa - 1 (?
2521 gtcggcccgg tcggttgevc.~ agac:catggg c,aactaccac: ccgcacggcg acgcgtcgat 2581 ctacgacagc ctggtgcgca~ tggc:cca<~ .rc-.:tggtr_gctg c:gctar_ccgc tggtggacgg 2641 ccagggcaac ttcggctcgc: caggcaatga c=cacrggcg gcyarga~yr a~a~c~Qaaac-2701 ~ggctgacc ccgttggc:ge~ tggagatctr~r_ gagggaaatc: gacgaggaga cagtcgattt 23S (macrolide/azithromycin ~rc~sistance) 23S-F amplification primer, 2i:>-mer~, hp2444-241:3 5' CGA AAT TCC TTG T'C(_i (:=it:.:rT AA SEQ ID NO. 46 3' 23S-R amplification primer, 20-mer, bp2683-2664 5' GTA TTT CAA CAA C'.GA (:'T(~ (A 3' SEQ ID NO. 47 23S-5s sequencing primer, 20-mf,r, bp2444-2463 5' CGA AAT TCC TTG TCCi (i(JT' A.A 3' S:EQ ID NO. 48 23S-3s sequencing primer, 2C!-mer, bp2683-2664 5' GTA TTT CAA CAA (:GA ~(~TC (T;A 3' SEQ ID NO. ~49 SEQ B7 NO. 50 2401 gccccagtaa arggrggt::g~ taactataac catcctaagg tag~gaaa r c-c-rt-Qr 2461 ~gttccga cc~tgcac~::~aa t:gc~cgtaacg a~ct:tcc~aac tgtc:tcaacc atagactcgg 2521. cgaaattgca ct:.acgagc::aa agatgc:t-gt tac:gc~gr~ggc aggacgaaaa gacc:ccggga 2581 cctt.cactac aactt;ggt;at t:ggtgttc~gg t.ac:ggt-.tgt gt:aggatagg tggc;agactt 2641. tgaagcacag .ac::gc~cvagv: t t c~tcrgg_~~rryrt-~rrgaaa rar~cactctg atcgtattgg To facilitate detection of the sequencing products using real-time fluorescence-based electrophoresis apparat.u:~ (for example, a~ 'Visible Genetics OPENGENETM sequencer), at least one of the sequencing l7rirners 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 cyanune dye such as (;'~'S.0 OR CY5.5.
The primers of the invention are suitably packaged in a kit. This kit will contain individually packagec:l 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), anti preferably includes the primer sets for a plurality of resistance genes, most preferably the primer sets for all ten resistance genes.
_ WO 00136142 PCT/CA99101177 -ll.-The suitable pr~ol.c:~co1 for the utilization of these primer sets in the evaluation ofM. tuberculosis in clinical ;samples utilizes P(.".R amplification, followed by cycle sequencing. DNA for use in the test is obtained. i=rorn a .sample of sputum (IOOuI-lOml). The sputum sample is processed according to Standard Infectious Disease/Public Health Laboratory practices (Mycobac~e~riology Bench Manual, Laboratory Services Branch, December 1997, Ontario Ministrwy of 1-Iealti~). The sputum sample is homogenized, decontaminated and concentr,~t~~d. Mycobacterial DNA is prepared directly from a portion of the concentrated cell sediment ( 100-:?OOuI) using standard DNA extraction methods or commercially available kits.
Amplification of the DNA is performed using the amplification primer sets described above. PCR reagent:. can be prepared for individual reactions, or may be prepared as a master mix which can be u,e;d fcsr multiple t<~sts e.g., 10 PC;R
reactions. Exemplary combinations of reagents are su mmarized in the 1E'ollowing table.
PCR mix 1 PCR 10 PCRs final conc. / PCR
genomic DNA (20ng~'u1) 1 Oul 20ng (~0. 5 fM) lOX PCR buffer I 2.5ui 25.Ou1 IX
2.5mM dNTP mix (l : l :l :1 y 2.5u~ 25 0ul 250uM
DMSO 1 3u1 l3.Oul 5%
Taq DNA polymerase (11J) 0.2u1 2.Oul 1 unit molecular grade water I e:i 5ul 165.Ou1 MTB gene primers ( I OuM) 1.0u1 1 O.OuI l Opmol per primer total volume per PCR 25.Ou1 If the master mix as shown in the column labeled I 0 PCRs is utilized, the mastermix contains all the necessary PCR reagerkt~ other than the gc.nomic DNA. In this example, 24.Ou1 of the mastermix is added to a PCR tube, that already contains I .Oul of genomic DNA, prior to the addition of the mineral oil overlay and placement in the thermocycler.
The genomic CI~NA preparation utilized must be of sufl'rcient quality and integrity for robust and reproducible PCR Suitable DNA preparation can be obtained using WO 00/36142 PCT/CA99/0117"
c! ._ the Gentra PuregeneTM DNA. i=.~alation kit. The kit components are appropriate for the isolation of genomic DNA from blood, fresh or ~TOZen tissue, archival material and paraf~m-embedded tissue.
Each primer pair is used to amplify a single gene region under the following conditions:
1. Denaturation 't4C 5 minutes 1 cycle 2. Denaturation '~4C 30 seconds Annealing 6t)C 3f> seconds 35 cycles Extension '72C 60 seconds 3. Extension 72C s minutes 1 cycle 4. Hold !~"C
The temperature change durin;the cycles of the step 2 is desirably set to ramp at a rate of 1 °C/sec.
