猪繁殖与呼吸综合征病毒野毒株与基因缺失弱毒疫苗株TJM-F92一步RT-PCR鉴别方法的建立

Development of one-step RT-PCR method for identification of wild virus strain and gene-deleted attenuated vaccine virus strain of porcine reproductive and respiratory syndrome virus

目的建立一种猪繁殖与呼吸综合征病毒(Porcine reproductive and respiratory syndrome virus,PRRSV)野毒株与基因缺失弱毒疫苗株TJM-F92一步RT-PCR鉴别方法。方法基于高致病性PRRSV TJ株及其基因缺失致弱疫苗株TJM-F92的nsp2基因序列设计1对引物,建立可鉴别不同高致病性PRRSV野毒株与基因双缺失弱毒疫苗株的一步法RT-PCR,并进行特异性、灵敏度、敏感性、重复性验证。结果高致病性PRRSV TJ株扩增出1 100 bp的条带,基因缺失弱毒疫苗株TJM-F92扩增出740 bp的条带,可对两毒株作出准确鉴别;建立的一步法RT-PCR可测出101TCID50/ml的PRRSV-TJ疫苗毒株,灵敏度较高;对已知阳性和阴性仔猪血清样品的检测结果100%相符,敏感性较高,且重复性较好。结论建立的一步法RT-PCR可用于接种TJM-F92疫苗猪群中PRRSV野毒感染与疫苗免疫猪的临床鉴别,有助于评价疫苗的免疫效果并及早发现野毒感染。

Objective To develop a one-step RT-PCR method for identification of wild virus strain and gene-deleted attenuated vaccine virus strain TJM-F92 of porcine reproductive and respiratory syndrome virus （PRRSV）. Methods A pair of primers were designed according to the nsp2 gene sequences of highly pathogenic PRRSV TJ strain and gene-deleted attenuated vaccine virus strain TJM-F92, based on which a one-step RT-PCR method for identification of various wild and gene-deleted attenuated strains of PRRSV was developed and verified for specificity, sensitiveness, sensitivity and reproducibility. Results The gene fragment at a length of 1 100 bp was amplified from TJ strain, while that at a length of 740 bp from TJM-F92 strain, indicating that the two strains were identified correctly. The developed one-step RT-PCR method showed high sensitiveness, by which PRRSV-TJ vaccine strain at 10t TCIDs0/ml was detected. Both the coincidence rates of determination results of known positive and negative serum samples of piglets were 100%, indicating high sensitivity of the method. The method also showed high reproducibility. Conchltsion The developed one- step RT-PCR method might be used for clinical identification of wild PRRSV infection and PPRSV vaccination in pigs inoculated by vaccine prepared with TJM-F92 strain, which was helpful to evaluation of immune effect of vaccine and early diagnosis of wild PRRSV infection.