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李坏死环斑病毒(Prunus necrotic ringspot virus,PNRSV)是世界部分范围内分布的有害生物,亦是我国重点关注的检疫对象。根据PNRSV各株系衣壳蛋白基因的保守序列,设计特异性引物和TaqMan荧光探针,进行了探针、引物和Mg2+浓度等反应体系和条件的优化实验,确定最佳的引物浓度为400 nmol/L、探针浓度为333 nmol/L、Mg2+离子浓度为5 mmol/L和dNTPs浓度为0.43 mmol/L时,其灵敏度达23个拷贝数。利用建立的实时荧光RT-PCR检测方法对PNRSV樱桃分离物进行了成功检测。这个方法具有灵敏、准确、简便、快速的特点,适合于李坏死环斑病毒的检测和鉴定。
Prunus necrotic ringspot virus (PNRSV) is a pest distributed in some parts of the world. It is also the quarantine target that China pays close attention to. According to the conserved sequence of capsid protein gene in each strain of PNRSV, specific primers and TaqMan fluorescent probes were designed, and optimized reaction conditions and conditions of probe, primer and Mg2 + concentration were determined. The optimal primer concentration was 400 nmol / L, the sensitivity was 23 copies when the probe concentration was 333 nmol / L, the Mg2 + concentration was 5 mmol / L and the dNTPs concentration was 0.43 mmol / L. The PNRSV cherry isolate was successfully detected by real-time fluorescence RT-PCR assay. This method is sensitive, accurate, simple and fast, suitable for the detection and identification of necrotic ring spot virus.