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WO2002022872A1 - Procede et kit de pcr multiplexe et oligonucleotides destines a la detection et l'identification de mycobacterium au moyen dudit procede - Google Patents

Procede et kit de pcr multiplexe et oligonucleotides destines a la detection et l'identification de mycobacterium au moyen dudit procede Download PDF

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Publication number
WO2002022872A1
WO2002022872A1 PCT/KR2001/001385 KR0101385W WO0222872A1 WO 2002022872 A1 WO2002022872 A1 WO 2002022872A1 KR 0101385 W KR0101385 W KR 0101385W WO 0222872 A1 WO0222872 A1 WO 0222872A1
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Prior art keywords
mycobacteria
oligonucleotide
seq
primers
multiplex pcr
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Ceased
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PCT/KR2001/001385
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English (en)
Inventor
Hee-Kyung Park
Hyun-Sook Park
Hyun-Jung Jang
Eun-Sil Song
Cheol-Min Kim
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SJHIGHTECH CO Ltd
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SJHIGHTECH CO Ltd
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Priority to AU2001278813A priority Critical patent/AU2001278813A1/en
Publication of WO2002022872A1 publication Critical patent/WO2002022872A1/fr
Anticipated expiration legal-status Critical
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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
    • 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
    • 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
    • C12Q2600/16—Primer sets for multiplex assays

