Novel potential diagnostic test for Mycobacterium tuberculosis complex using combined immunomagnetic separation (IMS) and PCR-CTPP
Sorasak IntorasootChayada Sitthidet TharinjaroenPonrut PhunpaeBordin Butr-IndrUsanee AnukoolKannaporn IntachaiSanthasiri OrrapinNapaporn ApiratmateekulSurachet ArunothongVorasak SuthachaiKanokwan SaengsawangPhadungkiat KhamnoiSupansa PataWatchara KasinrerkKhajornsak Tragoolpua
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To exploit immunomagnetic separation combined with PCR with confronting two-pair primers (IMS-PCR-CTPP) as a rapid method for detection of Mycobacterium tuberculosis complex (MTC) and identification of Mycobacterium bovis from sputum specimens.Monoclonal antibody (mAb) against the mycobacterial antigen, 85B (Ag85B), was coupled with magnetic particles for specific immunomagnetic separation (IMS) of Mycobacterium spp. Immunofluorescence assay indicated the capability of mAb to bind to Ag85B in both the recombinant and the native form. The IMS combined with PCR-CTPP targeting the mycobacterial lep B gene was further implemented using 133 sputum samples with acid-fast bacilli grading from negative to 3+. The results showed the sensitivity and specificity of IMS-PCR-CTPP vs gold standard culture method were 89·9 and 88·6% respectively.The IMS-PCR-CTPP method shortens the time for tuberculosis (TB) diagnosis from months to a day. This method is also suitable for investigation of MTC and epidemiological study of Myco. bovis in sputum specimens.This study is the first report emphasizing the combination of IMS and PCR-CTPP for the detection of MTC and simultaneous identification of Myco. bovis from sputum. It could be used for TB diagnosis in resource-limited countries with high TB burden.Keywords:
Immunomagnetic separation
Mycobacterium tuberculosis complex
Mycobacterium tuberculosis complex
Multiplex
Insertion sequence
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Bovine tuberculosis (BTB) is a zoonotic disease caused by Mycobacterium bovis, a member of the Mycobacterium tuberculosis complex (MTC). The quick and specific detection of this species is of extreme importance, since BTB may cause economic impacts, in addition to presenting imminent risks to human health. In the present study a nested real-time PCR test (nested q-PCR) was used in post-mortem evaluations to assess cattle carcasses with BTB-suspected lesions. A total of 41,193 cattle slaughtered in slaughterhouses located in the state of Mato Grosso, were examined. Of the examined animals, 198 (0.48%) showed BTB-suspected lesions. M. bovis was isolated in 1.5% (3/198) of the samples. Multiplex-PCR detected MTC in 7% (14/198) of the samples. The nested q-PCR test detected MTC in 28% (56/198) of the BTB-suspected lesions, demonstrating higher efficiency when compared to the multiplex-PCR and conventional microbiology. Nested q-PCR can therefore be used as a complementary test in the national program for control and eradication of bovine tuberculosis.
Mycobacterium tuberculosis complex
Bovine tuberculosis
Multiplex
Nested case-control study
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Mycobacterium tuberculosis complex
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We developed a novel multiplex PCR assay using dual-priming oligonucleotide primers targeting the RD1 gene for simultaneous identification of the Mycobacterium tuberculosis complex and M. bovis bacillus Calmette-Guérin (BCG). This assay would be useful both for detection of the M. tuberculosis complex and for differentiation of M. bovis BCG from pathogenic M. tuberculosis complex species.
Mycobacterium tuberculosis complex
Multiplex
Priming (agriculture)
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Objective To develop a new,rapid and easy method for species identification of Mycobacterium tuberculosis complex(MTBC).Methods Seven sets of primers(Mtbc1-7) for MTBC were used to amplify the genomic DNA from the reference strains and 96 clinical isolates.Results The specificities of 7 sets of primers were identified by PCR with 3 MTBC reference strains,9 non-mycobacterium reference strains and 27 non-tuberculosis mycobacterium(NTM) reference strains. The results showed that mycobacterium and non-mycobacterium were identified by Mtbc1;NTM and MTBC by Mtbc2;M.microti by Mtbc3;M.bovis and M.bovis BCG by Mtbc4;M.africanum subtypeI and M.tuberculosis by Mtbc5;M.bovis BCG by Mtbc4 and Mtbc6 together;M.canettii by Mtbc5 and Mtbc7 together.Of 96 clinical isolates,20 MOTT,3 M.microti,1 M.bovis,1 M.bovis BCG,3 M.africanum subtypeⅠ,1 M.caprae,3 M.canettii,64 M.africanum subtypeⅡ and M.tuberculosis were identified.Conclusion PCR is a rapid and effective method for the species identification of MTBC,and can be used for molecular epidemiology.
