番茄环斑病毒HC-RT-PCR-ELISA检测

Development of a Hybridization Capture HC-RT-PCR-ELISA Method for Reliable and Sensitive Detection of Tomato Ringspot Virus

番茄环斑病毒 (ToRSV)是我国对外检疫一类有害生物 ,目前国内尚无存在的报道。PCR技术是一种快速灵敏的植物病毒检测方法 ,但核酸内的聚合酶抑制物会导致漏检现象 ,而只通过凝胶电泳进行结果判定会出现假阳性 ,这两方面限制了PCR技术在对外检疫中的应用。利用共价结合在PCR管壁上的引物特异性杂交诱捕核酸粗提液中的靶标核酸 ,洗掉杂质及抑制物质 ,在同一管内作RT PCR ,凝胶电泳检测液相产物的同时对固相产物进行杂交检测 ,提高了结果的可靠性及灵敏度。利用所建立的HC RT PCR ELISA成功地从法国进口葡萄苗中检出ToRSV。

Tomato ringspot virus is a very important quarantine virus (on the A1 list of China),which has not been reported from China yet. RT PCR is a fast and sensitive method for ToRSV detection, but non detectable and false positive result is often occurred .A novel hybridization capture RT PCR ELISA was developed based on the above mentioned RT PCR, The main contributions to the development of the method are that a solid primer is successfully bound to PCR tube wall specially for aimed RNA capture in order to raise RNA purification and reduce time consumption. DIG labeled probe hybridization with solid PCR product was performed as well as electrophoresis of liquid product to reduce false positive result. The method was successfully used to detect the virus from the imported grape.