猪瘟病毒野毒株与疫苗株酶切检测方法的建立、优化及应用

为有效鉴别猪瘟病毒强毒株(Shimen)与弱毒疫苗株(HCLV),根据GenBank上已发表的猪瘟病毒囊膜糖蛋白E2基因高度保守区设计一对特异性引物,在其跨越区内部有Shimen株独有的限制性内切酶Bgl Ⅱ酶切位点,采取酶切RT-PCR产物的方法鉴别Shimen株和疫苗株,同时对该方法的特异性和敏感性进行检测。结果表明,应用该方法从Shimen株和疫苗株中均能扩增出一条大小为750 bp的特异性片段,疫苗株的RT-PCR产物不能被Bgl Ⅱ酶切,Shimen株的RT-PCR产物则被酶切为大小分别为520和230 bp的两条带。此方法可扩增猪瘟病毒的E2基因保守片段,对病毒RNA的最小检出量为3.96×10^-4 μg/mL。采用此方法检查了30例临床疑似猪瘟病料,结果3例感染猪瘟病毒强毒,21例为猪瘟弱毒疫苗株,其他为猪瘟阴性。

Development,Optimization and Application of Restriction Enzyme for Identification of Highly Virulent Classical Swine Fever Virus and Vaccine Strain

In order to identify the highly virulent strain (Shimen) and vaccine strain (HCLV) of classical swine fever virus (CSFV),a pair of specific primers was designed against conserved domain of envelop glycoprotein E2 gene of CSFV sequence published in GenBank. A specific site of BglⅡ for restriction enzyme digestion was found and placed within PCR primers. The shimen strain and vaccine strain of CSFV were distinguished using single digestion of RT-PCR products with Bgl Ⅱ. Specificity and sensitivity were detected in parallel. A specific 750 bp fragment was amplified from both Shimen strain and vaccine strain of CSF with this method. The RT-PCR products of vaccine strain could not be cut by Bgl Ⅱ,while Shimen strain could be cut into two fragments of 520 and 230 bp length respectively. The conserved E2 gene fragment of CSFV could be amplified specifically with this assay,and the detection limit was 3.96×10^－4 μg/mL of RNA. This assay was also evaluated with 30 clinical samples, 3 of them were detected with Shimen strain of CSF infection,21 of them were vaccine strains,others were CSFV negative.