title: Novel targeting of the lepB gene using PCR with  confronting two-pair primers for simultaneous  detection of Mycobacterium tuberculosis complex  and Mycobacterium bovis.
abstract: Tuberculosis (TB), caused by members of the Mycobacterium tuberculosis complex (MTC), is the leading cause of infectious disease-related mortality worldwide. The standard method for TB diagnosis usually requires long periods of mycobacteria cultivation, leading to delayed diagnosis, inefficient treatment and widespread occurrence of the disease. Therefore, a rapid method for the detection and differentiation of MTC from other mycobacteria is essential for disease diagnosis. Here, we describe the potential of using the type I signal peptidase (lepB) gene as a novel target for TB diagnosis, based on confronting two-pair primers PCR (PCRCTPP) that can detect MTC and simultaneously differentiate M. bovis. The limit of detection of the developed technique was equivalent to 12–120 bacilli. PCR-CTPP was highly specific to only MTC and M. bovis, and no cross-reaction was detected in 27 DNA of the non-tuberculous mycobacterial and bacterial strains tested. Thirty-nine blinded clinical isolates and 72 sputum samples were used to validate the PCR-CTPP in comparison with the standard mycobacterial culture method. The sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV) of PCR-CTPP were equal to 95, 100, 100 and 95 %, respectively, when tested with clinical isolates. Furthermore, upon testing with the sputum samples, the sensitivity, specificity, PPV and NPV were observed to be 84, 76, 90 and 67 %, respectively. Hence, this highly sensitive novel technique, which is rapid, easy to conduct and cost-effective, is a potential method for TB diagnosis and epidemiological studies, especially in resource-limited countries with a high TB burden.
authors.affiliation: Division of Clinical Microbiology, Department of Medical Technology, Faculty of Associated Medical Sciences, Chiang Mai University, Chiang Mai 50200, Thailand.
authors.forename: Chayada
authors.initials: C
authors.lastname: Sitthidet Tharinjaroen
authors.affiliation: Division of Clinical Microbiology, Department of Medical Technology, Faculty of Associated Medical Sciences, Chiang Mai University, Chiang Mai 50200, Thailand.
authors.forename: Sorasak
authors.initials: S
authors.lastname: Intorasoot
authors.affiliation: Division of Clinical Microbiology, Department of Medical Technology, Faculty of Associated Medical Sciences, Chiang Mai University, Chiang Mai 50200, Thailand.
authors.forename: Usanee
authors.initials: U
authors.lastname: Anukool
authors.affiliation: Division of Clinical Microbiology, Department of Medical Technology, Faculty of Associated Medical Sciences, Chiang Mai University, Chiang Mai 50200, Thailand.
authors.forename: Ponrut
authors.initials: P
authors.lastname: Phunpae
authors.affiliation: Division of Clinical Microbiology, Department of Medical Technology, Faculty of Associated Medical Sciences, Chiang Mai University, Chiang Mai 50200, Thailand.
authors.forename: Bordin
authors.initials: B
authors.lastname: Butr-Indr
authors.affiliation: Division of Clinical Microbiology, Department of Medical Technology, Faculty of Associated Medical Sciences, Chiang Mai University, Chiang Mai 50200, Thailand.
authors.forename: Santhasiri
authors.initials: S
authors.lastname: Orrapin
authors.affiliation: National Metal and Materials Technology Center, National Science and Technology Development Agency, Pathumthani 12120, Thailand.
authors.forename: Sirikwan
authors.initials: S
authors.lastname: Sangboonruang
authors.affiliation: Office of Disease Prevention and Control Region 10, Chiang Mai 50100, Thailand.
authors.forename: Surachet
authors.initials: S
authors.lastname: Arunothong
authors.affiliation: TB/HIV Research Foundation (THRF), Chiangrai 57000, Thailand.
authors.forename: Boonchai
authors.initials: B
authors.lastname: Chaiyasirinroj
authors.affiliation: Department of Medical Technology, Chaingrai Prachanukroh Hospital, Chiangrai 57000, Thailand.
authors.forename: Naowarat
authors.initials: N
authors.lastname: Kunyanone
authors.affiliation: Division of Clinical Immunology, Department of Medical Technology, Faculty of Associated Medical Sciences, Chiang Mai University, Chiang Mai 50200, Thailand.|Biomedical Technology Research Center, National Center for Genetic Engineering and Biotechnology, National Science and Technology Development Agency at the Faculty of Associated Medical Sciences, Chiang Mai University, Chiang Mai 50200, Thailand.
authors.forename: Watchara
authors.initials: W
authors.lastname: Kasinrerk
authors.affiliation: Division of Clinical Microbiology, Department of Medical Technology, Faculty of Associated Medical Sciences, Chiang Mai University, Chiang Mai 50200, Thailand.
authors.forename: Khajornsak
authors.initials: K
authors.lastname: Tragoolpua
mesh_terms: D001426:Bacterial Proteins / Q000235:genetics*; D003362:Cost-Benefit Analysis; D017931:DNA Primers; D004269:DNA, Bacterial / Q000235:genetics; D005798:Genes, Bacterial; D006801:Humans; D057230:Limit of Detection; D008565:Membrane Proteins / Q000235:genetics*; D009163:Mycobacterium bovis / Q000235:genetics; D009169:Mycobacterium tuberculosis / Q000235:genetics / Q000302:isolation & purification*; D020411:Oligonucleotide Array Sequence Analysis; D016133:Polymerase Chain Reaction; D012680:Sensitivity and Specificity; D012697:Serine Endopeptidases / Q000235:genetics*; D013183:Sputum / Q000382:microbiology; D014376:Tuberculosis / Q000175:diagnosis / Q000382:microbiology
chemical_list: D001426:Bacterial Proteins; D017931:DNA Primers; D004269:DNA, Bacterial; D008565:Membrane Proteins; D012697:Serine Endopeptidases; C026515:type I signal peptidase