一步法RT-PCR检测烟草环斑病毒试剂盒的研制与应用

Research and Application of One-step RT-PCR Kit in the Detection of Tobacco Ringspot virus

根据烟草环斑病毒基因组序列设计特异性引物,利用RT-PCR技术研制成一步法检测该病毒的试剂盒,并建立了操作程序。对试剂盒扩增到的预期产物进行测序验证,产物序列与目的序列间的同源性为97.8%,表明该试剂盒的准确性较高。试验结果还表明,该试剂盒在常温、4℃和-20℃下可分别保存2d、7d和60d;灵敏度是DAS-ELISA的100倍;质量控制试验结果一致,具有较高的重复性。

A one-step RT-PCR kit was developed for the detection of tobacco ringspot virus （TRSV） with the special primers,which were designed based on the published sequence of TRSV and application procedure. The sequence analysis showed 97.8 % nucleotide acid identity between the positive product of the kits and the published sequence, and the result showed that the reagents system and the application procedure were right. After stored at -20 ℃ for two months at last ,at 4 ℃ for one week or at room temperature for two days, the kits still could be used for the detection, which sensitivity was 100 times more than that of DAS-ELISA and the result of quality control was same.