通过DNA链置换扩增检测呼吸道标本中的结核分枝杆菌。
Detection of Mycobacterium tuberculosis in respiratory specimens by strand displacement amplification of DNA.
作者信息
Down J A, O'Connell M A, Dey M S, Walters A H, Howard D R, Little M C, Keating W E, Zwadyk P, Haaland P D, McLaurin D A, Cole G
机构信息
Becton Dickinson Research Center, Research Triangle Park, North Carolina 27709-2016, USA.
出版信息
J Clin Microbiol. 1996 Apr;34(4):860-5. doi: 10.1128/jcm.34.4.860-865.1996.
A total of 294 clinical respiratory specimens, including 75 with culture-positive results, were tested for the presence of Mycobacterium tuberculosis by strand displacement amplification (SDA) of DNA. A region of the IS6110 insertion element and an internal control sequence were amplified and then detected by a chemiluminescence assay. Receiver operator-characteristic curves were used to evaluate three methods for declaring specimens positive for M. tuberculosis. By the preferred method, SDA chemiluminescence results were converted to theoretical numbers of M. tuberculosis organisms. A positive threshold (PT) value, above which 95% of the SDA results were judged to be M. tuberculosis positive (sensitivity = 95%), was found to be 2.4 M. tuberculosis organisms per SDA reaction. The analogous PT value for 95% sensitivity on smear-positive specimens was 3.6 M. tuberculosis organisms per reaction. The PT of 2.4 M. tuberculosis organisms per reaction detected 100% of culture-positive, smear-positive specimens (sensitivity = 100%), while 95% sensitivity was achieved with a PT of 15.5 M. tuberculosis organisms per reaction. Specificities, which were calculated with respect to culture- and smear-negative specimens, ranged from 96% at a PT of 15.5 M. tuberculosis organisms to 84% at a PT of 2.4 M. tuberculosis organisms per reaction. The M. tuberculosis-negative specimens were also segregated according to whether the patients received antituberculosis chemotherapy. SDA specificity ranged from 90% (PT = 2.4 M. tuberculosis organisms) to 98% (PT = 15.5 M. tuberculosis organisms) for the M. tuberculosis-negative specimens from patients who had not received chemotherapy. SDA specificity in the M. tuberculosis-negative specimens from patients who received chemotherapy was lower (85 to 94%). This study represents the first large-scale demonstration of M. tuberculosis detection in clinical sputum specimens by isothermal DNA amplification with SDA.
共对294份临床呼吸道标本进行检测，其中75份培养结果呈阳性，通过DNA链置换扩增（SDA）检测结核分枝杆菌的存在情况。对插入序列IS6110的一个区域和一个内部对照序列进行扩增，然后通过化学发光测定法进行检测。采用受试者工作特征曲线评估判定标本结核分枝杆菌呈阳性的三种方法。通过首选方法，将SDA化学发光结果转换为结核分枝杆菌生物体的理论数量。发现一个阳性阈值（PT）值，高于该值时，95%的SDA结果被判定为结核分枝杆菌阳性（敏感性=95%），为每个SDA反应2.4个结核分枝杆菌生物体。涂片阳性标本95%敏感性的类似PT值为每个反应3.6个结核分枝杆菌生物体。每个反应2.4个结核分枝杆菌生物体的PT可检测出100%培养阳性、涂片阳性的标本（敏感性=100%），而每个反应15.5个结核分枝杆菌生物体的PT可实现95%的敏感性。针对培养和涂片阴性的标本计算的特异性范围为，每个反应15.5个结核分枝杆菌生物体时为96%，每个反应2.4个结核分枝杆菌生物体时为84%。结核分枝杆菌阴性标本还根据患者是否接受抗结核化疗进行了分类。对于未接受化疗患者的结核分枝杆菌阴性标本，SDA特异性范围为90%（PT=2.4个结核分枝杆菌生物体）至98%（PT=15.5个结核分枝杆菌生物体）。接受化疗患者的结核分枝杆菌阴性标本中SDA特异性较低（85%至94%）。本研究是首次通过SDA等温DNA扩增对临床痰标本中的结核分枝杆菌进行大规模检测的示范。
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