After amplifican~ion, :>..Oul from the 25.Ou1 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 sequerACing reaction. (comparable verification of sequencing purity is performed on each of the other amplification products Sequence anaty sis is e;arried 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 ~;en~e.
For initial sequence analysis of rpoB, the rpoB-Ss primer should be used. For confrrmatory sequence analysis the rpoB-3s primer should be used. For each template to be sequenced, aliquot 3.Ou1 of each of the nucleotide ternunation mixes into four separate tubes marked <A>, <C>, v~G> anci ~~"lrv=~ and store on ice until the sequencing mastermix is prepared.
rpoB template 2 Oul lOX VGI Sequenace TM butler 2 Sul _. WO 00/36142 PCT/CA99/0117T
-13v DMSO 3.Sul 2.5uM dye-sequencing primer 2 Ou1 PCR grade water 9 Oul X10 diluted The moc . 1 n c 0 5 ul total volume ~. ~? Oul Mix the DMSO and other corriponents in the mastermix well by repeated pipetting (5 times) with a micropipette. Store t'~~e mastermix on ic;e until ready to add to the nucleotide termination mixes.
Add 5.Ou1 of the mastermix to c,ach of the four marked tubes containing the nucleotide termination m~xe~;, Add ~.Oul lightweight mineral c>il to each of the four marked tubes containing the mastermix and nucleotide termination mixc~.s.
Store on ice u.mtil ready to load into the thermocycler.
paramgterc fir .nculg cycl c .d ~
I . Denaturation 94C 5 minutes I X
2. Denaturation 94C 30 seconds Annealing 60C 30 seconds 35X
Extension 7~C.' 60 seconds 3. Extension 7~C 5 minutes 1X
4. Hold ~''C.
The temperature change during; the cycles of the step 2 is desirably set to ramp at a rate of 1 °C/sec.
At the end of the cycle sequencing reaction add 6.Ou1 of the Stop Loading Dye directly to each of the four to b es to stop the :sequencing reaction. The sequencing samples are heated at 95°C for '~ minutes and then placed on ice before loading 2.Oul (from a total volume of l4ul) on the CLIPPERT"~' ~~equencer. 'The remainder of the sequencing reaction can be stored at -20°C for subsequent use.
._ WO 00/36142 _ 1.1 -The CL.IPPERT~'~ sequences is sex-up as described in the OPENGENE
Automated DNA Sequencin~;l .yystem User Manual. Run parameters for the CLIPPERT'M
sequences are 54°C/ 1300voltsl t).Ssec sampling/35min run/50% laser power. The samples loaded included 2 ul each of t.hc, Forward and reverse sequencing reaction products for the target gene, differentially labaled, for example with C1~'S.0 and CY5.5 cyanine dye labels.
Once the run is completed, tire base-called data is analysed by comparison of the test sequence to the rpoB sequence database in GEl\fELIBRAR.IANTM. This sequence alignment.
compares the test sequence to the standard control sequence and allows sequence ambiguities to be assessed. Once edited the°'test sequence ran be screened far antibiotic resistance-associated mutations using Cil=:1'vfELIBRARIA1'~'rM
3'estircg far multiple types oi~ 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 M. tubercz~Jnci.c' are only performed on AFB smear-positive sputum samples. 'These methods serve; as canfirrnatary tests for the presence ofM.
tuberculosis. In addition to these molecular hiological method;,, the culture-based procedures for M.
tuberculosis detection (Bac Tec liquid culture, agar plate and slant cultures) are performed in parallel. AFB smear-negative sputum samples 2~re processed with only the culture-based detection procedures (Figure 1 ).
In the present irmention bath AFB smear-positive and smear-negative sputum samples can be processed using; bath culture-!eased 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 X000 bacteria/ul the AFB stain will be negative. In addition to this is the observ~ltion that the decontamination procedure used to prepare the sputum sample usually kills l0'-20°io 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 a.re culture-positive for M.
tuberculosis (Ontario Public Health Laboratory). 'This level of myc;abacteria is easily detected by molecular biological methods and is t:h~~refore 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 Ivl.tuberculosis infections (rpoB, katG, rpsL/s12, PK, embB, pncA) WO 00/36142 PC'T/CA99/01177 lc,-and detect other species of nryc;cibacteria (23 S) in the absence of M.
tuberculosis (Figure 2).
Group I analyses are performed before both Group Il and Group III. Group I
analysis will provide information on the antibiotic resistance; status to rifampin (rpoB), isoniazid (katG), streptomycin (rpsL/sl2) and a.~:iithronrycin (23~>). In addition the rpoB
amplification indicates the presence ofM. tuberculcrsn,s and in the absf;nce of rpoB amplification the 23S sequence allows identification of most oi.' the clinically relevant mycobacterial species. Group II
analysis provides information care antibiotic resistance mutations in the "second-line"
antibiotics used to treat M. tr~b~erculosis infections namely, isoniazid (PR), ethambutol (embB), pyrazinamide (pncA) and civprofloxacin (gyrA). Group III contains gene targets in which mutations associated vvitt~ 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. tuherculo..w.s infections, and thus the associated antibiotic resistance mutation patterns. ~~ary geographically. As shown in Figure 2 the culture-based methods are performed in parallel. The molecular biological methods would permit the idenstification ofM: tuberculosis from both AFB smear-positive and smear-negative sln.~tum samples a.nd further provide information on the antibiotic resistance profile ~;~f t hese samples yell in advance of current culture-based methods. This information wcmuld be crucial tc~ the initiation of appropriate and effective antibiotic treatment regimens fiar t~~! tubercr~lrr.~is infections.