Definitions

  • the present invention relates to a new multiplex polymerase chain reaction (PCR) method and a multiplex PCR kit and oligonucleotides for rapid and specific identification of Mycobacteria species, and more particularly, to a multiplex PCR method capable of detecting Mycobacteria genus and identifying M. tuberculosis (MTB) and non-tuberculosis Mycobacteria (NTM) species thereof in a single PCR using genus-specific and species-specific primers derived from the internal transcribed spacer (ITS) sequences of Mycobacteria.
  • PCR polymerase chain reaction
  • Mycobacteria are major pathogens causing human diseases. In the world, 800 million people are infected every year by Mycobacte um tuberculosis and 300 million people among them die from the infection (Raviglione, M. O, D. E. Snider, and A. Kochi, "Global epidemiology of tuberculosis, morbidity and mortality of a worldwide epidemic," JAMA, 271 :220-226, 1995). Recently, as the number of peoples suffering from AIDS rapidly increases, infections caused by non-tuberculosis Mycobacteria (NTM) are gradually increasing (Barnes, P., A. B. Bloch, P. T. Davidson, and D. E. Snider, "Jr.
  • NTM non-tuberculosis Mycobacteria
  • Bottger "Toward a phylogeny and definition of species at the molecular level within the genus Mycobactehum," Int. J. Syst Bacteriol., 40:323-30 (1990b); Rogall T., T. Flohr, and E. C. Bottger, "Differentiation of Mycobactehum species by direct sequencing of amplified DNA," J. Gen. Microbiol., 136 (Pt9):1915-1920 (1990a)).
  • the 16S rRNA gene has similar nucleotide sequences among some species, there is a limitation in identifying species to a certain extent (Fox, G. E., J. D. and P. J. Jurtshum, "How close is close: 16S rRNA sequence identity may not be sufficient to guarantee species identity," Int. J. Syst. Bacteriol. 42: 166-170 (1992)).
  • IS6110 insertion element has multiple copy numbers in TB complexes of M. tuberculosis, M. africanum, M. bovis, and M. microti, and thus a PCR probe method using IS6110 as a target sequence is used.
  • Mycobactehum tuberculosis which do not have the IS6110 insertion element have been reported and thus may yield pseudo-negative test results (Yuen L. K., B. C. Ross, K. M. Jackson, and B. Dwyer, "Characterization of Mycobactehum tuberculosis strains from Vietnamese patients by Southern blot hybridization," J. Clin. Miclobiol., 31 :1615-1618 (1993)).
  • primers capable of amplifying the IS6110 insertion element and a detection kit therefor are commercially available as TB-PCR and TB Detection Kit (from Bioneer Co., Korea), application of these primers is restricted to certain species and the PCR kit can only detect the presence of TB complex.
  • NTM non-tuberculosis Mycobacteria
  • PCR polymerase chain reaction
  • PCR kit for use in the new PCR method in which detection of Mycobacteria genus and identification of M. tuberculosis (MTB) and non-tuberculosis Mycobacteria (NTM) species can be achieved at the same time through a single PCR using a minimum number of primers. It is a third object of the present invention to provide oligonucleotide primers for use in a new multiplex PCR method in which detection of Mycobacteria genus and identification of M. tuberculosis and non-tuberculosis Mycobacteria species can be achieved at the same time.
  • the first object of the present invention is achieved by a PCR method for simultaneously amplifying at least two target genes in a single tube through a single reaction using at least one oligonucleotide having a common sequence for the at least two target genes to be amplified, as a fixed primer(s), and at least two oligonucleotides each having gene-specific sequences as specific primers. It is preferable that the number of fixed primers is one and the number of specific primers is an integer from 2 to 6.
  • the second object of the present invention is achieved by a multiplex PCR kit comprising: at least one oligonucleotide having a common sequence for at least two target genes to be amplified, as a fixed phmer(s); and at least two oligonucleotides each having species-specific sequences as specific primers, wherein the fixed primer(s) and the specific primers are simultaneously reacted in a single tube.
  • the fixed primer is an oligonucleotide primer having a common sequence for Mycobacteria genus and the specific primers are oligonucleotide primers each having Mycobacteria species-specific sequences, such that identifying Mycobacteria and diagnosing infections caused by Mycobacteria are possible. More preferably, the fixed primer comprises the oligonucleotide having SEQ ID NO. 1 and the specific primers comprise at least two oligonucleotides having SEQ ID NOs. 2 through 6.
  • the fixed primer comprises the oligonucleotide having SEQ ID NO. 1 and the specific primers comprise the oligonucleotides having SEQ ID NOs. 2 and 3
  • detection of Mycobacteria genus and identification of M. tuberculosis can be achieved at the same time.
  • the specific primers comprise the oligonucleotides having SEQ ID NOs. 2 and 6
  • detection of Mycobacteria genus and identification of M. avium can be achieved at the same time.
  • the specific primers comprise the oligonucleotides having SEQ ID NOs. 2, 3 and 6, detection of Mycobacteria genus and identification of M. tuberculosis or M. avium can be achieved at the same time.
