title: Direct identification of mycobacteria from smear-positive sputum samples using an improved multiplex polymerase chain reaction assay.
abstract: The rapid identification of mycobacteria from smear-positive sputum samples is very important. To identify the Mycobacterium tuberculosis complex (MTBC) and frequently isolated nontuberculous mycobacteria strains directly from smear-positive sputum samples, an improved multiplex polymerase chain reaction (PCR) assay was developed. Nine pairs of primers targeting the 16S-23S rDNA internal transcribed spacer-1, hsp65, and the early secretory antigen (ESAT-6) gene sequences were developed, and their efficacy was evaluated in comparison to traditional culturing and 16S rRNA gene sequencing methods. A total of 200 smear- and culture-positive sputum specimens collected between November 2005 and May 2006 were used for the analysis. The results of the assay showed an accurate identification rate for acid-fast bacilli (AFB) 3+, AFB 2+, and AFB rare/1+ samples of 98%, 95%, and 53%, respectively. The improved multiplex PCR method saves time and has advantages for identifying mycobacteria from AFB 2+ and 3+ sputum samples. The method is suitable for use in countries with a high MTBC prevalence rate.
authors.affiliation: Department of Laboratory Medicine, Chang Gung Memorial Hospital, Taoyuan, Taiwan.
authors.forename: Ju-Hsin
authors.initials: JH
authors.lastname: Chia
authors.forename: Tsu-Lan
authors.initials: TL
authors.lastname: Wu
authors.forename: Lin-Hui
authors.initials: LH
authors.lastname: Su
authors.forename: An-Jing
authors.initials: AJ
authors.lastname: Kuo
authors.forename: Hsin-Chih
authors.initials: HC
authors.lastname: Lai
mesh_terms: D000942:Antigens, Bacterial / Q000235:genetics; D001426:Bacterial Proteins / Q000235:genetics; D015373:Bacterial Typing Techniques; D018834:Chaperonin 60 / Q000235:genetics; D017931:DNA Primers / Q000235:genetics; D004269:DNA, Bacterial / Q000032:analysis / Q000235:genetics; D021903:DNA, Ribosomal Spacer / Q000235:genetics; D006801:Humans; D060885:Multiplex Polymerase Chain Reaction / Q000379:methods*; D009161:Mycobacterium / Q000145:classification* / Q000235:genetics / Q000302:isolation & purification*; D009164:Mycobacterium Infections / Q000175:diagnosis* / Q000382:microbiology; D012336:RNA, Ribosomal, 16S / Q000235:genetics; D012338:RNA, Ribosomal, 23S / Q000235:genetics; D012680:Sensitivity and Specificity; D013183:Sputum / Q000382:microbiology*; D014376:Tuberculosis / Q000175:diagnosis* / Q000382:microbiology
chemical_list: D000942:Antigens, Bacterial; D001426:Bacterial Proteins; D018834:Chaperonin 60; D017931:DNA Primers; D004269:DNA, Bacterial; D021903:DNA, Ribosomal Spacer; C093276:ESAT-6 protein, Mycobacterium tuberculosis; D012336:RNA, Ribosomal, 16S; D012338:RNA, Ribosomal, 23S; C074119:heat-shock protein 65, Mycobacterium