Examples A pool of DI~,~ samples from antibiotic-sensitive M. tuberculosis isolates was obtained from the L,C>7C; I--Ieauth and Wc;lfares Canada. Ottawa, Ontario.
Wild-type sequence traces, for all gene targets hn~3~wn to harbor mutations in antibiotic-resistant M. tuberculosis, were generated.
A panel of 1_:)rd:'~ samples from five phenotypic streptomycin-resistant M.
tuberculosis isolates was obt;~ined lronr the Public Health Laboratory, Ontario Ministry of Health, 'Coronto, Ontario. ~Cr~ese Ir)NA samples were examined for antibiotic resistance-associated mutations in all l~~ antibiotic gene targets listed above.
Streptomycin resistance-associated mutations were dc;tected in the rpsL/s12 gene in four isolates.
Parallel antibiotic resistance-associated mutatic~rws in the rpoB ~:rifampin), kat(.i (isoniazid), PR (isoniazid), _. 1 ~ ..
embB (ethambutol), pncA (pyr~~inarnide) and g~rrA (ciprofloxacin) genes were also identified which underscores the impor~:a~~ce of examining all the gene targets for first-line antibiotics used in the treatment of A~ trrber«culosi.s. A sunnmary of the results is shown in 'Cable 1.
The following references are cited herein and are incorporated herein by reference for all states which aliow such incorporation.
DL Wiliiams et al. (1f94). Characterisation of rifampin resistance in pathogenic mycobacteria. Antimicrob Agents (:hemother 38: 2380-2386.
WH Haas et al. (19971. Molecular analysis of katG gene mutations in strains of Mycobacterium tuberculosis complex from Africa« Antimicrob Agents Chemother 41: 1b01-1603.
S Sreevatsan et al. (1!197). Analysis of the oxyR-ahpC region in isoniazid-resistant and -susceptible Mycobacterium tuberculosis complex organisms recovered from disea:;ed humans and animals in diverse localities. Antimicrob Agents Chemother 41i : 600-606.
A Teienti et al. (19941. C:enotypic assessment of isoniazid and rifampin resistance in Mycobacterium tutrerculosis: a blind study at the reference laboratory level.
Antimicrob Agents C'hemother 35: ''19-723.
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: 1134-640.
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-ti40.
MA Lety et al. (1997).. A single point mutation in the embB gene is responsible for resistance to etharnbutol in Mycobacterium smegmatis. Antimicrob Agents C',hemother 41: 2629-2633.
A Scorpio et al. (1997). Characaterisation of pncA mutations in pyrazinamide-resistant Mycobacterium tuberculosis. Antimicrob Agents Chemather 41: 540-543.
_ [7 _ C Xu et al. (1996). Fluoroquinilone resistance associated with specific gyrase mutations in clinical isolates of muitidrug-resistant Mycobacterium tuberculosis.
J Infect Disease 174- 1127-1130.
ICA Nash et al. ( 1990. 4Jenetic basis of macrolide resistance in Mycobacterium avium isolated from p;~tients with disseminated disease. Antimicrob Agents Chemother 39: 2625-2(i30.
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<110> Visible Genetics Inc.
Shipman, Robert <120> Method and Kit for the ~'~.°haracterization of Antibiotic-Resistance Mutaauons in Mycobacterium tuberculosis <130> VGEN.P-055-WO
<140>
<141>
<150> 60/111,794 <151> 1998-12-11 <160> 50 <170> PatentIn Ver. 2.1 <210> 1 <211 > 20 <212> DNA
<213> Mycobacterium tuberculosis <220>
<223> rpoB-F amplification primer <400> 1 tacggtcggc gagctgatcc 20 <210> 2 <211> 20 <212> DNA
<213> Mycobacterium tuberculosis <220>
<223> rpoB-R amplification primer <400> 2 tacggcgttt cgatgaaccc 20 2_ <210> 3 <211> 20 <212> DNA
<213> Mycobacterium tuberculosis <220>
<223> rpoB-Ss sequencing primer <400> 3 tacggtcggc gagctgatcc 20 <210> 4 <211> 20 <212> DNA
<213> Mycobacterium tubc:rnulosis <220>
<223> rpoB-3s sequencing primer <400> 4 tacggcgttt cgatgaaccc 20 <210>
<211 > 480 <212> DNA
<213> Mycobacterium tuberculosis <220>
