猴副流感病毒SV5的PCR检测方法建立及初步应用

目的]建立检测SV5的PCR方法并加以初步应用。方法]根据Genbank中报道的SV5序列,针对其中的SH基因设计引物进行PCR反应,扩增产物进行测序并用BLAST软件进行同源性比对,同时利用限制性内切酶的酶切反应以证实此PCR反应的特异性。在此基础上设计巢式PCR提高此方法的灵敏度。利用此方法对20份猴肾源细胞培养物和40份血清标本进行检测。结果]利用设计的引物扩增出的序列测序结果证实与报道的SV5SH基因相对位置的序列一致。AccIII限制性内切酶可对PCR产物进行特异性酶切。巢式PCR比一次PCR的敏感度有所提高。用此方法检测的20份猴肾源细胞培养物和40份血清标本结果为阴性。结论]本文首次初步建立了检测SV5病毒的PCR方法,排除实验室用20份猴肾源细胞培养物和40份血清标本SV5的污染。

Establishment and primary application of the PCR Method to detect simian virus 5

Objectives] To establish a PCR method to detect SV5 in biological material from monkeys. Methods] We have designed a pair of primers matched with SH gene of SV5 for PCR and sequenced the product fragments with BLAST soft to identify the homology and conformed by with AccIII restriction digestion. we also designed nested primers to improve the sensitivity of the PCR. The PCR technique was used to detect our cells and sera from Rhesus monkeys. Results] The PCR product was proved to be the frangment of the SH gene of SV5, and the PCR product was digested with the AccIII which is a specific site of the gene fragment. The sensibility of the examination was improved by nested PCR. All samples from monkeys were detected to be negative using this PCR method. Conclusions] We established the PCR method to detect SV5.