Nakahara K, Hataya T, Uyeda I
Department of Agrobiology and Bioresources, Faculty of Agriculture, Hokkaido University, Sapporo, Japan.
J Virol Methods. 1999 Jan;77(1):47-58. doi: 10.1016/s0166-0934(98)00135-9.
A simple, rapid method of nucleic acid extraction on a microcentrifuge tube scale for detecting viroids is presented. Five distinct citrus viroids (CVds), chrysanthemum stunt viroid (CSVd), hop stunt viroid (HSVd), hop latent viroid (HLVd) and potato spindle tuber viroid (PSTVd) were detected in their natural host plants by hybridization using cRNA probes and reverse transcription-polymerase chain reaction (RT-PCR). Nucleic acids (NA) were liberated from tissues by incubation in a buffer containing potassium ethyl xanthogenate (PEX) without tissue homogenization, and then precipitated with ethanol (NA-PEX). All the viroids except CVd-IV could be detected clearly in NA-PEX by hybridization. HSVd, HLVd and PSTVd could also be detected in NA-PEX by RT-PCR. Although CVds and CSVd could not be detected in NA-PEX by RT-PCR, they were detected after further purification: differential precipitation with 2-butoxyethanol and HCl treatment followed by ethanol-precipitation. In addition, PCR in the presence of tetramethylammonium chloride specifically amplified the cDNA of all five distinct CVds under the same temperature and cycle conditions. Since all the viroids could be detected in NA liberated by PEX, the amount of NA extracted by the method described here is sufficient for detecting viroids, enabling the processing of a large number of samples.
本文介绍了一种在微量离心管规模上用于检测类病毒的简单、快速核酸提取方法。使用cRNA探针和逆转录-聚合酶链反应（RT-PCR）杂交，在其自然寄主植物中检测到了五种不同的柑橘类病毒（CVds）、菊花矮化类病毒（CSVd）、啤酒花矮化类病毒（HSVd）、啤酒花潜隐类病毒（HLVd）和马铃薯纺锤块茎类病毒（PSTVd）。通过在含有乙基黄原酸钾（PEX）的缓冲液中孵育而不进行组织匀浆，从组织中释放出核酸（NA），然后用乙醇沉淀（NA-PEX）。除CVd-IV外，所有类病毒均可通过杂交在NA-PEX中清晰检测到。HSVd、HLVd和PSTVd也可通过RT-PCR在NA-PEX中检测到。虽然CVds和CSVd不能通过RT-PCR在NA-PEX中检测到，但在进一步纯化后可检测到：用2-丁氧基乙醇进行差异沉淀和HCl处理，然后乙醇沉淀。此外，在氯化铵存在下的PCR在相同温度和循环条件下特异性扩增了所有五种不同CVds的cDNA。由于所有类病毒均可在由PEX释放的NA中检测到，因此本文所述方法提取的NA量足以检测类病毒，从而能够处理大量样品。