<223> rpoB (rifarnpin resistance) PCTICA99/Ol 177 <400> 5 aaaccgacga catcgaccac ttcg~~c.aacc gccgcctgcl; tacggtcggc gagctgatcc 60 aaaaccagat ccgggtcggc atgtcgcgga tggagcggg;t ggtccgggag cggatgacca 120 cccaggacgt ggaggcgatc acac:cgcaga cgttgatcaa catccggccg gtggtcgccg 180 cgatcaagga gttcttcggc accagccagc tgagccaatt catggaccag aacaacccgc 240 tgtcggggtt gacccacaag cgcc~yactgt cggcgctgl;g gcccggcggt ctgtcacgtg 300 agcgtgccgg gctggaggtc cgc~;~acgtgc acccgtcgc:a ctacggccgg atgtgcccga 360 tcgaaacccc tgaggggccc aaca~tcggtc tgatcggctc gctgtcggtg tacgcgcggg 420 tcaacccgtt cgggttcatc ,gaasrc;ccgt accgcaagg~t ggtcgacggc gtggttagcg 480 <210> 6 <211> 20 WO 00/36142 PCT'/CA99/01177 <212> DNA
<213> Mycobacterium tuberculosis <220>
<223> katG-F amplification primer <400> 6 atggggctga tctacgtgaa 2«
<210> 7 <211> 20 <212> DNA
<213> Mycobacterium tuberculosis <220>
<223> katG-R amplification primer <400> 7 ggtgttccag ccagcgacgc 20 <210> 8 <211> 20 <212> DNA
<213> Mycobacterium tuberculosis <220>
<223> katG-Ss sequencing pranler <400> 8 atggggctga tctacgtgaa 20 <210> 9 <211> 20 <212> DNA
<213> Mycobacterium tubercv.losis <220>
<223> katG-3s sequencing primer <400> 9 ggtgttccag ccagcgacgc 20 WO 00/36142 PC'T/CA99/01177 _ 4 <210> 10 <211 > 660 <212> DNA
<213> Mycobacterium tuberculosis <220>
<223> katG (isoniazid resistance) <400> 10 gctcggcgat gagcgttaca gcggtaagcg ggatctggag aacccgctgg ccgcggtgca 60 gatggggctg atctacgtga acccggaggg gccgaacggc aacccggacc ccatggccgc 120 ggcggtcgac attcgcgaga cgtttcl;~;cg c,atggccatg aacgacgtcg aaacagcggc 180 gctgatcgtc ggcggtcaca ctttcf;gtaa gacccatggc ~c;cggcccgg ccgatctggt 240 cggccccgaa cccgaggctg ctccg~rigga gcagatggg,c ttgggctgga agagctcgta 300 tggcaccgga accggtaagg acgcgatcac cagcggcatc gaggtcgtat ggacgaacac 360 cccgacgaaa tgggacaaca gtttcc~rc:ga gatcctgtac p;gctacgagt gggagctgac 420 gaagagccct gctggcgctt ggcaat~~cac c:gccaaggac; ggcgccggtg ccggcaccat 480 cccggacccg ttcggcgggc caggg,cgctc cccgacgatg ctggccactg acctctcgct 540 gcgggtggat ccgatcta.tg agcggat4:ac gcgtcgctgg caggaacacc ccgaggaatt 600 ggccgacgag ttcgccaagg cctgg'.a~Gaa gctgatccac cgagacatgg gtcccgttgc 660 <210> 11 <211> 20 <212> DNA
<213> Mycobacterium tubi:rc~ulosis <220>
<223> PR-F amplification pricier <400> 11 accactgctt tgccgccacc 2t1 <210> 12 <211> 20 <212> DNA
<213> Mycobacterium tuberculosis <220>
<223> PR-R amplification primer <400> 12 ccgatgagag cggtgagctg 2t~
WO 00/36142 ~ PCT/CA99/01177 <210> 13 <21 I > 20 <212> DNA
<213> Mycobacterium tuber~;ulosis _ <220>
<223> PR-Ss sequencing p~°inaer <400> 13 accactgctt tgccgccacc 20 <210> 14 <211>20 <212> DNA
<213> Mycobacterium tuber<;ulosis <220>
<223> PR-3s sequencing prinu~,r <400> 14 ccgatgagag cggtgagctg 20 <210> 15 <211 > 420 <212> DNA
<213> Mycobacterium tuberculosis <220>
<223> oxyR-ahpC intergen~c :~e;gion (PRj <400> 15 atgccctggg ggtgcaccga gaccggcttc cgaccaccgc tcgccgcaac gtcgactggc 60 tcatatcgag aatgcttgcg gcactgct~~a accactgctt tgccgccacc gcggcgaacg I20 cgcgaagccc ggccacggcc ggctal;c°acc tcttggcggc; gatgccgata aatatggtgt gatatatcac ctttgcctga cagcgacttc acggcacgat ggaatgtcgc aaccaaatgc 240 attgtccgct ttgatgatga ggagagtcat gccactgcta accattggcg atcaattccc 300 cgcctaccag ctcaccgctc tcatcggr.~;g tgacctgtcc aaggtcgacg ccaagcagcc 360 cggcgactac ttcaccacta tcaccagt~;a cgaacacc;ca ggcaagtggc gggtggtgtt 420 <2I0> 16 <211> 20 <212> DNA
<213> Mycobacterium tube.rctrlosis <220>
<223> fabG-H amplification primer <400> 16 cctcgctgcc cagaaaggga 20 <210> 1~
<211> 20 <212> DNA
<213> Mycobacterium tuber~:wlosis <220>
<223> fabG-R amplification primer <400> 17 atcccccggt ttcctccggt 20 <210> 18 <211> 20 <212> DNA
<213> Mycobacterium tubt~rr,ulosis <220>
<223> fabG-Ss sequencing primer <400> 1 g cctcgctgcc cagaaaggga 20 <210> 19 <211> 20 <212> DNA
<213> Mycobacterium tuberculosis <220>
<223> fabG-3s sequencing primer <400> 19 atcccccggt ttcctccggt 20 WO 00/36142 PCT/CA99/011??
