猴副流感病毒SV5 PCR检测方法的建立与初步应用

Establishment of PCR Method for Detection of Simian Parainfluenza Virus SV5 and Its Preliminary Application

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。

Objective To establish a PCR method for detection of simian parainfluenza Virus SV5 in biological material from monkeys and explore its application. Methods A pair of primers matched with SH gene of SV5 were designed for PCR and product fragments were sequenced with software BLAST to identify their homology. Acc Ⅲ restriction endonuclease was used for specific digestion. Nested primers were designed to improve the sensitivity of PCR reaction. PCR technique was used to detect cells and sera from rhesus monkeys. Results The PCR product was proved to be identical to the SH gene of SV5 reported, and the PCR products could be cut with the Acc m . The sensitivity of the examination was improved by nested PCR. All samples from monkeys were detected to be negative using this PCR method. Conclusions We have preliminarily established a PCR method to detect SV5, and using this technique 20 samples of products from monkey renal cell culture and 40 monkey serum samples were confirmed to be not contaminated by SV5.