NESTED POLYMERASE CHAIN-REACTION ASSAY FOR THE DETECTION OF CYTOMEGALOVIRUS OVERCOMES FALSE POSITIVES CAUSED BY CONTAMINATION WITH FRAGMENTED DNA
NESTED POLYMERASE CHAIN-REACTION ASSAY FOR THE DETECTION OF CYTOMEGALOVIRUS OVERCOMES FALSE POSITIVES CAUSED BY CONTAMINATION WITH FRAGMENTED DNA
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DOI：
10.1002/jmv.1890300202
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发表时间：
1990-02-01
影响因子：
12.7
通讯作者：
GARRETT, CT
中科院分区：
文献类型：
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作者：
PORTERJORDAN, K;ROSENBERG, EI;GARRETT, CT
聚合酶链反应 (PCR) 技术为组织培养提供了一种有前景的替代方案，可快速、灵敏地检测巨细胞病毒 (CMV) 感染。然而，在含有水但不含 DNA 的样品中检测到高水平的背景扩增，使得边缘阳性样品的解释变得极其困难，并降低了测定的灵敏度。水或阳性样品的扩增信号可以通过 DNase 处理来消除，但不能通过各向异性膜过滤、高压灭菌或紫外线照射来消除。通过液体杂交检测，在与水的反应显示产物形成之前观察到 10 至 12 个循环的滞后时间。巢式 PCR 的使用消除了背景，并且在阳性样品的连续稀释中，液体杂交检测的灵敏度提高了 500 至 1000 倍。我们认为背景信号是由 DNA 小片段产生的，这些片段可能是通过高压灭菌病毒培养材料产生的。这些片段会逃脱过滤，并且重叠的 DNA 片段可以相互引发，形成完整的嵌合序列，然后进行扩增。适当控制每个步骤的循环次数的巢式 PCR 应该能够成功克服由 DNA 片段引起的假阳性，无论污染是否发生在收集地点、加工过程中或在执行测试的设施中。
The polymerase chain reaction (PCR) technique offers a promising alternative to tissue culture for the rapid and sensitive detection of cytomegalovirus (CMV) infection. However, high levels of background amplification detected in samples containing water but no DNA make interpretation of borderline positive samples extremely difficult and reduce the sensitivity of the assay. The signal from amplification of water or positive samples can be eliminated by DNase treatment, but not by filtration through anisotropic membrane, autoclaving, or ultraviolet irradiation. A lag time of 10 to 12 cycles is observed before the reactions with water will show product formation by liquid hybridization detection. The use of nested PCR eliminates the background and, in serial dilutions of a positive sample, shows a 500- to 1000-fold increase in sensitivity by liquid hybridization detection. We suggest that the background signal is arising from small fragments of DNA, which may be produced by autoclaving viral culture material. Such fragments would escape filtration, and overlapping fragments of DNA can prime one another to form complete mosaic sequences that will then amplify. Nested PCR, appropriately controlled for the number of cycles at each step, should successfully overcome such false positives caused by fragmented DNA, no matter if the contamination occurs at the collection site, in processing, or at the facility performing the test.