Tsai Y L, Tran B, Palmer C J
Environmental Sciences Laboratory, County Sanitation Districts of Orange County, Fountain Valley, California 92728.
Appl Environ Microbiol. 1995 Jan;61(1):363-6. doi: 10.1128/aem.61.1.363-366.1995.
It is important to determine the stability of naked viral RNA in seawater, since false-positive results can occur when reverse transcriptase-PCR (RT-PCR) is used to detect viruses if the RT-PCR amplifies free RNA instead of RNA from intact viruses. An acid guanidinium thiocyanate-phenol-chloroform method was used to extract total RNA from a filtered poliovirus cell culture suspension. The sensitivity of detection in this viral RNA study was 600 fg when RT-PCR was used. The extracted total RNA was seeded into filtered and unfiltered seawater, and the resulting preparations were incubated at 4 degrees C and at room temperature (23 +/- 1 degrees C). Our results showed that the seeded RNA was more stable in filtered seawater than in unfiltered seawater at both temperatures. The viral RNA could not be detected by the RT-PCR after 2 days of incubation in unfiltered seawater and after 28 days of incubation in filter-sterilized seawater. Therefore, because of the relatively short life of viral RNA in natural water, the detection of virus in environmental samples by the RT-PCR was mainly due to the presence of well-protected viral particles and not due to the presence of naked viral RNA.
确定裸露病毒RNA在海水中的稳定性很重要，因为当使用逆转录聚合酶链反应（RT-PCR）检测病毒时，如果RT-PCR扩增的是游离RNA而非完整病毒的RNA，就可能出现假阳性结果。采用酸性硫氰酸胍-苯酚-氯仿法从过滤后的脊髓灰质炎病毒细胞培养悬液中提取总RNA。在这项病毒RNA研究中，使用RT-PCR时检测灵敏度为600 fg。将提取的总RNA接种到过滤和未过滤的海水中，所得制剂在4℃和室温（23±1℃）下孵育。我们的结果表明，在这两个温度下，接种的RNA在过滤后的海水中比在未过滤的海水中更稳定。在未过滤的海水中孵育2天后以及在过滤除菌的海水中孵育28天后，RT-PCR均无法检测到病毒RNA。因此，由于病毒RNA在天然水中的寿命相对较短，通过RT-PCR检测环境样品中的病毒主要是由于存在保护良好的病毒颗粒，而非裸露病毒RNA的存在。
