title: Detection and discrimination of Mycobacterium tuberculosis complex.
abstract: A real-time quantitative polymerase chain reaction (qPCR) was developed for detection and discrimination of Mycobacterium tuberculosis (H37Rv and H37Ra) and M. bovis bacillus Calmette-Guérin (BCG) of the Mycobacterium tuberculosis complex (MTBC) from mycobacterial other than tuberculosis (MOTT). It was based on the melting curve (Tm) analysis of the gyrB gene using SYBR(®) Green I detection dye and the LightCycler 1.5 system. The optimal conditions for the assay were 0.25 μmol/L of primers with 3.1 mmol/L of MgCl(2) and 45 cycles of amplification. For M. tuberculosis (H37Rv and H37Ra) and M. bovis BCG of the MTBC, we detected the crossing points (Cp) at cycles of 16.96 ± 0.07, 18.02 ± 0.14, and 18.62 ± 0.09, respectively, while the Tm values were 90.19 ± 0.06 °C, 90.27 ± 0.09 °C, and 89.81 ± 0.04 °C, respectively. The assay was sensitive and rapid with a detection limit of 10 pg of the DNA template within 35 min. In this study, the Tm analysis of the qPCR assay was applied for the detection and discrimination of MTBC from MOTT.
authors.affiliation: Bacteriology Unit, Infectious Disease Research Center, Institute for Medical Research, Jalan Pahang, 50588 Kuala Lumpur, Malaysia. rahizzan@imr.gov.my
authors.forename: Rahizan
authors.initials: R
authors.lastname: Issa
authors.forename: Nurul Akma
authors.initials: NA
authors.lastname: Mohd Hassan
authors.forename: Hatijah
authors.initials: H
authors.lastname: Abdul
authors.forename: Siti Hasmah
authors.initials: SH
authors.lastname: Hashim
authors.forename: Valentinus H
authors.initials: VH
authors.lastname: Seradja
authors.forename: Athirah
authors.initials: A
authors.lastname: Abdul Sani
mesh_terms: D001431:Bacteriological Techniques / Q000379:methods*; D004269:DNA, Bacterial / Q000235:genetics; D006801:Humans; D025202:Molecular Diagnostic Techniques / Q000379:methods*; D009169:Mycobacterium tuberculosis / Q000145:classification* / Q000235:genetics / Q000302:isolation & purification*; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D044366:Transition Temperature; D014376:Tuberculosis / Q000175:diagnosis* / Q000382:microbiology*
chemical_list: D004269:DNA, Bacterial