EP1137803A1 - Procede et necessaire pour la caracterisation de mutations produites par une resistance aux antibiotiques chez le mycobacterium tuberculosis - Google Patents

Procede et necessaire pour la caracterisation de mutations produites par une resistance aux antibiotiques chez le mycobacterium tuberculosis

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Publication number
EP1137803A1
EP1137803A1 EP99957813A EP99957813A EP1137803A1 EP 1137803 A1 EP1137803 A1 EP 1137803A1 EP 99957813 A EP99957813 A EP 99957813A EP 99957813 A EP99957813 A EP 99957813A EP 1137803 A1 EP1137803 A1 EP 1137803A1
Authority
EP
European Patent Office
Prior art keywords
seq
nos
sequencing
primers
amplification
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP99957813A
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German (de)
English (en)
Inventor
Robert Shipman
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Visible Genetics Inc
Original Assignee
Visible Genetics Inc
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Visible Genetics Inc filed Critical Visible Genetics Inc
Publication of EP1137803A1 publication Critical patent/EP1137803A1/fr
Withdrawn legal-status Critical Current

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Classifications

    • 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
    • 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

Definitions

  • This application relates to a method and kit for the characterization of antibiotic resistance mutations in Mycobacterium tuberculosis, and particularly to the evaluation of such mutations in clinical samples.
  • M. tuberculosis can be resistant to all antibiotics that are currently used to treat tuberculosis patients.
  • Antibiotic resistance is due to acquired point mutations in target genes in the genome of M. tuberculosis. These point mutations render the organism insensitive to the action of the antibiotic by preventing it's uptake or activation, or by altering the antibiotic target.
  • the observed antibiotic resistance in M. tuberculosis is not due to an episome-encoded resistance gene transferred from one strain to another and, like other bacteria, is single-step (one point mutation), high-level resistance.
  • M. tuberculosis is carried out on DNA recovered from sputum samples handled according to Standard Infectious Disease/Public Health Laboratory practices.
  • the sputum sample is decontaminated and a cell sediment isolated. This cell sediment is the sample source for all routine procedures used in the detection and isolation of M. tuberculosis. Portions of this sample are used in BacTec cultures for selective growth of tuberculosis, agar plate/agar slant cultures for .
  • tuberculosis tuberculosis, acid-fast bacilli (AFB) smears for mycobacteria detection and molecular biological methods for the detection of M. tuberculosis and atypical mycobacteria.
  • AZA acid-fast bacilli
  • Mycobacterial DNA is prepared directly from the decontaminated sputum cell sediments according to standard procedures and this mycobacterial DNA is used in the various molecular biological detection procedures.
  • the methods presently in use for the detection of M. tuberculosis are either PCR-based or probe-based. These tests are used primarily on AFB smear-positive samples. Since the presence of M. tuberculosis has already been established by the AFB smear, these tests are used primarily in a confirmatory capacity as opposed to a diagnostic capacity. Furthermore, these tests provide no information on the potential antibiotic resistance of these M. tuberculosis samples.
  • Azithromycin 23 S rRNA sequence nucleotide 2568A e
  • Probe-based tests do exist for the determination of rifampin resistance inM tuberculosis (line probe assay-InnoTek), but these probes rely on prior knowledge of antibiotic resistance-associated mutations in the rpoB gene. Mutations outside the region covered by the probe or new mutations not included in the probe cocktail could still confer resistance, but would not be detected using this product in it's present form.
  • a method for detecting antibiotic-resistance mutations in clinical M. tuberculosis sputum samples which is capable of detecting mutations in all of the gene targets which confer antibiotic resistance. It is an object of the present invention to provide such a method. It is a further object of this invention to provide amplification and cycle sequencing primer sets, and kits containing such primer sets, for use in the characterization of antibiotic resistance mutations in M. tuberculosis.
  • Amplification and cycle sequencing primer sets have been developed for the detection and analysis of antibiotic resistance-associated mutations in defined regions of the rpoB (rifampin), katG (isoniazid), oxyR-ahpC PR (isoniazid), mabA (isoniazid), rpsL/sl2 (streptomycin), 16S/rrs (streptomycin), embB (ethambutol), pncA (pyrazinamide), gyrA (ciprofloxacin) and 23 S (azithromycin) genes.
  • rpoB rifampin
  • katG isoniazid
  • oxyR-ahpC PR isoniazid
  • mabA isoniazid
  • rpsL/sl2 streptomycin
  • 16S/rrs streptomycin
  • embB ethambutol