  • the third object of the present invention is achieved by an oligonucleotide having SEQ ID NO. 1 for use in detecting Mycobacteria genus, an oligonucleotide having SEQ ID NO. 3 for use in identifying Mycobacterium tuberculosis, an oligonucleotide having SEQ ID NO. 4 for use in identifying Mycobacterium fortuitum as a non-tuberculosis Mycobacteria, an oligonucleotide having SEQ ID NO. 5 for use in identifying Mycobacterium flavescens as a non-tuberculosis Mycobacteria, and an oligonucleotide having SEQ NO. 6 for use in identifying Mycobacterium avium as a non-tuberculosis Mycobacteria.
  • FIG. 1 is a schematic representation of locations of the oligonucleotide primers used in a multiplex polymerase chain reaction (PCR) method according to the present invention, and a map of PCR amplification products;
  • PCR polymerase chain reaction
  • FIG. 2 shows an electrophoresis image taken after a PCR using genus-specific primers ITSF and MYC6 for Panmycobacteria;
  • FIG. 3 shows an electrophoresis image taken after a PCR using species-specific primers MTB10, FOR12, FLA9, and MAC5 for Mycobacteria
  • FIG. 4 shows an electrophoresis image taken after a multiplex PCR according to the present invention on a M. tuberculosis (MTB) and six non-tuberculosis Mycobacteria (NTM) using primers ITSF, MTB10, FOR12, FLA9, MAC5, and MYC6
  • FIG. 5 is a schematic representation of the electrophoresis image of FIG. 4.
  • a multiplex polymerase chain reaction (PCR) method is developed for identifying Mycobacteria and diagnosing infections caused by the same, in which nucleotide sequences of an internal transcribed spacer (ITS) region between the 16S rRNA and 23S rRNA genes, which has genus- and species-specific sequences for Mycobacteria, are used as a target sequence.
  • ITS internal transcribed spacer
  • 23S rRNA genes which contain both conserved and polymorphic sequences for Mycobacteria, a genus-specific primer having the conserved sequence and species-specific primers having the polymorphic sequences are derived.
  • One conserved sequence region is used as a common reverse primer, and four species-specific primers combined with the one genus-specific primer are used as forward primers to obtain PCR products having different sizes.
  • M. tuberculosis (MTB) and non-tuberculosis Mycobacteria (NTM) species can be identified.
  • primers are designed such that PCR products obtained using the primers have different sizes for easy discrimination thereof by gel electrophoresis. All six primers are simultaneously applied to a single PCR to detect the Mycobacteria genus and identify M. tuberculosis and non-tuberculosis Mycobacteria species through the single PCR.
  • one primer pair is used for detection of one target strain.
  • 5 primer pairs i.e., 10 primers
  • the number of reactions required can be reduced by multiplex PCR, the number of primers required cannot be reduced even for the multiplex PCR.
  • the present inventor has researched to reduce the number of primers required to a minimum number and further develop primers having the same reaction conditions, which allows the primers to be simultaneously applied to the same PCR. As a result, detecting the Mycobacteria genus and identifying MTB and NTM species through a single PCR using only six primers can be realized.
  • the PCR primers according to the present invention are synthesized from an ITS sequence of Mycobacteria.
  • the multiplex PCR method according to the present invention for detecting the Mycobacteria genus and at the same time identifying MTB and NTM species through a single reaction uses six primers including species-specific forward primers and one common genus-specific reverse primer.
  • many restrictions are applied, such as ratios of A, G, C, and T of the primers, preventing formation of a dimer between the primers, and prohibiting three or more repetitions of the same sequence.
  • PCR conditions such as concentrations of template DNA, primers, dNTP, and Mg 2+ , reaction temperature, and reaction time, should be appropriate.
  • the reaction conditions should be further restricted when designing the primers.
  • the primers should be designed such that the amplification products have different sizes and can be distinguished from one another on a gel after the PCR.
  • PCR primers for use in detecting the Mycobacteria genus and identifying MTB and NTM species according to the present invention are designed to be appropriate for the multiplex PCR method. All six primers can be simultaneously applied to a single PCR, and thus the Mycobacteria genus can be detected and both MTB and NTM species can be identified through a single PCR with high sensitivity.
  • the PCR primers according to the present invention and the sizes of PCR products obtained using the primers are shown in Table 1 ,
  • the MYC6 (SEQ ID NO. 1 ) is the conversed sequence of Mycobacteria genus and its sense or antisense sequence can be used as a common reverse primer (fixed primer) in the present invention.
  • the ITSF (SEQ ID NO. 2) has the conserved sequence of the 16S RNA region, however is used as a specific primer in the present invention because it is a forward primer used for the detection of Mycobacteria.
  • the MTB10 (SEQ ID NO. 3), FOR12 (SEQ ID NO. 4), FLA9 (SEQ ID NO. 5) and MAC5 (SEQ ID NO. 6) are the specific sequences of MTB or NTM species and their sense or antisense sequences can be used as forward primers (specific primers) in the present invention.
  • FIG. 1 is a schematic representation of the map of amplification products from the multiplex PCR according to the present invention and locations of the primers used for the amplification.
  • Example 1 Incubation of Standard Mycobacteria Strains and Isolation of Genomic DNA
