The Establishment And Application Of Rapid Detecting Methods Of Potato Viruses

Potato viruses are the most important disease of potato, causing serious economic losses every year. At present, it is an important method for control of potato virus disease that the production and use of detoxification seed. The rapid and accurate potato virus detection technology is the key method to ensure virus-free seed potato quality.In this study, four pairs of virus-specific primers were designed based on the genome sequences of potato virus Y (PVY), potato virus A (PVA), potato virus S (PVS) and potato leafroll virus (PLRV). Using the total RNA which extracted from the potatoes with different viruses as template, four DNA fragments were amplified by RT-PCR with the precent of virus-specific primers. The four DNA fragments were transformed into Ecoli after being connected with a pGEM-T easy cloning vector and were sequenced. The sequencing analysis showed that the obtained DNA fragments were indeed the corresponding target gene fragments of the target viruses (the nucleotide homology is above99%). We established a convention RT-PCR technology for detecting the four vriues.The method of extracting potato virus RNA was optimized to solve the problem that failing to extract the RNA of potato tubers with high starch content by conventional plant RNA extraction kit. At first, a new method for potato tuber total RNA extraction was established. The RNA prepared by the method was in full compliance with the requirements of detecting the potato virus by RT-PCR. The method was rapid, simple, cheap, and was more suitable for the detection of a large number of samples. Based on the method above, a new buffer solution was developed. The viruses in the potato tuber were detected by RT-PCR after homogenized in the buffer solution, without extracting the potato tuber RNA. Therefore, it was a simpler, more efficient and cheaper detecting method than the above methods. The method has not been reported in China.The reaction system and response procedures of RT-PCR detection technology were optimized. A multiplex reverse transcription polymerase chain reaction (m-RT-PCR) was established for the simultaneous detection of PVS, PVY and PVA and was optimized from MgCl2concentration. The result showed that when the MgCl2concentration of5.5mmol/L, the amplification of the DNA fragments was the best. This method was faster, simpler and more efficient.The detoxification plantlets and detoxification seed from Henan and Heilongjiang province were detected with the established potato virus detection methods. The virus with the highest detection rate was PVY, the second was PVA in the samples from Henan province, the virus with the highest detection rate was PVS, the second was PLRV in the samples from Heilongjiang province.The viruses in the potato collected from many potato growing areas in China were detected with the methods established by us. The results showed that PVY was detected in the seven provinces including Heilongjiang, Liaoning and Henan; PVA was detected in Heilongjiang, Hunan, Fujian and Guizhou province; PVS was detected in the five provinces including Heilongjiang, Henan and Guizhou; PLRV was detected in Heilongjiang and Guizhou province. The occurrence of PVA in Guizhou province and PVS in Henan and Jilin province were reported for the first time.