The Development Of Molecular Detection Kit For Potato Virus Y And Investigation Of PVY Strains

Potato virus Y is a main factor that affects the potato production, leading to significant yield losses and quality degradation. The serious reduction can reach to 80 percent or even absolutely no production. One of the ways to deal with this problem is to get virus-free plantlets by meristem tip culture, but whether or not the plantlets recovered from meristem culture is virus-free needs confirmation. The proper detection mothod is the first requirement for the control of such diseases. Therefore, it is necessary of developing a rapid, sensitive and specific method for potato industry.Detection kits greatly promoted the development of potato industry. As a stable, efficient, easy to operate, and other advantages, molecular detection kit has become an important tool in the virus detection. When need to do potato virus detection by molecular detection kit, imported from abroad in the main mostly.Primers LIU-PVY1 and LIU-PVY2 were designed and synthesized based on PVY CP gene sequence. Using the virus specific primers, one fragment of 0.34 kb was amplified by RT-PCR using the total RNA of the PVY infected plant; however, for the healthy sample nothing was got, so the routine RT-PCR detection method of PVY is established. Based on the routine RT-PCR detection method, One-step RT-PCR detection method for PVY is established by mixing all the reagents used in RT and PCR and designing the reaction conditions used in RT and PCR reaction. One-step RT-PCR detection accordingly was also established.In the optimization of RT-PCR system: Established M-MLV concentration, r-Tag enzyme concentration, the quality and concentration of dNTP, Mg2+concentration and PCR reaction of the annealing temperature, time and frequency of circulation. One of the most important is the choice of primer. Primer NIE-PVY and PVYj are come from Canadian potato center, PVYSP is domestic reported primer and LIU-PVY is designed by Heilongjiang Institute of Agricultural Sciences. This research records reverse transcription-polymerase chain reaction, the stability, generality and specificity has carried on the comparison to four pair of primers of potatoes Y virus. The results show: The LIU-PVY and PVYSP have high stability, generality and specificity, suitably makes the mass sample detection of PVY.This experiment used SDS extraction, Trizol and proteinase K extraction of total RNA extraction in plant. Measured in the OD260 \ OD280 values all greater than 1.70, the order was: Trizol method> proteinase K method> SDS method. On the three methods for extracting RNA gel electrophoresis and RT-PCR product of electrophoresis analysis showed that the same Trizol method is superior to other methods. Moreover Trizol method simple easy, time-saving advantages, the final choice the Trizol method extracts total RNA in plant, and as a kit in the plant RNA extraction methods.Based on the detection methods of the potato virus Y, one-step RT-PCR detection kit is es- tablished. The result of usage of the kits show: The kit had high stability and specificity. The sensitivity of detection in leaves is about 100μg by ELISA and 15μg by NASH, while the sensitivity for the kit was about 6.25μg. Though the sensitivity of the kit was the same as routine RT-PCR, but the use of the kit was time-saving and cost-effective. 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.The investigation results of PVYO, PVYN and PVYC shows: The virus of PVYN and PVYC are distributed in Heilongjiang Province. On the whole, the disease rate of PVYO is 83.24percent, PVYN is 73.41percent, PVYC is 0percent. The disease rate of PVYO is higher than PVYN, the composite infection rate of PVYN and PVYC is 56.65percent.Using the virus specific primers SP3, SP4 and SP5, one 544bp gene fragment can be amplified by RT-PCR using the RNA from the plant infected by PVYN, one 672bp gene fragment can be amplified by RT-PCR using the RNA from the plant infected by PVYO and for the healthy sample nothing can be amplified, so the RT-PCR detection method of PVYN and PVYO is established.