  • Standard strains of Mycobacteria were obtained from the Korean Collection for Type Culture (KCTC) and the American Type Culture Collection (ATCC). The DNA of these strains were isolated using InstaGene matrix (Bio-Rad Co.). 200 ⁇ l InstaGene matrix was put into a 1.5 ml tube. A target strain was incubated in a solid medium (Ogawa plate), one scrapping of the strain layer was removed with an inoculating loop and suspended in the InstaGene Matrix contained in the tube. The suspension was reacted at 56°C for 30 minutes and mixed thoroughly for 10 seconds. The mixture was heated at 100°C for 8 minutes and mixed thoroughly for 10 seconds. The mixture was centrifuged at 12,000 rpm for 3 minutes and the supernatant was collected as a template DNA for PCR.
  • KCTC Korean Collection for Type Culture
  • ATCC American Type Culture Collection
  • ITSF SEQ ID NO. 2
  • MYC6 SEQ ID NO. 1
  • FIG. 2 is an electrophoresis image taken after the PCR performed using primers ITSF and MYC6 derived from the Mycobacteria genus-specific sequence.
  • lane M represents an index of molecular weight of 100 basepair (bp)
  • lane C represents distilled water used as a negative control
  • lane 1 represents M. tuberculosis
  • lane 2 represents M. flavescens
  • lane 3 represents M. avium
  • lane 4 represents M. fortuitum
  • lane 5 represents M. kansasii
  • lane 6 represents M. chelonae
  • lane 7 represents M. szulgai. It is apparent from FIG. 2 that only Mycobacterium microorganisms are amplified through the PCR using the primers ITSF and MYC6.
  • FIG. 3 is an electrophoresis image taken after a PCR performed using species-specific primers MTB10, FOR12, FLA9, and MAC 5 derived from Mycobacteria species-specific sequences.
  • images A, B, C, and D are for M. tuberculosis, M. flavescene, M. avium, and M. fortuitum, respectively.
  • Lane M represents an index of molecular weight of 100 bp
  • lane C represents distilled water used as a negative control
  • lane 1 represents M. tuberculosis
  • lane 2 represents M. flavescens
  • lane 3 represents M. avium
  • lane 4 represents M. fortuitum
  • lane 5 represents M.
  • lane 6 represents M. chelonae
  • lane 7 represents M. szulgai.
  • image A M. tuberculosis
  • image B M. flavescens
  • image C M. avium
  • image D M. fortuitum
  • a multiplex PCR method was carried out for MTB and six NTM species using primers ITSF, MTB10, FOR12, FLA9, MAC5, and MYC6 according to the present invention.
  • the reaction mixture contained 500 mM KCI, 100 mM Tris-HCI (pH 9.0), 1% Triton X-100, 2.5 mM each dNTP (dATP, dGTP, dTTP, and dCTP), 1.5 mM MgCI 2 , 1 U Taq DNA polymerase, a PCR enhancer, 10 pmols of each species-specific primer, and 30 pmols of the common reverse primer.
  • FIG. 4 is an electrophoresis image taken after the PCR performed for MTB and six NTM species using the primers ITSF, MTB10, FOR12, FLA9, MAC5, and MYC6.
  • lane M represents an index of molecular weight of 100 bp
  • lane C represents distilled water used as a negative control
  • lane 1 represents M. tuberculosis
  • lane 2 represents M. flavescens
  • lane 3 represents M. avium
  • lane 4 represents M. fortuitum
  • lane 5 represents M. kansasii
  • lane 6 preresents M. chelonae
  • lane 7 represents M. szulgai.
  • FIG. 4 represents M. szulgai.
  • Mycrobacteria genus-specific amplification occurs in all lanes 1 through 7. Amplification of one MTB occurs in lane 1 , M. flavescens in lane 2, M. avium in lane 3, and M. fortuitum in lane 4. For. three NTMs, M. kansasii, M. chelonae, and M. szulgai, only Mycobacteria genus-specific amplification not specific to the species occurs.
  • FIG. 5 is a schematic representation of the electrophoresis image of FIG. 4.
  • bands 1 , 3, 5, 7, 9, 10 and 11 represent the PCR amplification products by the Mycobacteria genus-specific primer pair ITSF-MYC6, which is common for the species
  • band 2 represents the PCR amplification product by M.
  • primer pair MTB10-MYC6 band 4 represents the PCR amplification product by M. /7avesce/7ce-specific primer pair FLA9-MYC6, band 6 represents the PCR amplification product by M. awi/m-specific primer pair MAC5-MYC6, and band 8 represents M. fortuitum-spec ⁇ f)c primer pair FOR12-MYC6.
  • FIGS. 4 and 5 it is evident that species-specific amplification can occur in a single tube with a plurality of mixed primers.
  • the species-specific primers according to the present invention have the same PCR conditions and thus they can be simultaneously applied to a single PCR.
  • MTB and NTMs can be identified through a single multiplex PCR.
  • the present invention is described with reference to the penta-multiplex PCR method in which a plurality of bacteria species can be identified through a single PCR in one test tube using one fixed primer which is common for the genus, and five genus- and species-specific primers, the present invention can be applied to any type of multiplex PCR including di-, tri-, tetra-, and hexa-multiplex PCRs within the scope of the present invention.
  • the Mycobacteria genus and four kinds of MTB and NTM species can be identified at the same time through a single PCR in one test tube using a minimum number of primers.
  • the multiplex PCR method according to the present invention ensures rapid and accurate diagnosis at low costs without conducting other biochemical tests.
  • the multiplex PCR method according to the present invention can identify at least two species through a single method, and thus it can be efficiently applied for the diagnosis of tuberculosis.
  • the multiplex PCR method according to the present invention is effective in detecting NTM at low costs.

