鸢尾黄斑病毒几种PCR检测方法的建立和比较研究

 以带有鸢尾黄斑病毒的烟草叶片为材料，研究建立了IYSV的普通RT PCR、免疫捕获RT PCR和SYBR Green Ⅰ实时荧光RT PCR方法，并比较了几种检测方法的灵敏度。结果表明，DAS ELISA检测灵敏度较低，为1 mg带毒叶片。而各种PCR方法的灵敏度均高于DAS ELISA 100倍以上，其中SYBR Green Ⅰ实时荧光RT PCR检测灵敏度最高，可从0.4 μg的带毒叶片中检出IYSV，而RT PCR的灵敏度为40 μg带毒叶片，IC RT PCR的检测灵敏度是RT PCR的4倍。鉴于DAS ELISA灵敏度较低，建议在用ELISA初筛时，如样品OD405值与阴性对照OD405值之比在2.0左右时需要再用分子方法加以确证，以防漏检。

Development and comparative study of several PCR-based approaches for detection of Iris yellow spot virus(IYSV)

RT-PCR,immuno-capture RT-PCR and SYBR Green Ⅰ real-time RT-PCR approaches were developed for detecting IYSV from infected leaves of tobacco(Nicotiana tabacium) plant.Comparative study on detective sensitivity of these PCR-based approaches was also carried out.In contrast to the double antibody sandwich(DAS)-ELISA that could only detect the virus from 1 mg of infected leaves,the developed PCR approaches showed more than 100 times higher sensitivity.In particular,SYBR Green Ⅰ real-time RT-PCR had the highest sensitivity and could detect IYSV from as low as 0.4 μg of the infected leaves,while RT-PCR detected the virus only from 40 μg of the infected leaves and IC-RT-PCR showed four times more sensitive than that of RT-PCR.Since the relative low sensitivity of DAS-ELISA,it is suggested that PCR approaches should be performed to avoid the possible omission,once the ratio of OD405 between sample and control is about 2.0 during the primary detection by ELISA.