Detection of Mycobacterium tuberculosis in respiratory specimens by strand displacement amplification of DNA
Detection of Mycobacterium tuberculosis in respiratory specimens by strand displacement amplification of DNA
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DOI：
10.1128/jcm.34.4.860-865.1996
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发表时间：
1996-04-01
影响因子：
9.4
通讯作者：
Cole, G
中科院分区：
文献类型：
--
作者：
Down, JA;OConnell, MA;Cole, G
采用DNA链位移扩增（SDA）检测294份临床呼吸道标本，其中培养阳性75份。IS6110插入元件的一个区域和一个内部控制序列被扩增，然后通过化学发光试验检测。采用受试者操作特征曲线对三种宣告标本结核分枝杆菌阳性的方法进行评价。通过优选的方法，将SDA化学发光结果转化为结核分枝杆菌的理论数量。阳性阈值（PT）为每个SDA反应2.4个结核分枝杆菌，高于该值95%的SDA结果被判定为结核分枝杆菌阳性（敏感性= 95%）。涂片阳性标本95%敏感性的类似PT值为每次反应3.6个结核分枝杆菌。每次反应2.4个结核分枝杆菌的检出率对培养阳性和涂片阳性标本的检出率为100%（灵敏度= 100%），而每次反应15.5个结核分枝杆菌的检出率可达到95%的灵敏度。根据培养和涂片阴性标本计算的特异性从15.5个结核分枝杆菌的检出率为96%到每次反应2.4个结核分枝杆菌的检出率为84%。结核分枝杆菌阴性标本也根据患者是否接受抗结核化疗进行分离。对于未接受化疗的结核分枝杆菌阴性标本，SDA特异性从90% （PT = 2.4个结核分枝杆菌）到98% （PT = 15.5个结核分枝杆菌）不等。接受化疗的患者结核分枝杆菌阴性标本的SDA特异性较低（85 - 94%）。本研究首次大规模展示了SDA等温DNA扩增在临床痰标本中检测结核分枝杆菌。
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.