Mycobacterium tuberculosis complex
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Mycobacterium bovis BCG vaccine strains were compared with Mycobacterium tuberculosis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. A 25-kDa protein observed in the BCG strains was absent in M. tuberculosis. Rabbit antibodies specific to the 25-kDa protein uniquely identified this protein in BCG strains but not in M. tuberculosis. It is suggested that the 25-kDa protein and polyclonal antibodies directed against this antigen can be exploited to distinguish BCG strains from M. tuberculosis.
Polyclonal antibodies
BCG vaccine
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Mycobacterium tuberculosis complex
Mycobacterium avium complex
Hybridization probe
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ABSTRACT Tuberculosis (TB) in humans is caused by members of the Mycobacterium tuberculosis complex (MTC). Rapid detection of the MTC is necessary for the timely initiation of antibiotic treatment, while differentiation between members of the complex may be important to guide the appropriate antibiotic treatment and provide epidemiological information. In this study, a multiplex real-time PCR diagnostics assay using novel molecular targets was designed to identify the MTC while simultaneously differentiating between M. tuberculosis and M. canettii . The lepA gene was targeted for the detection of members of the MTC, the wbbl1 gene was used for the differentiation of M. tuberculosis and M. canettii from the remainder of the complex, and a unique region of the M. canettii genome, a possible novel region of difference (RD), was targeted for the specific identification of M. canettii . The multiplex real-time PCR assay was tested using 125 bacterial strains (64 MTC isolates, 44 nontuberculosis mycobacteria [NTM], and 17 other bacteria). The assay was determined to be 100% specific for the mycobacteria tested. Limits of detection of 2.2, 2.17, and 0.73 cell equivalents were determined for M. tuberculosis / M. canettii , the MTC, and M. canettii , respectively, using probit regression analysis. Further validation of this diagnostics assay, using clinical samples, should demonstrate its potential for the rapid, accurate, and sensitive diagnosis of TB caused by M. tuberculosis , M. canettii , and the other members of the MTC.
Mycobacterium tuberculosis complex
Multiplex
Nontuberculous Mycobacteria
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Mycobacterium tuberculosis is the causal agent of tuberculosis in humans, however, the zoonotic transmission of Mycobacterium bovis from animals to humans, mainly immunocompromised individuals, is of great impact in public health. The necessity to design rapid and precise methods to differentiate between M. tuberculosis and M. bovis has lead to the development of strategies of genotypic identification. The aim of this study was to evaluate the discriminatory power of a multiplex PCR assay to differentiate between M. tuberculosis and M. bovis clinical isolates. Conditions for simultaneous amplification with oligonucleotidos targeted Rv0577 and Rv1510 genes sequences were standardized. Rv0577 is specific of M. tuberculosis complex mycobacteria and Rv1510 is located in polymorphic regions (RD4) of the mycobacterial genome of M. tuberculosis complex, but is absent in M. bovis and M. bovis BCG, allowing to differentiate between M. tuberculosis complex strains. To evaluate the specificity of the PCR assay, 46 clinical isolates were analyzed. The amplicon from Rv0577 gene was detected in all of the clinical isolates from M. tuberculosis complex, whereas the 1033 pb product from Rv1510 (RD4), was exclusively observed in M. tuberculosis and was absent in M. bovis and M. bovis BCG, showing the specificity of this PCR assay to identify mycobacteria of the M. tuberculosis complex and to differentiate between M. tuberculosis and M. bovis in a single-step assay. This multiplex PCR assay can be applied to the identification and differentiation of mycobacteria of M. tuberculosis complex in clinical samples, to the quality control of products derived from animals with infection suspicions and to develop studies on great scale of zoonotic transmission of M. bovis in susceptible populations.
Mycobacterium tuberculosis complex
Multiplex
Amplicon
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A new clonal complex of Mycobacterium bovis present at high frequency in cattle from west central African countries has been described as the African 1 (Af1) clonal complex. Here, the first intrafamilial cluster of human tuberculosis cases due to M. bovis Af1 clonal complex strains is reported. We discuss hypotheses regarding modes of transmission.
Mycobacterium tuberculosis complex
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