三叶半夏组培苗的病毒检测

Detection of Viruses from Tissue-culturing Plantlets of Pinellia ternata(Thunb.)Breit.

组织培养阶段植物细胞中的病毒含量较低,难以用常规方法进行可靠检测。本研究采用DAS-ELISA、RT-PCR和核酸杂交三种方法对不同来源的12个三叶半夏(Pinellia ternata(Thunb.)Breit.)组培苗样品中的黄瓜花叶病毒(Cucumber mosaic virus,CMV)和大豆花叶病毒(Soybean mosaic virus,SMV)进行比较检测。结果显示:经DAS-ELISA检测,4个样品携带CMV,4个样品携带SMV,1个样品同时携带2种病毒;经RT-PCR检测,5个样品携带CMV,6个样品携带SMV,1个样品同时携带2种病毒;经核酸杂交检测验证,其结果与RT-PCR的检测结果一致。可见,对于三叶半夏组培苗,DAS-ELISA检测灵敏度不够高,需用灵敏度更高和特异性更强的核酸杂交方法来对其进行病毒检测,确保检测结果准确可靠。

The concentration of viruses in plant cells are induced during tissue culturing periods, and it is difficult to use conventional methods for reliable detection. We used DAS-ELISA, RT-PCR and nucleic acid hybridization after PCR amplification to test Cucumber mosaic virus （CMV） and Soybean mosaic virus （SMV） from tissue-culturing plantlets of P. ternata Among twelve seedling lines, CMV was detected from four seedling lines, SMV was detected from four seedling lines, while we found one seedling line was complexly infected by both viruses. RT-PCR detection using primers corresponding to coat protein gene of a penellia strain of CMV and a penellia strain of SMV respectively, resulted five cases of CMV infection：and six cases of SMV infection, one seedling line of complex infection by both viruses. Using RT-PCR products for further confirmation by nucleic acid hybridization, the same results were obtained with RT-PCR. The above results show that the sensitivity of DAS-ELISA detection is not enough for detecting tissue-culturing plantlets of P. ternata. In order to ensure the accuracy and reliability of seedling quarantine, nucleic acid hybridization after amplification of viral RNAs, with higher sensitivity and specificity is required.