马铃薯S病毒PVS^O株系RT-LAMP检测方法的建立及应用

Establishment of Loop-mediated Isothermal Amplification Assay for Detection of Potato virus S Ordinary Strain

为建立一种马铃薯S病毒普通株系PVS^O快速、灵敏的RT-LAMP可视化检测方法。根据马铃薯S病毒普通株系的CP核苷酸序列设计4条引物,以感染PVS的马铃薯叶片总RNA为模板,采用一步法RT-LAMP,在62℃下反应60 min,扩增产物利用琼脂糖电泳分析和钙黄绿素染料显色判断结果,同时对其特异性和灵敏性进行测定。结果表明,建立的RT-LAMP方法可特异地对PVS^O进行检测,检测灵敏度为RT-PCR的100倍。对33份马铃薯样品检测表明,RT-LAMP与RT-PCR方法检测结果基本一致。因此建立的PVS^O的RT-LAMP可视化检测方法简便,特异性强,灵敏度高,适合PVS^O的快速检测。

The object of this study is to develop a One-step reverse transcription loop-mediated isothermal amplification（RT-LAMP）assay for rapid and sensitive detection of Potato virus S ordinary strain（PVS^O）. Four RT-LAMP primers were designed on the basis of the coat protein（CP）gene sequences of PVS^O,and template RNA from infected leaves were used for One-step RT-LAMP which were carried out under isothermal conditions at 62 ℃ for 60 minutes. RT-LAMP products were analyzed by electrophoresis in agarose gels followed by staining with Calcein. Then the specificity and sensitivity of RT-LAMP were testified. A rapid and specific RT-LAMP method for detection of PVS^O was established. Sensitivity of the RT-LAMP assay was 100-fold higher than ordinary RT-PCR method,and the testing results of 33 potato samples were basically consistent with RT-PCR. The RT-LAMP described in this study represents a sensitive,specific and rapid assay for the detection of PVS^O.