鸢尾黄斑病毒几种PCR检测方法的建立和比较研究  

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

作　　者：代欢欢[1,2] 杨翠云[2] 周琦[3] 于翠[2] 

机构地区：[1]海海洋大学,上海201306 [2]上海出入境检验检疫局食品中心,上海200135 [3]北京出入境检验检疫局,北京100026

出　　处：《植物病理学报》2012年第2期126-130,共5页Acta Phytopathologica Sinica

基　　金：国家质检总局科技项目(2011IK172)

摘　　要：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 GreenⅠ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 GreenⅠ实时荧光RT-PCR检测灵敏度最高,可从0.4μg的带毒叶片中检出IYSV,而RT-PCR的灵敏度为40μg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OD405值与阴性对照OD405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。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.

关 键 词：鸢尾黄斑病毒 PCR 检测方法 灵敏度 

分 类 号：S432[农业科学—植物病理学]