Study On Detection Of PVY And PLRV Using Taqman Real-Time Quantitative PCR

Potato is a stape food crop and is widely grown in China. One of the important factors affecting potato quantity and quality is the diseases caused by several potato viruses. Production of virus or viroid free seed-potato using tissue culture is the main measure of controlling potato virus diseases.?The traditional detection methods such as indicator plant,microscopic detection of inclusionbody,ELISA,DIA and nucleic acid hybridization,can not fulfill the requirements of the detection because they are more or less time-consuming,complicated in operation,less sensitive and too dangerous,while reverse transcription polymerase chain reaction (RT-PCR) has become the most adopted technology for virus detection since PCR was invented. This technology is sensitive,specific and rapid,which can fulfill the requirements of modern plant quarantine. Research purpose: to establish a rapid,sensitive and reliable duplex real- time RT- PCR assay strategy for Potato Virus Y (PVY) and Potato Leafroll Virus (PLRV) detection at same time in order to provide technical support for rapid and accurate identification and monitoring forecasting of PVY and PLRV.Research contents:1. Comparing the different RNA concentration and purity extracted by different extraction methods such as improved CTAB method,extraction kit method,soaking method,minicolumn centrifugal method and so on, the minicolumn centrifugal method could be used to extract qualified RNA from PVY/PLRV and reduce the false-negative rate for its better performance to exclude PCR inhibitors like plant pigments,protein and polysaccharide.2. Fluorescent Quantitive Polymerase Chain Reaction FQ-PCR) assay for detection of PVY and PLRV were established respectively. The PCR conditions were optimized to improve the sensitivity,specifity and repeatability. A standard curve was constructed and the result showed that the sensitivity of the method was 1500 corpies and the linear relation was fitted.3. To our knowledgement,this is the first report for real-time RT-PCR assay for the simultaneous detecton of PVY and PLRV. The sequence downloaded from Genbank was aligned by software and the specific primers and probes were designed in the conserved region of PVY,PLRV. The RT-PCR conditions were optimized to improve the sensitivity,specifity and repeatability. The relative quantification in real-time RT-PCR to diction of PVY and PLRV were established with this method,and the good results were achieved by using the method in the actual application.Results: A duplex real- time RT- PCR method was established for PYV and PLRV witch can detect and differentiate PVY and PLRV in one action. The sensitivity of the assay was 1500 template copies for PVY and PLRV,which is 100 times than that of the routine RT- PCR. The field samples were examined using the test system repeatedly and the results were reproducible. Different concentrations of PVY and PLRV mixed together still could be identified by this assay,which implied the assay could be applied to diagnosis simultaneous infection of PVY and PLRV.Conclusion: rapid,robust and reliable molecular-based detection systems were established with minicolumn centrifugal method and positive standard samples. The whole procedure from sample treatment to detection results cost only 4h in total, which fulfilled the requirements in practical detection. Therefore,the two detection methods could be applied to detect citrus seedlings and monitor the field dynamic changes of PVY and PLRV.