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
• • 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

Landscapes

• 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)
• Biophysics (AREA)
• Immunology (AREA)
• Microbiology (AREA)
• Molecular Biology (AREA)
• Biotechnology (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)

Applications Claiming Priority (1)

Publications (1)

Family

ID=4066068

Family Applications (1)

Country Status (2)

Families Citing this family (4)

Family Cites Families (2)

• 1997
  • 1997-12-30 EP EP97953602A patent/EP1044282A1/de not_active Withdrawn
  • 1997-12-30 WO PCT/BR1997/000087 patent/WO1999035284A1/en not_active Application Discontinuation

Non-Patent Citations (1)

Also Published As

Similar Documents

Legal Events