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WO1999035284A1 - Diagnostic, identification, et caracterisation de m. tuberculosis et autres mycobacteries par etude de retardement sur gel - Google Patents

Diagnostic, identification, et caracterisation de m. tuberculosis et autres mycobacteries par etude de retardement sur gel Download PDF

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Publication number
WO1999035284A1
WO1999035284A1 PCT/BR1997/000087 BR9700087W WO9935284A1 WO 1999035284 A1 WO1999035284 A1 WO 1999035284A1 BR 9700087 W BR9700087 W BR 9700087W WO 9935284 A1 WO9935284 A1 WO 9935284A1
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WO
WIPO (PCT)
Prior art keywords
mycobacteria
pcr
tuberculosis
dna
shift
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PCT/BR1997/000087
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English (en)
Inventor
Paulo César PEREGRINO FERREIRA
Erna Geessien Kroon
Maria Elizabeth Bernardes Margutti Pinto
Agdemir Wáleria ALEIXO
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Universidade Federal De Minas Gerais
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Publication date
Application filed by Universidade Federal De Minas Gerais filed Critical Universidade Federal De Minas Gerais
Priority to EP97953602A priority Critical patent/EP1044282A1/fr
Priority to PCT/BR1997/000087 priority patent/WO1999035284A1/fr
Publication of WO1999035284A1 publication Critical patent/WO1999035284A1/fr

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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
    • 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
    • C12Q1/6806—Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay
    • 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

