猪繁殖与呼吸综合征病毒美洲株与NSP2变异株二重PCR诊断方法的建立及应用

Establishment and application of multiplex PCR on detecting porcine reproductive and respiratory syndrome virus VR2332 and NSP2 strain

根据猪繁殖与呼吸综合征病毒(PRRSV)美洲型标准株VR2332保守序列与近年PRRSV自然变异株序列分析研究,设计合成了2对特异引物,分别扩增出225、427bp两条特异型基因片段,通过优化RT-PCR条件,建立了可同时检测PRRSV标准株和NSP2变异株的二重PCR诊断方法。应用该方法分别对广西不同地区的16份病、死猪的血液、肺等组织进行检测,结果15份PRRSV阳性,其中1份为标准株,14份为变异株。试验结果表明,PRRSV标准株与变异株二重PCR诊断方法具有高度特异性、敏感性和实用性,可用于临床诊断,为目前流行的猪高热病病原研究及广西PRRS的防制提供了一定科学依据。

According to the sequences of American type strain VR2332 and natural variant strain of PRRSV in recent years, two specific primers were designed and two specific gene fragments of 225 bp and 427 bp were amplified respectively. By adjusting RT-PCR conditions, multiplex PCR method was established for detecting PRRSV type strain and NSP2 variant strain simuhaneously. The blood and lung of sixteen samples isolated from diseased or dead porcine in different districts of Guangxi were detected by this method, and the results showed that 15 samples were positive, among them, one sample was American type strain （225 bp） and 14 samples were variant strains （427 bp）. These results indicated that multiplex PCR method had high specificity and sensitivity, which could be applied in clinical diagnosis and would provide bases on studying pathogen of epidemiological swine high fever and control of PRRSV in Guangxi.