Polymerase chain reaction for diagnosis of M-tuberculosis: Comparison of simple boiling and a conventional method for DNA extraction
Polymerase chain reaction for diagnosis of M-tuberculosis: Comparison of simple boiling and a conventional method for DNA extraction
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DOI：
10.1006/bmme.1996.0003
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发表时间：
1996-02-01
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影响因子：
--
通讯作者：
Stutman, HR
中科院分区：
文献类型：
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作者：
Afghani, B;Stutman, HR
虽然聚合酶链反应（PCR）已越来越多地用于临床标本中结核病（TB）的快速诊断，但关于DNA提取方案尚未达成共识。我们比较了一个简单的煮沸方法，一个传统的，劳动密集型的化学方法，使用溶菌酶和二氧化硅颗粒。煮沸法在30 min内完成，化学法至少需要6 h。在微生物实验室进行常规处理后，从77例患者中共获得82份临床标本（主要是呼吸道标本）。在通过每种方法提取DNA后，进行PCR以检测IS 6110的123-bp片段，并将结果与培养物进行比较。在培养阳性的20例标本中，煮沸法和化学法分别有17例（85%）和12例（60%）阳性。在62份培养阴性标本中，煮沸法和化学法分别有61份（98%）和57份（92%）为阴性。对于含有超过罕见AFB的涂片，煮沸和化学方法的灵敏度分别为100%和92%。我们的研究结果表明，煮沸法提取DNA是更敏感和不低于传统的化学方法的特异性。需要进行更大规模的研究，包括各种临床标本，以标准化PCR诊断M的最佳条件。结核(C)出版社：Academic Press，Inc.
Although the polymerase chain reaction (PCR) has been used increasingly for rapid diagnosis of tuberculosis (TB) in clinical specimens, no consensus exists regarding DNA extraction protocols. We compared a simple boiling method to a conventional, labor-intensive chemical method using lysozyme and silica particles. The boiling method was performed in less than 30 min; the chemical method required at least 6 h. A total of 82 clinical specimens (mostly respiratory) from 77 patients were obtained after routine processing from the microbiology laboratory. After DNA extraction by each method, PCR was performed to detect the 123-bp segment of IS6110, and results were compared to culture. Of 20 culture-positive specimens, 17 (85%) and 12 (60%) were positive by boiling and chemical methods, respectively. Of 62 culture-negative specimens, 61 (98%) and 57 (92%) were negative by boiling and chemical methods, respectively. The sensitivity was 100 and 92% for the boiling and chemical methods, respectively, for smears containing more than rare AFB. Our results suggest that boiling method of DNA extraction is more sensitive and no less specific than a conventional chemical method. Larger studies including a variety of clinical specimens are necessary to standardize the optimal conditions of PCR for diagnosis of M. tuberculosis. (C) 1996 Academic Press, Inc.