  • pncA pyrazinamide
  • gyrA cipr
  • the present invention uses a series of tests designed to detect antibiotic resistance-associated mutation in all gene targets for all antibiotics presently in use for the treatment of tuberculosis.
  • the tests are employed in a hierarchical manner on both AFB smear-positive or smear-negative samples to determine both the presence and antibiotic-resistance of M. tuberculosis in a given sample. This method permits the simultaneous determination of M. tuberculosis presence in a sample and the antibiotic-resistance profile to an entire panel of antibiotics.
  • Fig. 1 shows known testing protocols for M. tuberculosis
  • Fig. 2 shows a hierarchical assay scheme for evaluating M. tuberculosis type in accordance with the invention.
  • regions of the genome of tuberculosis associated with antibiotic resistance are amplified and sequenced using specifically designed amplification and sequencing primers.
  • Various techniques for amplification are known, including the basic PCR amplification techniques described in US Patent No. 4,683,202, which is incorporated herein by reference.
  • various techniques for sequencing are know, some of which require prior amplification and some of which do not. Included among known sequencing techniques are those disclosed in US Patents Nos. 5,834,189 and 5,789,168, which are incorporated herein by reference.
  • the primers of the invention can be used in any of these sequencing formats, although the invention is exemplified below using separate amplification and cycle-sequencing steps.
  • primers which have been optimized for the amplification and sequencing of regions associated with each of the ten known types of antibiotic resistance. These primer sets are shown below, along with the sequence of the genes that they are used to analyze. In the gene sequences, the locations of the primers are underlined.
  • rpoB rifampin resistance
  • katG isoniazid resistance
  • katG-F amplification primer 20-mer, bp722-741
  • PR-F amplification primer 20-mer, bp451-470
  • PR-R amplification primer 20-mer, bp687-668
  • mabA-F amplification primer 20-mer, bp56-75
  • rpsL/sl2 streptomycin resistance
  • 16S-R amplification primer 21-mer, bpl47-127 5' CGT CAC CCC ACC AAC AAG CTG 3' SEQ ID NO.27
  • pncA pyrazinamide resistance
  • pncA-F amplification primer 20-mer, bpl-20
  • gyrA fluoroquinilone/ciprofloxacin resistance
  • gyrA-F amplification primer 20-mer, bp2383-2402
  • At least one of the sequencing primers is preferably labeled with a flourescent label.
  • the label is selected for compatibility with the sequencing apparatus employed, and may be, for example, fluorescein or a cyanine dye such as CY5.0 OR CY5.5.
  • the primers of the invention are suitably packaged in a kit.
  • This kit will contain individually packaged amplification and sequencing primers sets for each resistance gene to be evaluated by the kit.
  • the kit of the invention includes at least 4 primers (two amplification and two sequencing primers), and preferably includes the primer sets for a plurality of resistance genes, most preferably the primer sets for all ten resistance genes.
  • the suitable protocol for the utilization of these primer sets in the evaluation of tuberculosis in clinical samples utilizes PCR amplification, followed by cycle sequencing. DNA for use in the test is obtained from a sample of sputum (lOOul-lOml).
  • the sputum sample is processed according to Standard Infectious Disease/Public Health Laboratory practices (Mycobacteriology Bench Manual, Laboratory Services Branch, December 1997, Ontario Ministry of Health).
  • the sputum sample is homogenized, decontaminated and concentrated.
  • Mycobacterial DNA is prepared directly from a portion of the concentrated cell sediment (100-200ul) using standard DNA extraction methods or commercially available kits.
  • PCR reagents can be prepared for individual reactions, or may be prepared as a master mix which can be used for multiple tests e.g., 10 PCR reactions. Exemplary combinations of reagents are summarized in the following table.
  • PCR mix 1 PCR 10 PCRs final cone. / PCR genomic DNA (20ng/ul) l.Oul 20ng
  • the mastermix contains all the necessary PCR reagents other than the genomic DNA.
  • 24.0ul of the mastermix is added to a PCR tube, that already contains 1.Oul of genomic DNA, prior to the addition of the mineral oil overlay and placement in the thermocycler.
  • the genomic DNA preparation utilized must be of sufficient quality and integrity for robust and reproducible PCR. Suitable DNA preparation can be obtained using the Gentra PuregeneTM DNA isolation kit.
  • the kit components are appropriate for the isolation of genomic DNA from blood, fresh or frozen tissue, archival material and paraffin- embedded tissue.
  • Each primer pair is used to amplify a single gene region under the following conditions:
  • the temperature change during the cycles of the step 2 is desirably set to ramp at a rate of C/sec.
