半巢式-RT-Realtime PCR检测李痘病毒

• 作 者: 粟智平 张静 黄迎波 李桂芬 耿金培 朱水芳
• 机 构: 烟台出入境检验检疫局,湖南出入境检验检疫局,中国检验检疫科学研究院,
• 出 处: 植物检疫
• 发表日期: 2012年03期
• 关键词: Realtime 李痘病毒 外壳蛋白基因 实时荧光 检疫性有害生物 探针 引物 检测 荧光探针 TaqMan

李痘病毒(Plum pox virus,PPV)是我国重要的植物检疫性有害生物。本研究根据PPV中CP基因(coat protein gene)的保守序列,设计了3条PCR引物和1条TaqMan探针,建立了半巢式-RT-RealtimePCR检测PPV的方法。该方法有机地结合了巢式PCR和实时荧光PCR技术;3条引物形成的2套PCR体系相互验证,有效提高了结果的准确性;荧光探针有效提高了检测的灵敏度。实验结果表明,本方法准确、灵敏、简便、快速,检出低限可达37fg/μL植物总RNA。

Plum pox virus (PPV) is an important plant quarantine pest in our country. In this study, three PCR primers and one TaqMan probe were designed according to the conserved sequence of coat protein gene in PPV, and a method of semi-nested-RT-Realtime PCR for detecting PPV was established. This method organically combines the nested PCR and real-time fluorescence PCR techniques. The two sets of PCR systems formed by the three primers are mutually validated, which effectively improves the accuracy of the results. The fluorescent probe effectively increases the sensitivity of the detection. The experimental results show that the method is accurate, sensitive, simple and rapid, and the detection limit is up to 37 fg / μL of plant total RNA.