Vodkin M H, Streit T, Mitchell C J, McLaughlin G L, Novak R J
Illinois Natural History Survey, Champaign.
Biotechniques. 1994 Jul;17(1):114-6.
An improved method for the extraction of viral RNAs was developed to facilitate the reverse transcription (RT)-PCR detection of mosquitoes infected with Western equine encephalitis virus or La Crosse virus. The solubilization method, which uses only EDTA and sodium dodecyl sulfate (SDS) followed by dilution of sample, allows accurate viral detection through the use of random hexamers for the RT followed by specific primers for the PCR. Identities of the reaction products were confirmed either by sequencing or restriction endonuclease digestion. Previous methods for the extraction of RNA for the coupled RT-PCR depended on combinations of guanidinium isothiocyanate, acid phenol, detergents and multiple centrifugations. Ideally, routine detection of viral RNAs for diagnostic purposes should bypass many of the above steps, while still providing a sensitive assay. Our level of detection is 1 infected mosquito in a group of 100.
为便于对感染西部马脑炎病毒或拉克罗斯病毒的蚊子进行逆转录（RT）-PCR检测，开发了一种改进的病毒RNA提取方法。该溶解方法仅使用乙二胺四乙酸（EDTA）和十二烷基硫酸钠（SDS），然后对样品进行稀释，通过使用随机六聚体进行逆转录，随后使用特异性引物进行PCR，从而实现准确的病毒检测。反应产物的同一性通过测序或限制性内切酶消化来确认。先前用于RT-PCR联用的RNA提取方法依赖于异硫氰酸胍、酸性酚、去污剂和多次离心的组合。理想情况下，用于诊断目的的病毒RNA常规检测应绕过上述许多步骤，同时仍提供灵敏的检测方法。我们的检测水平为100只蚊子中有1只受感染的蚊子。
