摘要： 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。
关键词:
CSFV,
PRRSV,
JEV,
多重二温式PCR
Abstract: 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 annea.1ing 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 withthe 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.
Key words:
lassical Swine fever virus,
Porcine reproductive and respiratory syndrome virus,
Japanese enceph.alitis virus：Multiplex two—step RT-PCR
中图分类号:
路斌, 王一成, 吴润, 袁秀芳, 徐丽华, 李军星, 王朝文. 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[J]. 华北农学报, 2010, 25(6): 38-43. doi: 10.7668/hbnxb.2010.06.008.
LU Bin, WANG Yi-cheng, WU Run, YUAN Xiu-fang, XU Li-hua, LI Jun-xing, WANG Zhao-wen. Establishment and Application of Multiplex Two-step RT-PCR for Detection of Swine Main RNA Virus Pathogens[J]. ACTA AGRICULTURAE BOREALI-SINICA, 2010, 25(6): 38-43. doi: 10.7668/hbnxb.2010.06.008.