<210> 20 <211> 360 <212> DNA
<213> Mycobacterium tuberculosis <220>
<223> fabG (isoniazid resistance;) <400> 20 agcgcgacat acctgctgcg caattc~,tag ggcgtcaata cacccgcagc cagggcctcg 60 ctgcccagaa agggatccgt catggtc;gaa gtgtgctgag tcacaccgac aaacgtcacg 120 agcgtaaccc cagtgcgaaa gttcc:c~;c~cg gaaatcgcag, c;cacgttacg ctcgtggaca 1$0 taccgatttc ggcccggccg cggcgagacg ataggttgtc ggggtgactg ccacagccac 240 tgaaggggcc aaacccccat tcgtatcc.cg ttcagtcctg gtt:accggag gaaaccgggg 300 gatcgggctg gcgatcgcac agc~;g~a~;gc tgccgacggc cacaaggtgg ccgtcaccca 360 <210> 21 <211> 20 <212> DNA
<213> Mycobacterium tuberculosis <220>
<223> s12-F amplification primer <400> 21 cggtagatgc caaccatcca 20 <210> 22 <211> 20 <212> DNA
<213> Mycobacterium tubc:rcaulosis <220>
<223> s12-R ampliLicatior~ primer <400> 22 gcatcagccc ttctccttct 20 <210> 23 <211> 20 <212> DNA
<213> Mycobacterium tuberculosis <220>
<223> s12-Ss sequencing primer <400> 23 cggtagatgc caaccatcca 20 <210> 24 <211> 20 <212> DNA
<213> Mycobacterium tubc;rc~;ulosis <220>
<223> sl2-3s sequencing pricrcer <400> 24 gcatcagccc ttctccttct 20 <210> 25 <211 > 420 <212> DNA
<2I3> Mycobacterium tuberculosis <220>
<223> rpsL/sl2 (streptomycin resistance) <400> 25 cggtagatgc caaccatcca gcagctggtc cgcaagggtc gtcgggacaa gatcagtaag 60 gtcaagaccg cggctctgaa gggca~gcccg cagcgtcgt~; gtgtatgcac ccgcgtgtac 120 accaccactc cgaagaagcc gaactcggcg cttcggaagg~ ttgcccgcgt gaagttgacg 180 agtcaggtcg aggtcacggc gtacattccc ggcgagggc:c~ acaacctgca ggagcactcg 240 atggtgctgg tgcgcggcgg ccgg~tgaag gacctgcctg; gtgtgcgcta caagatcatc 300 cgcggttcgc tggatacgca gggtgts;aag aaccgcaaa.c aggcacgcag ccgttacggc 360 gctaagaagg agaagggctg atgcc:acgca. aggggccc~;c gcccaagcgt ccgttggtca 420 <210> 26 <211> 21 <212> DNA
<213> Mycobacterium tube~r~:ulosis <220>
<223> 16S-F amplificatic>n ~:rime;r PCT/CA99/0117?
<400> 26 ,,1 ggtgatctgc cctgcacttc g ' <210> 27 <211> 21 <212> DNA
<213> Mycobacterium tubm-culosis <220>
<223> 16S-R amplification ~~rimer <400> 27 21 cgtcacccca ccaacaagct g <210> 28 <211> 21 <212> DNA
<213> Mycobacterium tuberculosis <220>
<223> 16S-Ss sequencing lorimer <400> 28 21 ggtgatctgc cctgcacttc g <210> 29 <211=~ 21 <212=> DNA
<213:> Mycobacterium cuheerculosis <220:>
<223> 16S-3s sequencing, primer <400> 29 21 cgtcacccca ccaacaagct 1;
<210> 30 <211> 147 <212> DNA
<21'3> Mycobacteriunn. tuberculosis <220>
<223> 16S/rrs (streptomycin resistance;) <400> 30 cgtgggtgat ctgccctgca cttcgggat~ agcctgggaa actgggtcta ataccggata 60 ggaccacggg atgcatgtct tgtggtgg~~a. agcgctttag cggtgtggga tgagcccgcg 120 gcctatcagc ttgttggtgg ggtgacg 14 <210> 31 <211> 21 <212> DNA
<213> Mycobacterium tubercuiosis <220>
<223> embB-F amplification primer <400> 31 cggcaagctg gcgcaccttc a 21 <210> 32 <211> 21 <212> DNA
<213> Mycobacterium tubercurosis <220>
<223> embB-R amplification primer <400> 32 agccagcaca ctagcccggcl; 21 <210> 33 <211> 21 <212> DNA
<213> Mycobacterium tuberc:u~losis <220>
<223> embB-Ss seuqencing primer PCTlCA99/01177 <400> 3 3 cggcaagctg gcgcaccttc a 21 _ 11 <210> 34 <211> 21 <212> DNA
<213> Mycobacterium tuberculosis <220>
<223> embB-3s sequencing pri~rner <400> 34 agccagcaca ctagcccggc g 21 <210> 35 <211> 300 <212> DNA
<213> Mycobacterium tuberculosis <220>
<223> embB (ethambutol resistance) <400> 35 cggcatgcgc cggctgattc cggcaa;~ctg gcgcaccttc accctgaccg acgccgtggt 60 gatattcggc ttcctgctct ggcatgtc~~t cggc.gcgaat tc~;tcggacg acggctacat 120 cctgggcatg gcccgagtcg ccgaccacgc cggctacat~; tccaactatt tccgctggtt 180 cggcagcccg gaggatccct tcggct;ggta ttacaacctg caggcgctga tgacccatgt 240 cagcgacgcc agtctgtgga tgcg~;c~t~;cc agacctggcc gccgggctag tgtgctggct 300 <210> 36 <211> 20 <2I2> DNA
<213> Mycobacterium tuberculosis <220>
<223> pncA-F amplification larimer <400> 36 atgcgggcl,~t tgatcatcgt 20 <210> 37 <211> 20 <212> DNA
<213> Mycobacterium tuberculosis _ 1~
<220>
<223> pncA-F amplification yrimer <400> 37 tcaggagctg caaaccaact 20 <210> 38 <211> 20 <212> DNA
<213> Mycobacterium tuberculosis <220>
