反转录聚合酶链反应检测猪繁殖与呼吸综合征病毒的研究

STUDIES ON THE DETECTION OF PORCINE PRODUCTIVE AND RESPIRATORY SYNDROME VIRUS(PRRSV) USING REVERSE TRANSCRIPTION POYMERASE CHAIN REACTION(RT-PCR)

本研究成功地建立了检测猪繁殖与呼吸综合征病毒 (PRRSV)的反转录 聚合酶链反应 (RT PCR)技术。根据PRRSV两个标准毒株 (美洲ATCCVR 2 332株及欧洲LV株 )膜蛋白和核衣壳蛋白的编码序列差异 ,自行设计合成了各自的引物 ,对两标准毒株及广州分离株 (CDG9512 )进行扩增 ,获得了预期长度约为 1kb的扩增片段。酶切鉴定 ,结果进一步证实两标准毒株间基因差异显著 ,分离株与美洲株更为接近 ,而不同于欧洲株。对猪伪狂犬病病毒、细小病毒、猪瘟病毒、传染性胃肠炎病毒、流行性腹泻病毒及空白对照进行扩增 ,结果均为阴性。该技术能检测到病毒达 10 2 .5TCID50的水平。表明RT PCR对PRRSV的检测不仅具有特异、敏感、快速诊断的优点 ,而且还可以对毒株的基因类型加以鉴别。在RT PCR技术检测PRRSV方法上也进行了改进与探讨。

In this study,a RT PCR technique was successfully developed for detecting PRRSV.Two pairs of primers were designed and synthesized respectively,according to the variations between the putative membrane (M) protein and nucleoecapsid(N) protein genes of U.S. strain (ATCC VR 2332) and European strain (LV).Viral RNAs of two standard strains and Guangzhou isolate(CDG9512)were reverse transcripted and amplified by PCR,a single band with the expected size of about 1 kb was obtained.The result of restriction endonuclease analysis confirmed the existence of striking difference between the two standard strains and the genomic pattern of CDG9512 isolate was similar to that of U.S.strain but differ from European strain.No amplification was observed when using RNAs extracted from HCV,TGEV,PDEV and uninfected HS 2 H cell or DNAs from PPV and PRV.However,10 2.5 TCID50 PRRSV could be detected by the technique.These results demonstrated that RT PCR was not only a specific,sensitive and rapid method for diagnosis of PRRS,but also a way to differentiate the genotypes of various PRRSV isolates.In addition,the methods of RT PCR technique for detecting PRRSV were improved and tested.