Gengvinij N, Pattanakitsakul S N, Chierakul N, Chaiprasert A
Department of Microbiology, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok Noi, Bangkok, Thailand.
Southeast Asian J Trop Med Public Health. 2001 Mar;32(1):114-25.
One-tube nested PCR was developed for diagnosis of pulmonary tuberculosis using sequences based on thel6SrRNA gene. The usage of primers 16SOL, 16SOR, 16SIL and 16SIR with optimized conditions could detect 555 bp DNA band from 21 species, 41 strains of mycobacteria and one isolate of Nocardia asteroides. It also revealed a specific 306 bp DNA band from 59 strains of M. tuberculosis complex. Cross amplification was observed in M. marinum, M. ulcerans and a few isolates of M. fortuitum complex. The developed method could detect as little as 100 fg of M. tuberculosis DNA. The PCR mixtures could be stored at 0 degrees C for 2 months or at -20 degrees C for at least 20 months without decrease in sensitivity. Using one-tube nested PCR for detection of M. tuberculosis compared with acid fast staining and culture results from 153 sputum specimens revealed 88.6% sensitivity and 89.2% specificity in smear positive specimens and 93.2% sensitivity and 85.0% specificity in culture positive specimens.
开发了一种单管巢式PCR方法，用于基于16S rRNA基因序列诊断肺结核。使用经过优化条件的引物16SOL、16SOR、16SIL和16SIR，可从21种、41株分枝杆菌和1株星形诺卡菌中检测到555 bp的DNA条带。它还从59株结核分枝杆菌复合群中显示出一条特定的306 bp DNA条带。在海分枝杆菌、溃疡分枝杆菌和少数偶然分枝杆菌复合群菌株中观察到交叉扩增。所开发的方法可检测低至100 fg的结核分枝杆菌DNA。PCR混合物可在0℃保存2个月或在-20℃保存至少20个月而不降低灵敏度。使用单管巢式PCR检测结核分枝杆菌，与153份痰标本的抗酸染色和培养结果相比，涂片阳性标本的灵敏度为88.6%，特异性为89.2%；培养阳性标本的灵敏度为93.2%，特异性为85.0%。
