SYBR Green Ⅱ荧光定量PCR结合熔解曲线鉴别不同亚型兔病毒性出血症病毒方法的建立

摘要

The real-time fluorescence PCR in combination of unique melting curves was developed to distinguish Rabbit hemorrhagic disease virus(RHDV) and RHDV2.A pair of specific primers were designed according to the published GenBank database of RHDV and RHDV2 capsid protein VP60 and labeled with SYBR Green fluorescent dye Ⅱ.The melting temperatures of RHDV and RHDV2 were (86.3 + 0.1) ℃ and (85.1-0.1) ℃,respectively,and single specific peaks were observed.These results indicated that the method developed here was able to quickly identify RHDV and RHDV2.%本研究建立了一种荧光定量PCR(Real-time fluorescence quantitative PCR)结合熔解曲线区分不同亚型兔病毒性出血症病毒(Rabbit hemorrhagic disease virus,RHDV)方法.根据GenBank数据库中已公布的不同亚型RHDV(经典RHDV和RHDV2)的衣壳蛋白VP60序列保守区设计了1对特异性引物,利用SYBR Green Ⅱ荧光染料,在实时荧光定量PCR方法的基础上结合熔解曲线进行分析,经典RHDV和RHDV2熔解温度(Tm)分别为(86.3±0.1)℃和(85.1±0.1)℃,扩增产物的熔解曲线分析均只出现单特异峰.结果表明,本研究建立的方法能够快速地鉴别经典RHDV和RHDV2.