Indentification And Detection Of Potato Virus And Viroid In Urumqi Region By Molecular Approach

Urumqi region is an important potato chip, potato production base in Xinjiang. The potato virus disease was common and serious. It was the main obstacle in production of potato. One of the ways to deal with this problem was to get virus-free plantlets by meristem tip culture, but whether or not the plantlets recovered from meristem culture was virus-free needs confirmation. The proper detection method was the first requirement for the control of such diseases. Our research in order to indentification virus types and develop a procedure for effective detection of potato virus. For potato virus free of material selection and cultivation under a potato chips, and the implementation of quarantine inspection and transportation virus disease effective prevention measures to provide scientific basis and technical support.Through investigation symptoms and electron microscopy observation, the viruses were amplified by RT-PCR, cloning and sequence analysis. It dentified clear the infection of the main agent of Urumqi region potato viruses had PVX, PVY, PVA, PLRV, PSTVd. The rate of infection showed that the first were PVY and PLRV, the second were PVX and PVA, last was PSTVd. Results of homology analyses showed that five in six PVY separation were PVYO, one was PVYN. The Urumqi region isolates compared with PSTVd Wisconsin was 98.4%, with France X76844 was 98.8%, and were most highly with other isolates in nucleotide sequences, but had some differences with other foreign isolates.One-step multiplex reverse transcriptionpolymerase chain reaction (m-RT-PCR) was developed for the simultaneous detection of potato virus X, potato virus Y, potato virus A and potato leafroll virus. The primers for PVX (732bp), PVY (422bp), PVA (132bp) and PLRV (336bp) fragments were designed based on the coat protein gene. The limit for Virus RNA about 7.8pg/μL. Detection of AMV, PVM, PVS, TMV and PSTVd were negative. The results showed that the one-step multiple RT-PCR was more fast,sensitive, efficient than two-step multiple RT-PCR.Two pairs of primers, two TaqMan probes, which marked with different fluorescent reporters, were designed and synthesized based on conserved nucleotide sequence of CP gene of several PVA and PVY isolates were designed and synthesized separately. The reaction parameters such as the concentration of two pair of primers, two TaqMan probes and the reaction buffer were optimized to develop a duplex real-time PCR assay for the stimultaneous detection of PVA and PVY. The duplex real-time PCR assay was found to be specific and no positive results were observed when nucleic acid from PVX, PLRV, AMV and TMV were used as multiplex real-time PCR templates. The lower limit of detection for PVA and PVY was 0.51fg/μL, and its sensitivity was 102 times higher than that of the conventional RT-PCR.One setp m-RT-PCR detection kit was assmebled according to systems of m-RT-PCR, reaction conditions and reagent concentrations. And the kit was used for some samples detected. The results showed that the kit was a specific, sensitive, efficient and cheap technique. So the kit can be used as a useful tool for routine testing, and selection of virus-free potatoes, especially when a large number of samples are detected.