A METHOD FOR THE DIAGNOSIS, IDENTIFICATION AND CHARACTERIZATION OF M. TUBERCULOSIS AND OTHER MYCOBACTERIA BY SHIFT MOBILITY ASSAY

Abstract: The present invention relates to a method for diagnosis, identification and characterization of M. tuberculosis or any other mycobacteria by using PCR and shift mobility assay (SMA). The method is based both on microheterogeneities observed within the 16S rRNA sequences established for mycobacteria and on nucleotide gaps and mismatches which cause a decrease in the electrophoretic mobility of DNA heteroduplex in polyacrylamide gels. The PCR strategy is based on the use of a specific primers for mycobacteria genera and divergence in sequences found in 16S rRNA coding gene. The difference in nucleotide sequence was enough to identify mycobacteria species, since a remarkable shift between single stranded and homoduplex bands in PAGE were observed among mycobacteria tested, when Mycobacterium tuberculosis was used as standard. The shift of PCR product of bacteria other than mycobacteria was observed above the single stranded band in PAGE, both in standard cultures and clinical specimens. SMA can thus provide a fast and sensitive method for detection and classification of mycobacteria in clinical samples as well as pure culture.

Description: A METHOD FOR THE DIAGNOSIS. IDENTIFICATION AND CHARACTERIZATION OF M TUBERCULOSIS AND OTHER MYCOBACTERIA BY SHIFT MOBILITY ASSAY. TECHNICAL FIELD OF THE INVENTION 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 BACKGROUND OF THE INVENTION Tuberculosis (TB) and other mycobactenal illnesses remain the number one cause of infectious diseases related deaths worldwide Annual increases of 10% per year have been reported (Bloom, B. R., and C. J. Murray Science 257 1055-1064, 1992 , Centers for Disease Control MMWR 40 58, 1991) in part related to the epidemic of HTV At the same time an increasing number of atypical presentations of AIDS patients with Mycobacterium tuberculosis (MTB) are also seen, with a vast increase in extra pulmonary diseases (Persing, D.H., J Clin Microbiol 29 1281-1285, 1991) Of even greater concern than the increase in absolute numbers of mycobactenal infections is the emergence of previously rare strains, collectively termed mycobacteria other than tuberculosis (MOTT) Moreover, of particular importance, is the emergence of MTB multi drug-resistant, which causes over 90% mortality in immunocompromised hosts (Shankar, P., N. Manjunath, R. Lakshmi, B. Aditi, P. Seth, and Shrini as Lancet 335 423, 42 1990) The conventional diagnosis of tuberculosis based on the gold standard, cuhure-grown organisms, is extremely sensitive (10-100 organisms per ml present for growth) but is expensive and time-consuming (2-8 weeks), because of the slow doubling rate (Vaneechoutte M., H. Beenhouwer, G. Claeys, G. Verschraegen. A. de Rouck, N. paepe, A. Alachouni, and F. Portaels J Clin Microbiol 31 2061-2065 1993) of acid-fast bacilli (AFB) Direct staining procedures are rapid but lack sensitivity and specificity since Legionella and Nocardia are stained as well (Rogall, T., J. Wolters, T. Flohr, and E.C. Boettger Int J Syst Bacteπol 40 323- 330, 1990) By overcoming the inherent limitations of stain and culture techniques, molecular methods have great potential for the rapid diagnosis of mycobacteria The polymerase chain reaction (PCR) was reported (Cousins, D.V., S.D. Wilton, B.R. Francis, and B.L. Gow I Clin Microbiol 30 255-258, 1992) to be useful in the direct diagnosis of tuberculosis infection directly from a variety of clinical specimens (Bocart, D., D. Lecossier, A. Lassence, D. Valeyre, J. P. Battesti, and AJ. Hance Am Rev Respir Dis 145 1142-1148,1992 , Brisson- Noel, A., B. Gicquel, D. Lecossier, V. Levy-Frebault, X. Nassif, and AJ. Hance Lancet li 1069-1071, 1989) with a diversity of genetic elements used as target templates The rapid detection of MTB by PCR and the high degree of sensitivity in clinical samples afforded by DNA amplification have been descπbed by vanous investigators (Cousins, D.V., S.D. Wilton, B.R. Francis, and B.L. Gow J Clin Microbiol 30 255-258, 1992 , Del Portillo, P., L.A. Murilio, and