Research On Real-time Fluorescent Quantitative RT-PCR For Detection Of PVY And PVA

Potato virus disease is an important disease of potato that causes serious economic losses every year. At present, the use of virus-free seed potato has been an important method for controlling the potato viruses. Potato virus detection technology that is fast and accurate is the key method to guarantee virus-free seed potato quality.In this study, it made a preliminary identification that what kind of virus the potato samples with different symptoms of the virus species was. Desigh two pairs of virus-specific primers according to the gene sequences of potato virus Y (PVY) and potato virus A(PVA). Do conventional RT-PCR identification with the virus-specific primers and the total RNA of potato samples as template.Using ELISA kit, the samples which has been identified of PVY were made a strain identification. And make an analysis of molecular variability between different PVY isolates of serotypes of serum, in order to identify PVY nucleotide mutation sites of various strain specificity, and design specific primers. Using the specific primers of different serum and taking the total RNA of potato samples as template.,do conventional RT-PCR identification and Real-time PCR identification.Under the stress of potato virus,make a study of the stability reference gene expression and screen internal standard gene. According to the gene sequence of potatoes, design seven reference genes and do real-time PCR reaction of virus potato samples of total RNA in normal section, necrosis and non-toxic potato samples and using geNorm program, analysis on the processing of the reaction result and stability of reference genes.On the basis of PVY genome and PVA genome sequences which have been published in GenBank database and in accordance with design principles of the standard fluorescent quantitative PCR primer, design2pairs of specific primers by Primer Premier5and screen out the most stable reference gene in potato as control under the stress of the virus to do a study of the real-time fluorescent RT-PCR detection to quantitate PVY and PVA.