  • rpoB-5s primer For initial sequence analysis of rpoB, the rpoB-5s primer should be used. For confirmatory sequence analysis the rpoB-3s primer should be used. For each template to be sequenced, aliquot 3.0ul of each of the nucleotide termination mixes into four separate tubes marked ⁇ A>, ⁇ C> ⁇ G> and ⁇ T> and store on ice until the sequencing mastermix is prepared.
  • thermocycler Store on ice until ready to load into the thermocycler.
  • the temperature change during the cycles of the step 2 is desirably set to ramp at a rate of l°C/sec.
  • the samples loaded included 2 ul each of the forward and reverse sequencing reaction products for the target gene, differentially labeled, for example with CY5.0 and CY5.5 cyanine dye labels.
  • the base-called data is analysed by comparison of the test sequence to the rpoB sequence database in GENELLBRARIANTM. This sequence alignment compares the test sequence to the standard control sequence and allows sequence ambiguities to be assessed. Once edited the test sequence can be screened for antibiotic resistance- associated mutations using GENELLBRARIANTM.
  • Fig. 2 Testing for multiple types of antibiotic-resistance mutations can be carried out using a hierarchical assay, as summarized in Fig. 2.
  • Fig. 2 At present molecular biological methods for the detection of tuberculosis are only performed on AFB smear-positive sputum samples. These methods serve as confirmatory tests for the presence ofM tuberculosis.
  • the culture-based procedures forM tuberculosis detection (BacTec liquid culture, agar plate and slant cultures) are performed in parallel.
  • AFB smear-negative sputum samples are processed with only the culture-based detection procedures ( Figure 1).
  • both AFB smear-positive and smear-negative sputum samples can be processed using both culture-based and molecular biological methods.
  • a limitation of the AFB stain methodology is it's limit of detection. If a sputum sample has a mycobacterial concentration of less than 5000 bacteria/ul the AFB stain will be negative.
  • the decontamination procedure used to prepare the sputum sample usually kills 10-20% of the mycobacteria present. This would suggest that two-thirds of the AFB smear-negative samples potentially contain mycobacteria.
  • 10-20%> of the AFB smear-negative samples are culture-positive forM tuberculosis (Ontario Public Health Laboratory). This level of mycobacteria is easily detected by molecular biological methods and is therefore incorporated in the present invention.
  • the hierarchy proposed incorporates tests that specifically detect M. tuberculosis (rpoB), detect mutations in genes associated with resistance to the "first-line" antibiotics used to treat M. tuberculosis infections (rpoB, katG, rpsL/sl2, PR, embB, pncA) and detect other species of mycobacteria (23 S) in the absence ofM tuberculosis ( Figure 2).
  • Group I analyses are performed before both Group II and Group III. Group I analysis will provide information on the antibiotic resistance status to rifampin (rpoB), isoniazid (katG), streptomycin (rpsL/sl2) and azithromycin (23 S).
  • rpoB amplification indicates the presence ofM tuberculosis and in the absence of rpoB amplification the 23 S sequence allows identification of most of the clinically relevant mycobacterial species.
  • Group II analysis provides information on antibiotic resistance mutations in the "second-line" antibiotics used to treat M tuberculosis infections namely, isoniazid (PR), ethambutol (embB), pyrazinamide (pncA) and ciprofloxacin (gyrA).
  • Group III contains gene targets in which mutations associated with antibiotic resistance are infrequently found.
  • This protocol permits specific gene targets to be examined according to the local treatment procedures since the both antibiotics used to treat M tuberculosis infections, and thus the associated antibiotic resistance mutation patterns, vary geographically.
  • the culture-based methods are performed in parallel.
  • the molecular biological methods would permit the identification ofM tuberculosis from both AFB smear- positive and smear-negative sputum samples and further provide information on the antibiotic resistance profile of these samples well in advance of current culture-based methods. This information would be crucial to the initiation of appropriate and effective antibiotic treatment regimens forM tuberculosis infections.
  • a panel of DNA samples from five phenotypic streptomycin-resistant M tuberculosis isolates was obtained from the Public Health Laboratory, Ontario Ministry of Health, Toronto, Ontario. These DNA samples were examined for antibiotic resistance- associated mutations in all 10 antibiotic gene targets listed above. Streptomycin resistance- associated mutations were detected in the rpsL/sl2 gene in four isolates.
  • Chemother 41 2629-2633. h A Scorpio et al. (1997). Characaterisation of pncA mutations in pyrazinamide- resistant Mycobacterium tuberculosis. Antimicrob Agents Chemother 41: 540-
  • katG.1 (isoniazid) agc513acc, Ser513Thr agc513acc, Ser513Thr agc513acc, Ser513Thr wt wt
  • gyrA (ciprofloxacin) agc95acc, Se.95Thr agc95acc, Ser95Thr agc95acc, Ser95Thr agc95acc. Ser95T r agc95acc, Ser95Thr