Definitions

  • the present invention relates to a method for detect, identify and classify Mycobacterium tuberculosis or any other mycobacteria, by using a urea-poliacrylamide gel (UP AGE) to distinguish between heteroduplex and homoduplex shift bands obtained by mixing PCR products derived from 16S rRNA coding gene fragment of mycobacteria
  • the method is based on divergence in sequences found in 16S rRNA to identify mycobacteria species, since a remarkable shift of heteroduplex bands are obtained between single stranded and homoduplex bands in UP AGE
  • PCR polymerase chain reaction
  • PCR reaction probes and primers for the insertion fragment IS986 or IS6110 (Cave, M.D., K.D. Eisenach, P.F. MacDermot, J. II Bates, and J.T. Crawford Mol Cel Probes 5 73-80, 1991 , Chevrel-DellagiJ).
  • A. Abderrahman, R. Haltiti, H. Koubaji, B. Gicquel, and K. Dellagi J Clin Microbiol 31 2446-2450, 1993) have been used and many different strategies have been proposed to confirm the identity of amplified DNA products
  • One common strategy for detecting and speciating microorganisms is specific gene-probe hybridization and PCR targeted to 16S rRNA subunit (B ⁇ ddinghaus, B., T.
  • Figure 1 shows a photography of ethidium bromide-stained of PCR amplification products with primers F-285 e ZR-244 derived from DNA of patients with M. tubercidosis and E. coli standard culture.
  • the PCR products (5 ⁇ l) were fractioned on a 1% gel agarose at 100 volts for 40 min. and showed an expected 360bp fragment.
  • Lane M PEL phage DNA digest with Hind III ; lane 2, M. tuberculosis ; lane 3, no DNA template; lane 4 to 13 show fragments amplified from clinical samples DNA.
  • Figure 2 shows a photography of a shift mobility assay of 360bp PCR fragment product derived from 16S rRNA coding gene of various bacteria using as standard M. tubercidosis .
  • the PCR product fragment of various bacteria were mix (2.5 ⁇ l v/v) with mycobacteria, denatured (95°C), re-annealed and run in PAGE at 100 volts for 60 min.
  • Figure 3 shows a photography of a shift mobility assay of 1030pb PCR fragment derived from 16S rRNA coding gen of Mycobacteria.
  • a PCR fragments from tuberculosis (standard) and various mycobacteria were mixed (2.5 ⁇ l v/v). After denaturation (95°C). re-annealed reactions were run in PAGE at 200 volts for 60min..
  • lane 6 M tuberculosis + M smegmatis, lane 7, M smegmans, lane 8, M tuberculosis + M kansasn, lane 9, M kansasn, lane 10, M tuberculosis + M scrofulaceum, lane 11, -V/ scrofulaceum
  • Figure 4 shows a photography of effect of fortuitum denved 16S rRNA 1030pb PCR product concentration in the shift mobility assay detection
  • a PCR fragments from standard M tuberculosis (1460 ⁇ g) and M fortuitum were mix (2 5 ⁇ l v/v) and denatured (95°C) After re- annealed reactions were run in 5% PAGE with 3% of urea in TBE buffer at 200 volts for 60 min
  • Lane 1 M tuberculosis lane 2, M fortuitum (1500 ⁇ g), lane 3, M tuberculosis + M fortuitum (1500 ⁇ g), lane 4, M tuberculosis + M fortuitum (800 ⁇ g), lane 5, M tuberculosis + M fortuitum (400 ⁇ g), lane 6, M tuberculosis + M fortuitum (200 ⁇ g)
  • Figure 5 shows a photography of effect of DNA template concentration in the heteroduplex mobility assay of 1030pb PCR fragment denved from rDNA of M tubercidosis e M fortuitum
  • a PCR fragments from M tuberculosis (standard) and M fortuitum denved from different DNA template concentration were mixed (2 5 ⁇ l v/v) denatured (95°C) re-annealed and run in PAGE at 200 volts for 60 min Lane 1, M tuberculosis (lOO ⁇ g).
  • Figure 6 shows the identification of M avium by heteroduplex mobility shift assay in clinical sample of patient suspected of mycobactena infection
  • the assay was peformed by using M tuberculosis and M fortuitum as a standard A PCR fragments from M tuberculosis or M fortuitum were mix (2 5ml v/v) to clinical sample #1 and #2 denatured (95°C) re-annealed and run in PAGE at 200 volts for 60 min
  • Clinical sample #1 showed in lanes 1 to 4 is a typical profile pathem of M tuberculosis and and clinical sample #2 ( lanes 5 to 8) of M avium Lane 1, sample clinical #1, lane 2, sample clinical #1 + M tuberculosis , lane , sample chnical #1 + M avium, lane 4, sample clinical #1 + M fortuitum , lane 5, sample clinical #2, lane 6, sample clinical #2 + M tuberculosis , lane , sample clinical #2 +M
  • Figure 7 shows a diagram of 16S R A and regicms where nucleotide sequences are different between some mycobactena species and E coh DETAILED DESCRIPTION OF THE INVENTION
  • an object of the present invention to provide a method of identification, classification or diagnosis for Mycobacterium tuberculosis or other mycobactena that uses the Shift Mobility Assay (SMA)
  • SMA Shift Mobility Assay
  • the method outlined here allowed identification of mycobacterium species from culture media or clinical samples based on heteroduplexes formed from 16S rRNA coding genes
  • the method is based on heteroduplexes migration in polyacrylamide gels observed after denaturing and reannealing mixtures of PCR products denved from vanous sources of 16S rRNA genes using M tuberculosis or M bovis as standard (Figure 1) When two non divergent sequence fragments were mixed two bands were observed in polyacrylamide gel one in the bottom (homoduplex) and other in the middle of the gel, which corresponds to the single stranded DNA ( Figure 2) due to minor differences in pnmer concentration, heteroduplexes with reduced mobility are detected when annealed DNA has 1 -2% divergent nucleo
  • a vanety of commercially available primers in the 16S rRNA coding gene region may be used for PCR amplification
  • the follow examples for illustrative purposes only, are desc ⁇ bed The examples llustrate the present invention and are not intended to limit it in spint or scope
  • EXAMPLE 1 Bacterial cultures Bactenal strains as Mycobacterium bovis, Mycobacterium avium, Mycobacterium scrofulaceum, Mycobacterium kansasn Mycobacterium smegmatis Mycobacterium fortuitum or other than mycobactena (Pseudomonas aeruginosa. Staphylococcus sp , E coll , Klebsiela sp e Proteus mirabilis) obtained from the Lowestein's, agar plate or liquid culture media
  • EXAMPLE 2 DNA extraction DNA was extracted after lysing 0 1 - 1000 ⁇ g of solid microorganism from pure culture with 100 - 500 ⁇ l of lysis buffer solution (TE pH 6 0-8 0, 0 1-2 0% of lysozyme, 0 1-10 % of Tween 80) for 30-60 min at 15-30°C SDS was then added to 1-10% plus 50- 500 ⁇ g/ml of proteinase K and the test tube kept for 40-120 mm at 37- 55 ⁇ C From clinical samples, ahqu ⁇ ts of fresh sputum were treated with N-acetyl,L-cyste ⁇ ne (0 5-2 0 mg/ml) for 40- 60 min, at room temperature, followed by treatment with same volume of lysis buffer as descnbed above DNA was extracted twice with phenol chloroform lsoamyhc alcohol as described by Boddinghaus et al (B ⁇ ddinghaus, B., T.
  • DNA amplification DNA was amplified by using three ohgonucleotides primers inside 16S rRNA of M tuberculosis were used two reverse designated pnmer ZR-244 (CCCACTGCTGCCTCCCGTA) located at nucleotides 298 to 317, MYC-264 located at positions 1027 to 1046 (TGCACACAGGCCACAAGGGA) and a foward pnmer designated F- 285 (AGAGTTTGATCCTGGCTCAG) conesponding to position 8 to 28
  • the resulting PCR product was 1030bp long when primers F-285 and MYC-264 were used and 360bp long for F- 285 and ZR-244 PCR reaction containing 0 l-900 ⁇ g of template DNA as indicated, 2 0-6 0 mM of MgCl2, 4-6 pM of each pnmer in 50-150 mM Tnsma (Sigma, SP, Brazil), pH 7 2-8 3, 50 to 200 ⁇ M of

