Bi H G, Saito A, Koide M, Ishimine T, Futenma M, Yamashiro Y, Kusano N, Kawakami K
First Department of Internal Medicine, Faculty of Medicine, University of the Ryukyus.
Kansenshogaku Zasshi. 1995 Mar;69(3):272-9. doi: 10.11150/kansenshogakuzasshi1970.69.272.
An insertion sequence repeated multiple times in the chromosome of Mycobacterium tuberculosis was used as a target for amplification using the polymerase chain reaction (PCR) assay for detecting Mycobacterium tuberculosis in clinical specimens. The sequences of primers were 5'-CCTGCGAGCGTAGGCGTCGG-3' (primer 1) and 5'-CTCGTCCAGCGCCGCTTCGG-3' (primer 2). One cycle of amplification consisted of denaturing at 94 degrees C for 2 min, primer annealing at 68 degrees C for 2 min, and extension at 72 degrees C for 2 min. DNA (5 fg) extracted from M. tuberculosis was detected by gel electrophoresis and Southern blot hybridization after 40 cycles of amplification. The amplification products were not obtained by DNA extracted from M. kansasii, M. intracellulare, M. avium, M. fortuitum, Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, Legionella pneumophila and Staphylococcus aureus; only from the M. tuberculosis complex. PCR results were compared with conventional cultural, pathological and microscopic findings in the detection of M. tuberculosis in 112 clinical specimens. There were 25 specimens that were positive for M. tuberculosis by cultural or pathological examination, of which 20 (80%) were positive by PCR. PCR detected the organism in 5 (83%) of 6 smear-positive specimens and 15 (79%) of 19 smear-negative specimens in which culture or pathology revealed M. tuberculosis. In addition, 2 smear-negative specimens and 8 smear-negative and culture-negative specimens were positive by PCR. These 10 samples were collected from the patients suspected as having tuberculosis by the clinical diagnosis based on the clinical history, characteristic radiographs, a positive PPD skin test and the effectiveness of anti-tuberculous drugs.(ABSTRACT TRUNCATED AT 250 WORDS)
结核分枝杆菌染色体中多次重复的插入序列被用作聚合酶链反应（PCR）检测的扩增靶标，用于检测临床标本中的结核分枝杆菌。引物序列为5'-CCTGCGAGCGTAGGCGTCGG-3'（引物1）和5'-CTCGTCCAGCGCCGCTTCGG-3'（引物2）。一轮扩增包括在94℃变性2分钟、在68℃引物退火2分钟以及在72℃延伸2分钟。扩增40轮后，通过凝胶电泳和Southern印迹杂交检测从结核分枝杆菌中提取的DNA（5 fg）。从堪萨斯分枝杆菌、胞内分枝杆菌、鸟分枝杆菌、偶然分枝杆菌、大肠杆菌、肺炎克雷伯菌、铜绿假单胞菌、嗜肺军团菌和金黄色葡萄球菌中提取的DNA未获得扩增产物；仅从结核分枝杆菌复合群中获得。在112份临床标本中检测结核分枝杆菌时，将PCR结果与传统培养、病理和显微镜检查结果进行了比较。通过培养或病理检查，有25份标本结核分枝杆菌呈阳性，其中20份（80%）通过PCR呈阳性。PCR在6份涂片阳性标本中的5份（83%）以及19份涂片阴性标本中的15份（79%）检测到该菌，这些涂片阴性标本的培养或病理检查显示为结核分枝杆菌。此外，2份涂片阴性标本以及8份涂片阴性且培养阴性的标本通过PCR呈阳性。这10份样本是从根据临床病史、特征性X线片、PPD皮肤试验阳性以及抗结核药物疗效被临床诊断怀疑患有结核病的患者中采集的。（摘要截短于250字）