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  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Analytical Chemistry (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Organic Chemistry (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Health & Medical Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Microbiology (AREA)
  • Immunology (AREA)
  • Molecular Biology (AREA)
  • Biotechnology (AREA)
  • Biophysics (AREA)
  • Physics & Mathematics (AREA)
  • Biochemistry (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Abstract

Cette invention concerne des ensembles d'armoceurs de séquençage de cycle et d'amplification qui permettent de détecter et d'analyser des mutations associées à une résistance aux antibiotiques dans des régions prédéterminées des gènes rpoB (rifampine), katG (isoniazide), oxyR-ahpC PR (isoniazide), mabA (isoniazide), rpsL/s12 (streptomycine), 16S/rrs (streptomycine), embB (éthambutol), pncA (pyrazinamide), gyrA (ciprofloxacine) et 23S (azithromycine) du Mycobacterium tuberculosis. Ces amorceurs peuvent être utilisés dans un procédé de détection et de caractérisation de Mycobacterium tuberculosis dans un échantillon. Ce procédé consiste à obtenir un échantillon d'expectoration que l'on suspecte de contenir le M. tuberculosis, à effectuer une première procédure de séquençage, avec ou sans amplification préalable, sur l'échantillon afin de détecter la présence de M. tuberculosis et, si ce dernier est présent, à évaluer les gènes rpoB, katG, rpsL/s12 et 23S afin de déceler la présence d'une résistance aux antibiotiques entraînant des mutations. Si (c) l'on détecte la présence de M. tuberculosis au cours de l'étape (b), on effectue une seconde procédure de séquençage, avec ou sans amplification préalable, sur l'échantillon afin d'évaluer les gènes additionnels et de déceler la présence d'une résistance aux antibiotiques entraînant des mutations.
EP99957813A 1998-12-11 1999-12-10 Procede et necessaire pour la caracterisation de mutations produites par une resistance aux antibiotiques chez le mycobacterium tuberculosis Withdrawn EP1137803A1 (fr)

Applications Claiming Priority (3)

Application Number Priority Date Filing Date Title
US11179498P 1998-12-11 1998-12-11
US111794P 1998-12-11
PCT/CA1999/001177 WO2000036142A1 (fr) 1998-12-11 1999-12-10 Procede et necessaire pour la caracterisation de mutations produites par une resistance aux antibiotiques chez le mycobacterium tuberculosis

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EP1137803A1 true EP1137803A1 (fr) 2001-10-04

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EP (1) EP1137803A1 (fr)
JP (1) JP2002532103A (fr)
AU (1) AU1543000A (fr)
CA (1) CA2354234A1 (fr)
WO (1) WO2000036142A1 (fr)

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KR100419897B1 (ko) * 2000-09-14 2004-03-03 김상재 아이나 내성 결핵균 검사용 프로브
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
EP2179041A4 (fr) * 2007-06-22 2010-12-22 Ibis Biosciences Inc Compositions et procédés permettant d'identifier des caractéristiques de sous-espèces de mycobacterium tuberculosis
CA2768768C (fr) 2009-06-23 2021-08-24 Gen-Probe Incorporated Compositions et procedes pour detecter un acide nucleique a partir de mollicutes
JP6976167B2 (ja) * 2014-06-13 2021-12-08 キュー−リネア エービー 微生物の検出及び特徴付け方法
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
US20190323066A1 (en) * 2018-04-20 2019-10-24 Longhorn Vaccines And Diagnostics, Llc Rapid Methods for the Detection of Microbial Resistance
WO2021195794A1 (fr) * 2020-04-01 2021-10-07 Universidad Católica Del Maule Procédé et système de diagnostic moléculaire pour la détection de différentes souches de mycobacterium tuberculosis
GB202013928D0 (en) * 2020-09-04 2020-10-21 Quadram Inst Bioscience Method and compositions for drug resistance screening
CN112526119B (zh) * 2020-11-13 2023-02-07 北京元恩生物技术有限公司 一种阿奇霉素酶联免疫检测试剂盒及其应用
KR102617096B1 (ko) * 2021-11-09 2023-12-29 주식회사 엔젠바이오 결핵균 검출 및 약제 내성 여부 확인을 위한 유전자 증폭용 조성물 및 이의 용도

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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
PT771360E (pt) * 1994-06-09 2004-07-30 Innogenetics Nv Metodo para a deteccao do espectro de resistencia a antibioticos de especies mycobacterium
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

Non-Patent Citations (1)

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Title
See references of WO0036142A1 *

Also Published As

Publication number Publication date
JP2002532103A (ja) 2002-10-02
WO2000036142A1 (fr) 2000-06-22
AU1543000A (en) 2000-07-03
CA2354234A1 (fr) 2000-06-22
WO2000036142A9 (fr) 2001-07-12

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