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  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Organic Chemistry (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Analytical 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)
  • Chemical Kinetics & Catalysis (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Abstract

L'invention concerne un procédé de diagnostic, d'identification et de caractérisation de M. tuberculosis, ou autre mycobactérie, par amplification en chaîne par polymérase (ACP) et étude de retardement sur gel. Le procédé repose sur les micro-hétérogénéités observées dans les séquences 16S ARNr établies pour les mycobactéries et sur les écarts de séquences nucléotidiques et les défauts d'adaptation qui entraînent une diminution de la mobilité électrophorétique d'hétéroduplex d'ADN dans les gels de polyacrylamide. La technique d'ACP repose sur l'utilisation d'amorces spécifiques pour les mycobactéries et sur les écarts de séquences observés dans le gène codant 16S ARNr. L'écart de séquence nucléotidique a suffi à identifier les mycobactéries, puisqu'on a constaté un retardement remarquable entre les bandes simple brin et homoduplex sur PAGE, parmi les mycobactéries testées, lorsque M. tuberculosis a été utilisée comme standard. On a observé un retardement pour le produit d'ACP des bactéries autres que les mycobactéries au-dessus de la bande simple brin de PAGE, en culture standard et sur spécimens cliniques. Ainsi, l'étude de retardement sur gel constitue un procédé rapide et sensible de détection et de classification des mycobactéries dans des échantillons cliniques et en culture pure.
PCT/BR1997/000087 1997-12-30 1997-12-30 Diagnostic, identification, et caracterisation de m. tuberculosis et autres mycobacteries par etude de retardement sur gel WO1999035284A1 (fr)

Priority Applications (2)

Application Number Priority Date Filing Date Title
EP97953602A EP1044282A1 (fr) 1997-12-30 1997-12-30 Diagnostic, identification, et caracterisation de m. tuberculosis et autres mycobacteries par etude de retardement sur gel
PCT/BR1997/000087 WO1999035284A1 (fr) 1997-12-30 1997-12-30 Diagnostic, identification, et caracterisation de m. tuberculosis et autres mycobacteries par etude de retardement sur gel

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PCT/BR1997/000087 WO1999035284A1 (fr) 1997-12-30 1997-12-30 Diagnostic, identification, et caracterisation de m. tuberculosis et autres mycobacteries par etude de retardement sur gel

