鉴别猪伪狂犬病病毒的双重PCR方法的建立及初步应用

为了检测猪伪狂犬病病毒(pseudorabies virus,PRV)的感染情况,并进行强弱毒株的鉴别诊断,本研究建立了快速、简便、灵敏度高、特异性强的鉴别猪PRV的双重PCR方法。针对PRVgE和gB基因序列,分别设计了2对特异性引物,通过对退火温度(50~60℃,按照1℃递增)、引物浓度(0.2~1.4μL,依次增加0.2μL)的优化,结果表明,双重PCR反应的最佳退火温度为56℃、最适引物添加量为1μL。特异性试验结果表明,该方法可以扩增出PRVgE(316bp)和gB(432bp)的目的片段,对PCV2、PTV、CSFV、PPV、PRRSV、大肠杆菌的DNA或cDNA均无扩增。敏感性试验结果表明,PRVgE和gB的最低核酸检出量分别为4.4×10³和3.3×10³拷贝/μL,与单一PCR方法的敏感性相近。应用该方法对广东、广西地区临床送检的56份组织样品进行检测,检测结果显示,强毒感染阳性率为53.6%(30/56),阴性率为46.4%(26/56),未发现有弱毒感染。

Establishment and Primary Application of Duplex PCR Assay to Detect Procine Pseudorabies Virus

In order to detect the procine pseudorabies virus (PRV) infection and differentiate virulent and avirulent PRV, a duplex PCR method was established with two pairs of specific primers designed based on the conserved sequences of gE and gB genes of PRV.Optimize the reaction conditions by adjusting concentration of primers from 0.2 to 1.4 μL, each time adding 0.2 μL and temperature of annealing from 56 to 60 ℃, added 1 ℃ each time, and so on.The results showed that the suitable annealing temperature was 56 ℃ and the suitable primer dose was 1 μL.Two special fragments of 316 bp (PRV gE) and 432 bp (PRV gB) were amplified by the optimized duplex PCR.The specific tests showed that no PCR products were detected for PCV2, PTV, CSFV, PPV, PRRSV and E.coli.The sensitivity test showed that DNA used for the duplex PCR might be as low as 4.4×10³ copy/μL for PRV gE and 3.3×10³ copy/μL for PRV gB.The sensitivity of the duplex PCR was close to the single PCR.Fifty-six clinical samples of sick pigs from different areas in Guangdong and Guangxi province were detected by the duplex PCR and the virulent detection rate was 53.6% (30/56) for PRV and the negative rate was 46.4%(26/56), and there was no attenuated PRV infection.