<223> pncA-Ss sequencing primer <400> 38 atgcgggcgt tgatcatcgt 20 <210> 39 <211> 20 <212> DNA
<213> Mycobacterium tubcrc;ulasis <220>
<223> pncA-3s sequencing primer <400> 39 tcaggagctg caaaccaact 20 <210> 40 <211> 561 <212> DNA
<213> Ivlycobacterium tub<;rc;ulosis <220>
<223> pncA (pyrazinamide, r~:sistarace) <400> 40 atgcgggegt tgatcatcgt cgacgt~~;cag aacgacttct gcgagggtgg ctcgctggcg 60 gtaaccggtg gcgccgcgct ggccc;~cgcc atcagcgact acctggccga agcggcggac 120 taccatcacg tcgtggcaac caaggael~tc cacatcgacc cl;ggtgacca cttctccggc 180 acaccggact attcctcgtc gtggccaccg cattgcgtca ~;cggtactcc cggcgcggac 240 ttccatccca gtctggacac gtcggcaatc gaggcggtgt ;ctacaaggg tgcctacacc 300 WO 00/36142 PC'TICA99/01177 __ ggagcgtaca gcggcttcga aggagtc:~;ac gagaacggca cgccactgct gaattggctg 360 cggcaacgcg gcgtcgatga ggtcgatgtg gtcggtattg c:caccgatca ttgtgtgcgc 420 cagacggccg aggacgcggt acgcairtggc ttggccacca gggtgctggt ggacctgaca 480 gcgggtgtgt cggccgatac cacc~ytc:gcc gcgctggagg agatgcgcac cgccagcgtc 540 gagttggttt gcagctcctg a 561 <210> 41 <211> 20 <212> DNA
<213> Mycobacterium tuberculosis <220>
<223> gyrA-F amplification prumer <400> 41 cagctacatc gactatgcga 20 <210> 42 <211> 20 <212> DNA
<213> Mycobacterium tubercsrlosis <220>
<223> gyrA-K amplification l~r~imer <400> 42 gggcttcggt gtacctcatc 20 <210> 43 <211> 20 <212> DNA
<213> Mycobacterium tuberculosis <220>
<223> gyrA-Ss sequencing primer <400> 43 cagctacatc gactatgcga 20 <210> 44 <211> 20 <212> DNA
<213> Mwcobacterium tuberc~~losis <220>
<223> gyrA-3s sequencing priuner <400> 44 gggcttcggt gtacctcatc 20 <210> 45 <211 > 420 <212> DNA
<213> Mycobacterium tuberculosis <220>
<223> gyrA (fluoroquiniloneiciprofloxacin resistance) <400> 4~
cgaccggatc gaaccggttg acatcg;a~;ca ggagatgcag cgcagctaca tcgactatgc 60 gatgagcgtg atcgtcggcc gcgcgctgcc ggaggtgcgc gacgggctca agcccgtgca 120 tcgccgggtg ctctatgcaa tgttcgatt~; cggcttccgc ccggaccgca gccacgccaa 180 gtcggcccgg tcggttgccg agaccatggg caactaccac c;cgcacggcg acgcgtcgat 240 ctacgacagc ctggtgcgca tggcc;cagcc ctggt.cgctg cgctacccgc tggtggacgg 300 ccagggcaac ttcggctcgc caggcw~itga cccaccggcg gcgatgaggt acaccgaagc 360 ccggctgacc ccgttggcga tggaga4tgct gagggaaatc l;acgaggaga cagtcgattt 420 <210> 46 <211> 20 <212> DNA
<213> Mycobacterium tuberculosis, <220>
<223> 23S-F amplification primer <400> 46 cgaaattcct tgtcgggtaa 20 <210> 47 <211> 20 <212> DNA
<213> Mycobacterium tuberculosis WO 00/36142 ~ 5 PCT/CA99/01177 <220>
<223> 23S-R amplification primer <400> 47 gtatttcaac aacgactcca 20 <210> 48 <21 I > 20 <212> DNA
<213> Mycobacterium tuberculosis <220>
<223> 23S-Ss sequencing primer <400> 48 cgaaattcct tgtcgggtaa 20 <210> 49 <211> 20 <212> DNA
<213> Mycobacterium tuberculosis <220>
<223> 23S-3s sequencing primer <400> 49 gtatttcaac aacgactcca 20 <210> 50 <211> 300 <212> DNA
<213> Mycobacterium tuberculosis <220>
<223> 23S (macrolidelazithromycin resistance) <400> 5 G
gccccagtaa acggcggtgg taacta~taac catcctaagg tagcgaaatt ccttgtcggg 60 taagttccga cctgcacgaa tggcgtaacg acttcccaac tgtctcaacc atagactcgg 120 cgaaattgca ctacgagtaa agatgctcgt tacgcgcggc aggacgaaaa gaccccggga 180 ccttcactac aacttggtat tggtgttcgg tacggtttgt gtaggatagg tgggagactt 240 tgaagcacag acgccagttt gtgtgg~~~~tc gttgttgaaa taccactctg atcgtattgg 300
Claims (14)
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, said sequencing procedure generating sequencing fragments for evaluation of the ~poB, katG, rpsL/s~2 and 23S genes for the presence of antibiotic-resistance inducing mutations when M. tuberculosis is present in the sample, wherein primers for the sequencing of the rpoB gene are selected such that the generation of sequencing produces for this gene is indicative of the presence of M. tuberculosis in the sample; and (c) if M. tuberculosis is detected as a result of generation of sequencing products for the rpoB gene is step (b), performing a second sequencing procedure, with or without prior amplification, on the sample to evaluate at least one additional M.
