title: Identification of non-tuberculous mycobacteria by real-time PCR coupled with a high-resolution melting system.
abstract: Non-tuberculous mycobacteria (NTM) are increasingly important opportunistic pathogens responsible for a variety of clinical diseases. The aim of this study was to evaluate a novel technique, real-time PCR coupled with high-resolution melting analysis (real-time PCR-HRMA), for NTM identification. Two pairs of unique primers targeted to the 16S rRNA gene and the 16S-23S internal transcribed spacer region were selected for further evaluation. A total of 149 mycobacterial clinical isolates were subjected to analysis using the real-time PCR-HRMA system. Overall, 134 NTM identified by the 16S rRNA full-gene sequencing method were categorized into four major groups: Mycobacterium avium complex, Mycobacterium chelonae group, Mycobacterium gordonae and Mycobacterium fortuitum group. Of the 134 prevalent mycobacterial isolates, 101 mycobacteria (75.4 %) could be identified correctly by the real-time PCR-HRMA system. The individual sensitivities for the M. avium complex, M. chelonae group, M. gordonae and M. fortuitum groups were 90.9, 89.1, 100 and 36.8 %, respectively. The specificity of identifying these groups varied from 96.4 to 100 %. When identification failed, mostly it was attributable to various species in the M. fortuitum group. The real-time PCR-HRMA system is therefore a rapid and sensitive method for identifying prevalent NTM in a clinical laboratory.
authors.affiliation: Graduate Institute of Pathology, National Defense Medical Center, Taipei, Taiwan, ROC.|Division of Clinical Pathology, Department of Pathology, Tri-Service General Hospital and National Defense Medical Center, Taipei, Taiwan, ROC.
authors.forename: Cherng-Lih
authors.initials: CL
authors.lastname: Perng
authors.affiliation: Department of Clinical Pathology, Taipei City Hospital Renai Branch, Taipei, Taiwan, ROC.
authors.forename: Hsing-Yu
authors.initials: HY
authors.lastname: Chen
authors.affiliation: Graduate Institute of Medical Science, National Defense Medical Center, Taipei, Taiwan, ROC.|Division of Clinical Pathology, Department of Pathology, Tri-Service General Hospital and National Defense Medical Center, Taipei, Taiwan, ROC.
authors.forename: Tzong-Shi
authors.initials: TS
authors.lastname: Chiueh
authors.affiliation: Division of Infectious Disease, Fong-Yuan Hospital, Taichung, Taiwan, ROC.|Graduate Institute of Medical Science, National Defense Medical Center, Taipei, Taiwan, ROC.
authors.forename: Wei-Yao
authors.initials: WY
authors.lastname: Wang
authors.affiliation: Division of Clinical Pathology, Department of Pathology, Tri-Service General Hospital and National Defense Medical Center, Taipei, Taiwan, ROC.
authors.forename: Chun-Ting
authors.initials: CT
authors.lastname: Huang
authors.affiliation: Graduate Institute of Medical Science, National Defense Medical Center, Taipei, Taiwan, ROC.|Division of Clinical Pathology, Department of Pathology, Tri-Service General Hospital and National Defense Medical Center, Taipei, Taiwan, ROC.
authors.forename: Jun-Ren
authors.initials: JR
authors.lastname: Sun
mesh_terms: D001431:Bacteriological Techniques / Q000379:methods*; D017931:DNA Primers / Q000235:genetics; D004269:DNA, Bacterial / Q000235:genetics; D004275:DNA, Ribosomal / Q000235:genetics; D021903:DNA, Ribosomal Spacer / Q000235:genetics; D006801:Humans; D025202:Molecular Diagnostic Techniques / Q000379:methods*; D009165:Mycobacterium Infections, Nontuberculous / Q000175:diagnosis* / Q000382:microbiology; D009170:Nontuberculous Mycobacteria / Q000145:classification / Q000235:genetics / Q000302:isolation & purification*; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D044366:Transition Temperature
chemical_list: D017931:DNA Primers; D004269:DNA, Bacterial; D004275:DNA, Ribosomal; D021903:DNA, Ribosomal Spacer