猪主要RNA病毒病多重二温式RT-PCR检测方法的建立及初步应用

Establishment and Application of Multiplex Two-step RT-PCR for Detection of Swine Main RNA Virus Pathogens

通过对Genbank登录的CSFV、PRRSV和JEV的核苷酸序列进行比对分析,找出CSFV的E2基因,PRRSV的Nsp2基因和JEV的E基因为相对保守区域[1]。利用生物学软件在保守序列分别设计一对引物,通过对PCR反应条件的优化,确定了最佳引物浓度、最佳Mg2+浓度和最佳退火温度,建立了多重二温式PCR方法检测CSFV、PRRSV和JEV。其扩增的目的片断大小分别为CSFV(482 bp)、PRRSV(576 bp)和JEV(375 bp),将传统三温式PCR过程中的退火与延伸合并为一步,从而大大节省临床检测时间,同时又能通过一个反应体系对CSFV、PRRSV及JEV三种猪主要RNA病毒病进行检测。用50份临床病料对本研究多重PCR技术和单项PCR技术进行对比验证,结果显示,两者的总符合率为93%以上。表明建立的多重PCR检测方法,具有特异、快速、准确的特点,可用于对这3种病毒的同时检测和鉴别诊断。

To develop a multiplex RT-PCR test for detection of swine main RNA virus pathogens,three sets of primers were designed based on the conserved domains of E2 gene of CSFV,Nsp2 gene of PRRSV and E gene of JEV.The conditions of the PCR test were optimized,such as Mg2＋ and the concentration of the primers,and annealing and extension step were merged into one step as well.The multiplex two-step PCR test amplified product with size of 576,482 and 375 bp specific for PRRSV,CSFV and JEV respectively.To evaluate the multiplex RT-PCR assay,50 clinical samples were detected.The data showed that the multiple PCR method being 93% coincidence with the single PCRs for the presence of CSFV,PRRSV and JEV.The optimized multiplex RT-PCR is both sensitive,specific and rapid,providing a new method for the clinical diagnosis and epidemiological research of the three diseases.