tuberculosis gene for the presence of antibiotic-resistance inducing mutations.
(a) obtaining a sputum sample suspected of containing M. tuberculosis, (b) performing a first sequencing procedure, with or without prior amplification, on the sample, said sequencing procedure generating sequencing fragments for evaluation of the ~poB, katG, rpsL/s~2 and 23S genes for the presence of antibiotic-resistance inducing mutations when M. tuberculosis is present in the sample, wherein primers for the sequencing of the rpoB gene are selected such that the generation of sequencing produces for this gene is indicative of the presence of M. tuberculosis in the sample; and (c) if M. tuberculosis is detected as a result of generation of sequencing products for the rpoB gene is step (b), performing a second sequencing procedure, with or without prior amplification, on the sample to evaluate at least one additional M.
tuberculosis gene for the presence of antibiotic-resistance inducing mutations.
2. The method of claim 1, wherein the second sequencing procedure evaluates PR, ~mbB pncA and gyrA genes for the presence of antibiotic-resistance mutations.
3. The method of claim 1, further comprising the step of performing a third sequencing procedure when M. tuberculosis was detected in step (b), separate from the first and second sequencing procedures, to evaluate 16S/~s 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 ka~G 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/s12 is performed using amplification primers as et forth in Seq. ID Nos.
21 and 22 and sequencing primers as set forth in Seq. ID Nos. 23 and 24.
21 and 22 and sequencing primers as set forth in Seq. ID Nos. 23 and 24.
7. The method of any of claims 1 to 6, wherein the second sequencing procedures for 23S 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 procedures for PR is performed using implification 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 procedures 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 procedures for ~mbB is performed using amplification primers as set forth in Seq. ID Nos. 31 and 32 and sequencing primers as set forth in Seq. ID Nos. 33 and 34.
11. The method of any of claims 1 to 10, wherein the second sequencing procedures 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 procedures for 16S/~s is performed using amplification primers as set forth in Seq. ID Nos. 26 and 27 and sequencing primers as set forth in Seq. ID Nos. 28 and 29.
-~~-
-~~-
13. The method of any of claims 2 to 12, wherein the third sequencing procedures 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 pairs of amplification primers and matched pairs of sequencing primers for amplification and sequencing the at least the rpoB, katG, rpsL/s12 and 23S genes of M. tuberculosis, characterized in that the amplificaiton and sequencing primer pairs include at least one combination of primer pairs 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. ID Nos. 33 and 34;
(h) amplification primers of Seq. ID Nos. 36 and 37 in combination and sequencing primers of Seq. ID Nos. 38 and 39;
(i) amplification primers of Seq. ID Nos. 41 and 42 in combination and sequencing primers of Seq. ID Nos. 43 and 44;
(j) amplification primers of Seq. ID Nos. 46 and 47 in combination and sequencing primers of Seq. ID Nos. 48 and 49.
(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. ID Nos. 33 and 34;
(h) amplification primers of Seq. ID Nos. 36 and 37 in combination and sequencing primers of Seq. ID Nos. 38 and 39;
(i) amplification primers of Seq. ID Nos. 41 and 42 in combination and sequencing primers of Seq. ID Nos. 43 and 44;
(j) amplification primers of Seq. ID Nos. 46 and 47 in combination and sequencing primers of Seq. ID Nos. 48 and 49.
