USE OF A REAMPLIFICATION PROTOCOL IMPROVES SENSITIVITY OF DETECTION OF MYCOBACTERIUM-TUBERCULOSIS IN CLINICAL-SAMPLES BY AMPLIFICATION OF DNA
USE OF A REAMPLIFICATION PROTOCOL IMPROVES SENSITIVITY OF DETECTION OF MYCOBACTERIUM-TUBERCULOSIS IN CLINICAL-SAMPLES BY AMPLIFICATION OF DNA
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DOI：
10.1128/jcm.29.4.712-717.1991
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发表时间：
1991-04-01
影响因子：
9.4
通讯作者：
HANCE, AJ
中科院分区：
文献类型：
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作者：
PIERRE, C;LECOSSIER, D;HANCE, AJ
我们比较了结核分枝杆菌定量培养和聚合酶链式反应检测结核分枝杆菌复合体DNA的敏感性和特异性，对82例临床标本进行了检测。使用了两种扩增方案，一种是标准扩增方案，它扩增编码65-kDa抗原的一段基因，另一种方案是用第二套嵌套寡核苷酸引物重新扩增初始扩增产物。虽然标准扩增法在18份培养100CFU/ml的标本中有18份阳性，在35份培养阴性的结核病患者标本中有4份阳性，但在6份培养100CFU/ml的标本中只有1份阳性。这种敏感性的缺乏不能用原始样本中存在的Taq聚合酶抑制剂来解释。24例培养阳性标本中24例阳性，35例培养阴性的肺结核患者标本中13例阳性(与标准扩增法和常规培养法相比，总敏感性为63%，P<；0.02)。使用标准扩增方法时，23例未被诊断为结核病的患者中有两例阳性，但再扩增方法没有发现额外的假阳性结果(总体特异度为91%)。我们得出结论，在不牺牲特异性的情况下，重新扩增方案的使用提高了临床样本中分枝杆菌DNA检测的敏感性。这种方法的敏感性似乎优于标准培养技术。
We have compared the sensitivity and specificity of quantitative mycobacterial culture against results obtained by using the polymerase chain reaction for the detection of DNA from organisms of the Mycobacterium tuberculosis complex in 82 clinical specimens from patients suspected of having tuberculosis. Two amplification protocols were used, a standard amplification protocol, which amplifies a segment of the gene coding for the 65-kDa antigen, and a protocol in which the initial amplification products are reamplified with a second set of nested oligonucleotide primers. Although the standard amplification protocol gave positive results for 18 of 18 samples which grew > 100 CFU/ml and gave positive results in 4 of 35 specimens from patients with tuberculosis which were negative by culture, only 1 of 6 samples which grew < 100 CFU/ml was positive. This lack of sensitivity could not be explained by the presence of inhibitors of Taq polymerase present in the original samples. In contrast, the reamplification protocol gave positive results for 24 of 24 samples which were positive by culture as well as for 13 of 35 samples from patients with tuberculosis which were negative by culture (overall sensitivity, 63%, P < 0.02, compared with the standard amplification protocol and routine culture). Two of 23 samples from patients not diagnosed as having tuberculosis gave positive results when the standard amplification protocol was used, but no additional false-positive results were seen with the reamplification protocol (overall specificity, 91%). We conclude that the use of a reamplification protocol improves the sensitivity of detection of mycobacterial DNA in clinical samples without sacrificing specificity. The sensitivity of this approach appears to be superior to that of standard culture techniques.