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US10/330,359 Continuation US20030219778A1 (en) 2000-08-29 2002-12-30 Method for the diagnosis, identification and characterization of M. tuberculosis and other mycobacteria by shift mobility assay

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Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2001044511A3 (fr) * 1999-12-15 2002-02-21 Gen Probe Inc Procedes et compositions pour la detection d'especes du complexe micobacterium avium
ES2190877A1 (es) * 2001-06-26 2003-08-16 Univ Santiago Compostela Metodo gedap (genotyping based on diagnostic amplification products) para detectar y/o prevenir errores de genotipado a partir de los productos de amplificacion de un locus polimorfico.
GB2435326A (en) * 2006-02-15 2007-08-22 Cell Analysis Ltd Heteroduplex analysis of non-human analytes
US7294489B2 (en) 1999-12-17 2007-11-13 Gen-Probe Incorporated Nucleic acid amplification and detection of Mycobacterium species

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1997016564A1 (fr) * 1995-11-01 1997-05-09 Chiron Diagnostics Corp. Detection moleculaire amelioree du mycobacterium tuberculosis faisant intervenir l'element d'insertion is6110
US5652106A (en) * 1993-06-04 1997-07-29 The United States Of America As Represented By The Secretary Of The Department Of Health And Human Services Rapid amplification-based subtyping of mycobacterium tuberculosis

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5652106A (en) * 1993-06-04 1997-07-29 The United States Of America As Represented By The Secretary Of The Department Of Health And Human Services Rapid amplification-based subtyping of mycobacterium tuberculosis
WO1997016564A1 (fr) * 1995-11-01 1997-05-09 Chiron Diagnostics Corp. Detection moleculaire amelioree du mycobacterium tuberculosis faisant intervenir l'element d'insertion is6110

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
PATENT ABSTRACTS OF JAPAN, Vol. 15, No. 403, (C-875), 1991; & JP 3164199 A (SHIMA KENKYUSHO K.K.) 16 July 1991. *

Cited By (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2001044511A3 (fr) * 1999-12-15 2002-02-21 Gen Probe Inc Procedes et compositions pour la detection d'especes du complexe micobacterium avium
US6747141B2 (en) 1999-12-15 2004-06-08 Gen-Probe Incorporated Methods and compositions for detection of mycobacterium avium complex species
US7361746B2 (en) * 1999-12-15 2008-04-22 Gen-Probe Incorporated Methods and compositions for detection of Mycobacterium avium complex species
US7294489B2 (en) 1999-12-17 2007-11-13 Gen-Probe Incorporated Nucleic acid amplification and detection of Mycobacterium species
US7879581B2 (en) 1999-12-17 2011-02-01 Gen-Probe Incorporated Nucleic acid amplification and detection of mycobacterium species
ES2190877A1 (es) * 2001-06-26 2003-08-16 Univ Santiago Compostela Metodo gedap (genotyping based on diagnostic amplification products) para detectar y/o prevenir errores de genotipado a partir de los productos de amplificacion de un locus polimorfico.
WO2003001176A3 (fr) * 2001-06-26 2004-03-04 Univ Santiago Compostela Methode de genotypage base sur des produits d'amplification de diagnostic (gedap) servant a la detection et/ou la prevention d'erreurs de genotypage a partir des produits d'amplification d'un locus polymorphique
ES2190877B2 (es) * 2001-06-26 2004-05-16 Universidade De Santiago De Compostela Metodo gedap (genotyping based on diagnostic amplification products) para detectar y/o prevenir errores de genotipado a partir de los productos de amplificacion de un locus polimorfico.
US7402382B2 (en) 2001-06-26 2008-07-22 Universidade De Santiago De Compostela GEDAP method (genotyping based on diagnostic amplification products) for detecting and/or preventing genotyping errors from amplification products of a polymorphic focus
GB2435326A (en) * 2006-02-15 2007-08-22 Cell Analysis Ltd Heteroduplex analysis of non-human analytes

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