M.E. Patarroyo J Clin Microbiol 29 2163-2168, 1991 , Folgueira, L., R. Delgado, E. Palenque, and A.R. Noriega J Clin Microbiol 31 1019-1021, 1993) However, there are a number of difficulties to be overcome before these methods can be used as a routine diagnosi test (Garcia-de-Lomas J. and D. Navarro J 16 S43-S48, 1997) Nevertheless, PCR analysis of chromosomal DNA is a simple and rapid method for identifying MTB to the species level and in clinical specimens ( Shawar, R.M., F.A.K. El Zaatari, A. Nataraj, and J.E. Clarridge J Clin Microbiol 31 61-65, 1993) 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 (Bδddinghaus, B., T. Rogall, T. Flohr, H. Blδcker, and E.C. Bδttger J Clin Microbiol 28 1751-1759, 1990), which is coded in mycobactena by multiple gene (10 genes for 16S RNA) copies per cell The 16S subunit contains sequences that have highly conserved regicms virtually identical in all bacteria (Figure 7), interspersed with other sequences found to be genus- and/or species-specific (Victor, T., R. DuToit, and P.D. VanHeiden. J Clin Microbiol 30 1514-1517, 1992) Conserved regions can be used to amplify rRNA from all prokaryotes or from broadly-related groups of bacteria, while the genus and species-specific interspersed sequence domains can be used to construct specific ohgonucleotide probes to detect and differentiate resulting PCR products, or to directly probe mycobacteria in clinical samples by hybndization Most often, PCR products are visualized with ethidium bromide staining on an agarose gel (Figure 1), with final confirmation on Southern blot Previously, colonmetπc PCR hybndization assays have been investigated as an alternative to gel electrophoresis and Southern blot analysis of PCR-amphfied DNA Such gels and Southern blots have proven to be time- consuming and tedious for typical clinical laboratones as well Solid-phase colonmetπc PCR assays capture denatured amplification products on probes bound to nylon membranes (as in reverse dot blots) (Plikaytis, B.B., K.D. Eisenach, J.T. Crawford, and T.M. Shinnick Molec Cell Probes 15 215-9, 19 1 , Sambrook, KJ., Fritsch, E.F., Maniatis, T , In Molecular Cloning. A Laboratory Manual USA Cold Spring Harbor Laboratory Press, 1989 ) or to micrσtiter plates (Thierry, D., M.D. Cave , K.D. Eisenach, J.T. Crawford, J.H. Bates, B. Gicquel, and J.L. Guesdon Nucleic Acids Res 18 188-189, 1990) However, the sensitivity of these assays suffer due to the tendency of the denatured PCR product strands to reassociate and exclude ohgonucleotide probes, and steanc interference between the bound ohgonucleotides and the solid support which impede hybndization to nucleic acids in solution (Plikaytis, B.B., K.D. Eisenach, J.T. Crawford, and T.M. Shinnick Molec Cell Probes 15 215-9, 1991) Other difficulties encountered using existing techniques include vanabihty from sample preparation and detection of PCR products (Nolte, F.S., B. Metchock, J.E. McGowan, A. Edwards, O. Okwumabaa, C. Thurmond, P.S. Mitchell, B. Pilkaytis, and T. Shinnick J Clin Microbiol 31 1772-1782, 1993), technically unmanageable steps to enhance detection, false-positive reactions due to the amplification of contaminating DNA and inhibition of DNA amplification (Noordhoek, G.T., A.HJ. Kolk, G. Bjune, D. Catty, J.W. Dale, P.E.M. Fine, P. Godfrey-Fausset, S-N. Cho, T. Shiinick, S.B. Svenson, S. Wilson, and J.D.A. van Embden J Clin Pathol 48 810-814, 1995) These difficulties argue for searching more precise, sensitive, reproducible and low cost assays for MTB diagnosis We report here a method for detection and identification of mycobacteria using PCR strategies targeted to mycobactenal 16S rRNA coding gene and the heteroduplex mobility assay previously developed for analysis of HTV (Delwart, E. L., Herring, B., Rodrigo, A. G. and Mullins, J. I PCR Meth and Appl , 1995 , Delwart, E. L., Shepaer, E. G., Louwagie, J, McCutchan, F. E. Grez, M., Waigmann, H. R., and Mullins, J. I. Science. 