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Abstract

L'invention concerne un nouveau procédé de réaction en chaine de la polymérase (PCR) multiplexe, ainsi qu'un kit et des oligonucléotides de PCR multiplexe avec lesquels les espèces du genre Mycobacterium peuvent être spécifiquement et rapidement identifiées grâce au procédé précité. Selon ledit procédé, au moins deux gènes cibles sont simultanément amplifiés dans un tube unique par une seule réaction au moyen d'au moins un oligonucléotide présentant une séquence commune pour les deux gènes cibles à amplifier, en tant qu'amorce fixe, et au moins deux oligonucléotides comportant des séquences spécifiques aux gènes en tant qu'amorces spécifiques. Le kit de PCR multiplexe utilise une amorce oligonucléotidique possédant une séquence conservée de Mycobacterium comme amorce fixe, et au moins deux amorces oligonucléotidiques qui sont spécifiques aux espèces du genre Mycobacterium, en tant qu'amorces spécifiques. L'utilisation du kit PCR multiplexe permet d'identifier au moins deux espèces du genre Mycobacterium par une réaction PCR unique. Le kit de PCR multiplexe peut s'appliquer de manière efficace et à faible coût dans le diagnostic de M. tuberculosis et la détection de Mycobacterium non tuberculosis.
PCT/KR2001/001385 2000-09-15 2001-08-14 Procede et kit de pcr multiplexe et oligonucleotides destines a la detection et l'identification de mycobacterium au moyen dudit procede Ceased WO2002022872A1 (fr)

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KR10-2000-0054166A KR100433260B1 (ko) 2000-09-15 2000-09-15 멀티플렉스 ｐｃｒ 방법 및 이를 이용한 마이코박테리아동정용 키트 및 올리고 뉴클레오티드
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DE10215238C1 (de) * 2002-04-06 2003-08-14 Cytonet Gmbh & Co Kg Nachweis von Mykobakterien in klinischem Material
WO2003080871A2 (fr) 2002-03-26 2003-10-02 Hain Lifescience Gmbh Procede et differenciation du complexe mycobacterium tuberculosis
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KR100454585B1 (ko) * 2001-10-09 2004-11-02 주식회사 에스제이하이테크 마이코박테리아의 균주 감별, 결핵균의 스트레인 감별과항생제 내성을 검출하기 위한 프로브를 포함하는마이크로어레이와 이를 이용한 검출 방법 및 진단 키트
CN116064853A (zh) * 2022-08-17 2023-05-05 浙江迪谱诊断技术有限公司 一种试剂盒及其应用

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US9982255B2 (en) 2013-03-11 2018-05-29 Kailos Genetics, Inc. Capture methodologies for circulating cell free DNA
US11162094B2 (en) 2013-03-11 2021-11-02 Kailos Genetics, Inc. Capture methodologies for circulating cell free DNA
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CN103555828B (zh) * 2013-10-10 2016-04-06 上海市肺科医院 分枝杆菌快速药敏检测方法及检测靶标
CN109975543A (zh) * 2019-03-01 2019-07-05 中国疾病预防控制中心传染病预防控制所 分枝杆菌Ku蛋白的应用
CN109975543B (zh) * 2019-03-01 2022-02-11 中国疾病预防控制中心传染病预防控制所 分枝杆菌Ku蛋白的应用

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