Applications Claiming Priority (3)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| US11179498P | 1998-12-11 | 1998-12-11 | |
| US60/111,794 | 1998-12-11 | ||
| PCT/CA1999/001177 WO2000036142A1 (en) | 1998-12-11 | 1999-12-10 | METHOD AND KIT FOR THE CHARACTERIZATION OF ANTIBIOTIC-RESISTANCE MUTATIONS IN $i(MYCOBACTERIUM TUBERCULOSIS) |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA2354234A1 true CA2354234A1 (en) | 2000-06-22 |
Family
ID=22340482
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA002354234A Abandoned CA2354234A1 (en) | 1998-12-11 | 1999-12-10 | Method and kit for the characterization of antibiotic-resistance mutations in mycobacterium tuberculosis |
Country Status (5)
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|---|---|
| EP (1) | EP1137803A1 (en) |
| JP (1) | JP2002532103A (en) |
| AU (1) | AU1543000A (en) |
| CA (1) | CA2354234A1 (en) |
| WO (1) | WO2000036142A1 (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2021195794A1 (en) * | 2020-04-01 | 2021-10-07 | Universidad Católica Del Maule | Molecular diagnosis method and system for the detection of different strains of mycobacterium tuberculosis |
Families Citing this family (15)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| KR100419897B1 (en) * | 2000-09-14 | 2004-03-03 | 김상재 | Probes for detection of isoniazid resistant mycobacterium tuberculosis |
| US7247430B2 (en) | 2001-08-14 | 2007-07-24 | Institut Pasteur | Compositions and methods for detecting multidrug resistant strains of M. tuberculosis having mutations in genes of the mutT family |
| GB0324650D0 (en) * | 2003-10-22 | 2003-11-26 | Acolyte Biomedica Ltd | Using nucleic acids for clinical microbiology testing |
| RU2343197C2 (en) * | 2004-12-20 | 2009-01-10 | Московский городской научно-практический центр борьбы с туберкулезом | Method of diagnostics of sensitivity of mycobacterium tuberculosis strains to fluoroquinolones |
| EP2179041A4 (en) * | 2007-06-22 | 2010-12-22 | Ibis Biosciences Inc | Compositions and methods for identification of subspecies characteristics of mycobacterium tuberculosis |
| AU2010264468B2 (en) | 2009-06-23 | 2015-01-22 | Gen-Probe Incorporated | Compositions and methods for detecting nucleic acid from Mollicutes |
| RU2409680C1 (en) * | 2009-07-20 | 2011-01-20 | Московский городской научно-практический центр борьбы с туберкулёзом Департамента здравоохранения города Москвы | Diagnostic technique for of mycobacterium tuberculosis strain sensitivity to aminoglycosides |
| CN106661606B (en) | 2014-06-13 | 2021-12-21 | Q-莱纳公司 | Method for detecting and characterizing microorganisms |
| GB201507026D0 (en) | 2015-04-24 | 2015-06-10 | Linea Ab Q | Medical sample transportation container |
| GB2554767A (en) | 2016-04-21 | 2018-04-11 | Q Linea Ab | Detecting and characterising a microorganism |
| EP3781702A4 (en) * | 2018-04-20 | 2022-01-19 | Longhorn Vaccines and Diagnostics, LLC | METHODS FOR DETECTION OF AUTOLOGOUS BLOOD DOPING |
| US20220145366A1 (en) * | 2020-06-17 | 2022-05-12 | The Translational Genomics Research Institute | Early detection of drug-resistant mycobacterium tuberculosis |
| GB202013928D0 (en) * | 2020-09-04 | 2020-10-21 | Quadram Inst Bioscience | Method and compositions for drug resistance screening |
| CN112526119B (en) * | 2020-11-13 | 2023-02-07 | 北京元恩生物技术有限公司 | Azithromycin enzyme-linked immunoassay kit and application thereof |
| WO2023085783A1 (en) * | 2021-11-09 | 2023-05-19 | 주식회사 엔젠바이오 | Gene amplification composition for detecting and determining drug resistance of mycobacterium tuberculosis complex, and uses thereof |
Family Cites Families (3)
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|---|---|---|---|---|
| US5643723A (en) * | 1994-05-26 | 1997-07-01 | Roche Molecular Systems, Inc. | Detection of a genetic locus encoding resistance to rifampin in mycobacterial cultures and in clinical specimens |
| DE69532675T2 (en) * | 1994-06-09 | 2005-02-24 | Innogenetics N.V. | PROCESS FOR DETECTION OF THE ANTIBIOTIC RESISTANCE SPECTRUM OF MYCOBACTERIUM SPECIES |
| US5834189A (en) * | 1994-07-08 | 1998-11-10 | Visible Genetics Inc. | Method for evaluation of polymorphic genetic sequences, and the use thereof in identification of HLA types |
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1999
- 1999-12-10 WO PCT/CA1999/001177 patent/WO2000036142A1/en not_active Ceased
- 1999-12-10 AU AU15430/00A patent/AU1543000A/en not_active Abandoned
- 1999-12-10 JP JP2000588389A patent/JP2002532103A/en active Pending
- 1999-12-10 CA CA002354234A patent/CA2354234A1/en not_active Abandoned
- 1999-12-10 EP EP99957813A patent/EP1137803A1/en not_active Withdrawn
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2021195794A1 (en) * | 2020-04-01 | 2021-10-07 | Universidad Católica Del Maule | Molecular diagnosis method and system for the detection of different strains of mycobacterium tuberculosis |
Also Published As
| Publication number | Publication date |
|---|---|
| JP2002532103A (en) | 2002-10-02 |
| WO2000036142A1 (en) | 2000-06-22 |
| WO2000036142A9 (en) | 2001-07-12 |
| AU1543000A (en) | 2000-07-03 |
| EP1137803A1 (en) | 2001-10-04 |
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