262: 1257-1261, 1993). This new approach is fast, simple and can produce information not easily obtained when compared with other methods described so far. Thus, combining molecular methods and PAGE we developed a method which resulted in an integrated strategy to achieve sensitive and specific diagnosis of mycobacteria. Combination of these approaches has wide application in detecting microorganisms and in detection of gene products important to clinical genetic and neoplastic disease. BRIEF DESCRIPTION OF THE DRAWINGS These and other objects, features and many attendant advantages of the invention will be better understood upon a reading of the following detailed description when considered in connection with the accompanying drawings wherein: 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 (5μl) 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 (2.5μl v/v) with mycobacteria, denatured (95°C), re-annealed and run in PAGE at 100 volts for 60 min. Lane 1, Proteus mirabilis ; lane 2, Proteus mirabilis + M. tuberculosis ; lane 3, Proteus mirabilis + M. avium ; lane 4, Proteus mirabilis +M. fortuitum ; lane 5, Pseudomonas aeruginosa; lane 6, Pseudomonas aeruginosa + M tuberculosis ; lane 7, Pseudomonas aeruginnosa + M. Avium ; lane 8, Pseudomonas aeruginosa + M. fortuitum ; lane 9, Klebisiela sp ; lane 10, Klebisiela + M. tuberculosis ; lane 11, Klebisiela + M. avium ; lane 12 . Klebisiela sp + M fortuitum. 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 (2.5μl v/v). After denaturation (95°C). re-annealed reactions were run in PAGE at 200 volts for 60min.. Lane 1, M. tuberculosis ; lane 2. tuberculosis + M. avium ; lane 3, M. avium ; lane 4, M.tuberculosis + M. fortuitum ; lane 5, M. fortuitum , 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 (1460ηg) and M fortuitum were mix (2 5μl 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 (1500ηg), lane 3, M tuberculosis + M fortuitum (1500ηg), lane 4, M tuberculosis + M fortuitum (800ηg), lane 5, M tuberculosis + M fortuitum (400ηg), lane 6, M tuberculosis + M fortuitum (200ηg) 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 5μl v/v) denatured (95°C) re-annealed and run in PAGE at 200 volts for 60 min Lane 1, M tuberculosis (lOOηg). lane 2, M fortuitum (lOOηg), lane 3, M tubercidosis (lOηg) + M fortuitum (lOηg), lane 4, M tuberculosis (5ηg) + M fortuitum (5ηg), lane 5, M tuberculosis (lηg) + M fortuitum (lηg), lane 6, M tuberculosis (0 lηg) + M fortuitum (0 lηg), lane 7, M tuberculosis (0 Olηg) + M fortuitum (0 Olηg) 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 avium , lane 8, sample clinical #2 + M fortuitum 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 It is, therefore , 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) 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 nucleotide sequences or when gaps were presented in one relative to a second sequence (Figure 7) Heteroduplexes were found above the single stranded DNA bands (close to the top of the gel) and between the homoduplexes (bottom of the gel) and single stranded bands The HMA of amplified PCR products from mycobacteria rDNA denved from culture is shown m Fig 2 and 4 Specific heteroduplexes were formed between species, even when more than two species were presented in the mixture Heteroduplex formation was also observed by mixing mycobactena and other species of bacteria (Fig 3), but these heteroduplexes migrated slower than the single stranded DNA Faster migrating heteroduplexes with mobilities faster than ssDNA were observed among all mycobactena species compared (Fig 4, 5 and 6) A vanety of commercially available primers in the 16S rRNA coding gene region may be used for PCR amplification In order that this invention may be better understood the follow examples for illustrative purposes only, are descπbed 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 μg of solid microorganism from pure culture with 100 - 500μl 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- 500μg/ml of proteinase K and the test tube kept for 40-120 mm at 37- 55^C From clinical samples, ahquσts of fresh sputum were treated with N-acetyl,L-cysteιne (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 (Bδddinghaus, B., T. Rogall, T. Flohr, H. Blδcker, and E.C. Bδttger J Clin Microbiol 28 1751-1759, 1990) ressuspended