南芥菜花叶病毒的几种PCR检测方法的建立和比较研究

Development and comparative studies of several PCR for detecting Arabis mosaic virus

以进境种球中截获的带毒洋水仙和郁金香为试验材料,建立了ArMV的免疫捕获RT-PCR、巢式PCR和Real-timePCR方法,并比较了几种检测方法的灵敏度。DAS-ELISA的检测灵敏度较低,为1 mg洋水仙或10 mg郁金香带毒种球,而各种PCR方法的灵敏度可高于DAS-ELISA 100倍以上,其中Real-time PCR检测的灵敏度最高,可从20 ng洋水仙或2μg郁金香的带毒种球中检出ArMV。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。

Immuno-capture RT-PCR, nested PCR and real-time PCR approaches were developed for detection of Arabis mosaic virus （ArMV） from the imported Narcissus and Tulip bulbs, as well as comparative study on detective sensitivities. The sensitivity of double antibody sandwich （DAS） -ELISA for detection of ArMV was relative low, which only detected the virus from 1 mg infected Narcissus or 10 mg Tulip bulbs, respectively. In contrast, the developed PCR approaches showed more than 100 times higher sensitivity. Specially, realtime PCR possessed the highest sensitivity and could detect the virus from as low as 20 ng Narcissus or 2 ug Tulip bulbs. When ELISA method is used for primary detection and the ratio of OD405 values between sample and control is about 2.0, it is suggested that the molecular approaches should be performed to avoid the possible omission.