Pest Analysis For The Importation Of Potato Seed From Germany And Research On The Detection Of Potato Virus With RT-PCR

The yield and planting area of potato in china take the first place in the world.But the average yield of potato far drops behind the countries developed in potato producing.We need introducing eximious potato seed to improve the yield and quality of potato in china. According to international tradition,we should do the relative pest risk analysis before introducing seed to protect the security of agriculture and zoology.One of the important factors affecting potato quantity and quality is the diseases caused by several potato viruses.Producting of virus free or viroid free seed-potato by tissue culture method is the main measure of controlling potato virus diseases.Introducing potato seed and developing virus free seed-potato need the detection of potato virus which is rapid,accurate and sensitive.Through the research of the pest risk analysis for the importation of potato seed from Germany,We forecast the risk of introducing potato seed and establish relative measures to reduce the risk.We also establish a technique using the muliplex reverse transcription polymerase chain reaction(m-RT-PCR) to detect several viruses simultaneously.1,Pest analysis for the importation of potato seed from Germany consists of there stages(1) Initiating the Process for Analyzing:According to guideline for pest risk analysis constituted by FAO,our research initiated the PRA by spreading pathway and indicated the pests associated with potato from Germany were 179 kinds.(2) Assessing Pest Risk:According to the geographical and regulatory criteria,the economic importance,the possibility for pests to spread by the seed,we made a primary assessment and compartmentalize the pests into regulated pest(RP) and non-regulated pest (NRP).From aspects of introduction potential,establishment potential,spread potential after establishment and potential importance,we made a detailed assessment for RP, defined 21 kinds of quarantine pest(QP) and 19 kinds of regulated non-quarantine pest (RNQP). (3) Managing Pest Risk:We listed risk management options for reducing risk to an acceptable level,then evaluated the efficacy and impact of the various options.2,Research on the detection of potato virus with RT-PCR(1) Design of the primers for potato viruses:The primers were designed based on the complete genome of PVA and the coat protein gene of PVY and PLRV.We adopt the simplex,duplex and multiplex reverse transcription polymerase chain reaction to detect PVY,PVA and PLRV and the amplification fragment by RT-PCR were 400bp(PVY), 300bp(PVA)and 222bp(PLRV).(2) The preparation for viruses RNA:Our research adopt three methods which include LiC1 method,RNAprep Plant Kit and soaking-solution method to preparate potato viruses RNA.The result showed that the RNA prepared by LiC1 method and RNAprep Plant Kit are more pure and intact than the RNA prepared by soaking-solution method.But the RNA prepared by these three methods are all qualified for RT-PCR.(3) Development of RT-PCR system:Reverse transcription(RT) stage and polymerase chain reaction(PCR) stage were researched and optimized in simplex,duplex and multiplex RT-PCR system.The result showed that the dNIPs concentration on RT stage and Mg²⁺ concentration on PCR stage had a great impact on the detection.The developed simplex, duplex and multiplex RT-PCR system could be used to detect PVY,PVA and PLRV.(4) Test of amplified products:The amplified cDNA of PVY,PVA and PLRV were extracted from the gel and cloned into vector PGM-T respectively.PCR test showed that the cDNA had been transferred into the vector(the positive white colony).The plasmids were extracted and sequenced,the compared with those in NCBI BLAST,and the result showed that the sequences of amplification products were correct.