in deionized H2O or any other hydrophyhc solvent and storage at -20 to -80^C until use DNA was measured by spectrophotometry EXAMPLE 3 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-900μg 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 200μM of each dNTP (Pharmacia, Sao Paulo, Brazil), 2 5-5 0 units of Taq DNA polymerase (Promega, Belo Horizonte, MG, Brasil) and the reaction was performed in a final volume of 20- lOOμl PCR reactions were earned out in a Perkin Elmer 4800 thermal cycler (Perkin Elmer/Cetus) for 35-40 cycles, using one sec ramp setting time between steps in a cycle of 93- 96°C for 1-3 min , 50-65 °C for 1-5 min , 70-72°C for 1-5 mm and a 10-20 min 65-72°C extension step was linked to die last cycle The annealing temperature for primers F-285 and MYC-264 was 45-70°C instead of 37-50°C used for primers F-285 and ZR-244 Positive

Claims: controls were 0 3μg of DNA from M bovis (BCG) Two negative controls were included one with no DNA template and the other with E coll DNA (0 5-2μg) in the PCR reaction and no amplification products were observed in these reactions PCR products were evaluated by electrophoresis on 2-6 % agarose gels and stained with ethidium bromide EXAMPLE 4 Shift mobility assay The shift mobility assay was performed by mixing 2 5-10μl of PCR products denved from standard M tubercidosis PCR product with 2 5-10μl of PCR product from other species or clinical samples plus lul of 10X annealing buffer (l-5mM NaCl, 100- 500mM Tns-Cl pH7 2-8 0 and 20-40 mM EDTA) The mixture was heated 90-96°C for 2-5 mm then cooled to 25-45^C in the thermocycler Heteroduplexes were electrophoresed on 3-6 % poKacrvlamide gels (25-30 0 8-1 0 Acrylamide Bis) and 3-7% of urea Gels were run at 100-200 volts when the 1030bp fragment PCR products were used and at 50-100 volts for 360bp fragments both for 1 hour, IX TBE buffer in a mini gel (Hoeffer mini-gel SE 200) apparatus Gels were than fixed and stained widi silver (Stain Plus, Bio-Rad, USA) While the present invention has been descnbed in connection with examples, it will be understood that modifications and vanations apparent to those ordinary skill in the art are within the scope of the present invention WHAT IS CLAIMED IS: I method for diagnosis, identification and characteπzation of M tuberculosis or any other mvcobacteπa by using PCR and shift mobility assav (SMA) in urea-pohacrylamide gel electrophoresis. compπsing the following steps 1) culture. 2) DNA extraction, 3) DNA amplification 4) Shift mobility assay 2 The method accordmg claim 1, wherem the the step 1 of culture can optional 3 The method accordmg claim 1, wherem the DNA extraction (2) from culture (1) or clinical samples can be made with phenol/chlorophorm extraction method or any other method 4 The method accordmg claim 1, wherem the DNA amplification can be made by us g any primer in the region of 16S rRNA codmg gene, or usmg any other gene than 16S rRNA coding gene 5 The method accordmg claim 1, wherem die shift mobility assav are made use of urea in pohacrvlamide gel electrophoresis to detect, visualize or identify by PCR products denved from sequences of 16S nbossomal R A codmg gene and for detection of heteroduplex complex observed between smgle strand (middle of migration) and homoduplex complex bands (bottom of the gel) , 6 The method accordmg claim 1, wherem use of M tuberculosis or any other mycobactena as standard to identify by shift mobility assay PCR products denved from sequences of 16S nbossomal RNA codmg gene or any other gene

Inventors: ALEIXO AGDEMIR WALERIA, BERNARDES MARGUTTI PINTO MARIA, GEESSIEN KROON ERNA, PEREGRINO FERREIRA PAULO CESAR, ALEIXO, AGDEMIR, WALERIA, BERNARDES MARGUTTI PINTO, MARIA, ELIZABETH, GEESSIEN KROON, ERNA, PEREGRINO FERREIRA, PAULO, CESAR
Applicants: ALEIXO AGDEMIR WALERIA, BERNARDES MARGUTTI PINTO MARIA, GEESSIEN KROON ERNA, PEREGRINO FERREIRA PAULO CESAR, UNIV MINAS GERAIS, ALEIXO, AGDEMIR, WALERIA, BERNARDES MARGUTTI PINTO, MARIA, ELIZABETH, GEESSIEN KROON, ERNA, PEREGRINO FERREIRA, PAULO, CESAR, UNIVERSIDADE FEDERAL DE MINAS GERAIS
Application year: 1997
Main classification: C12Q1
IPC codes: C12Q1