建立基于TaqMan探针的李坏死环斑病毒实时荧光RT-PCR检测方法

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。

Development of real-time fluorescent RT-PCR assays for the detection of prunus necrotic ringspot virus with a TaqMan probe

Prunus necrotic ringspot virus (PNRSV) is considered a quarantine pathogen of fruit trees disease in some parts of the world, and also a plant quarantine virus issued by Chinese government. A pair of primer and a TaqMan probe based on the conserved nucleotide sequence of coat protein gene of different PNRSV strains were designed and synthesized. Then through optimizing the concentration of primers, probe, Mg2+ and dNTPs, a real-time fluorescent RT-PCR was developed for detection of PNRSV in fruit trees. when optimal concentrations of primers, probe, Mg2+ and dNTPs was 400 nmol/L, 333 nmol/L, 5 mmol/L and 0.43 mmol/L, the assay for specific detection was highly sensitive, which could detect the template concentration as low as 23 copies, and could detect PNRSV in leaves tissues of cherry trees successfully. This reliable, sensitive, quick and easy-handling method is suitable for detection and identification of Prunus necrotic ringspot virus.