Establishment Of Multiplex PCR Detection For Four Potato Viral Diseases

The potato (Solanum tuberosum L.), which is a major food combine grain andvegetables, occupies a pivotal position in food production and economic development in ourcountry. But with the expansion of cultivated area and continuous cropping make frequentoccurrence of increasingly serious potato disease, which is the most common viral diseasehazards. Potato virus is known up to35, of which there is a genuine hazard6-7species.Virus infection not only affects plant growth season, and even lead to the degradation ofgermplasm. The main hazard of potato virus is common mosaic virus (Potato virus X, PVX),heavy mosaic virus (Potato virus Y, PVY), latent mosaic virus (Potato virus S, PVS) and leafcurl virus (Potat Leaf Roll virus, PLRV), these viruses often mixed infection, cause greaterharm. Currently, there were not any effective drugs to treatment of viral diseases in the world,virus-free seed potato production and application of prevention of viral diseases become animportant measure. Establish rapid, accurate and sensitive virus detection techniques forseed quality testing and seed market supervision have important practical significance.Currently, there are some virus detections, such as indicator plant, electron microscopy,immunological methods and methods for plant molecular biology. Plants need to observe theindicates the specified symptoms of a particular plant, sometimes because of specifiedsymptoms were not obvious and difficult to identify, but also the detection period is too longto meet the actual needs. Due to electron microscopy detection techniques require expensiveequipment and skilled operators, general laboratory cannot meet. Currently, enzyme-linkedimmunosorbent assay (ELISA) detection method is commonly used, but limited to lowconcentration of the host phloem and viruses (such PLRV). ELISA detection method is notsensitive enough remained. It occasionally false positive or false negative result, there werealso shortcomings the operation cumbersome and time-consuming. PCR technology is rapid,sensitive and accurate, and its related technologies have been extensively used in virusdetection, In production, potato often infected with mix potato virus, requiring to theimplementation of a variety of viruses detected in the same reaction simultaneously to meetthe needs of rapid detection. Developed on the basis of conventional PCR multiplex reversetranscription PCR (Multiplex Reverse Transcription PCR, RT-mPCR) technology, which not only retains the conventional PCR with specificity and sensitivity, but also reduce steps andsave reagents, important the variety of viruses can be detected in the same reaction.Nowadays, there are two to three kinds of synchronous detection RT-mPCR potato virusreported, but no more than three or more virus detection reports. Therefore, this study potatoPVX, PVY, PVS and PLRV synchronous detection of targets, screening four kinds ofconserved sequence of the virus coat protein, according to the principle of multiplex PCRprimers designed to optimize the multiplex PCR reaction system and the reaction conditions,the establishment of these four molecular detection of virus diseases synchronization system.The main results obtained are:1. Four potato coat protein genes (PVX, PVY, PVS and PLRV) were cloned. Establishmolecular detection reference for four kinds of viruses. Reference domestic PCR detectionof potato disease related literature retrieval National Center for Biological Information(NCBI) sequences of four kinds of molecules in the virus detection molecule is determined(Coat Protein, CP) for the virus coat protein gene. Four viruses amplified fragment offull-length CP gene by RT-PCR; connection with the cloning vector, import E.coli DH5α,was created four kinds of viral clones CP gene. After sequencing, clones of four kinds ofviral CP gene sequence homology with the reported one in more than96%. Clonescontaining four kinds of virus CP gene for mPCR system to set up and provide a stablesample testing positive control.2. Screening and validated four kinds of virus CP gene amplification conserved regionstability. By NCBI BLASTN and BIOEDIT comparison to find conserved sequences of eachvirus CP gene, according to multiplex PCR primer design principles primers were designedfor each virus CP gene conserved sequence containing four kinds of clones virus CP gene asa template. Amplified421,202,516and330bp as specific fragment; through the detectionof different pathogens is verified by specific primers.3. Create a durable four kinds of potato virus synchronization RT-mPCR detectionsystem. Through optimization of the reaction system and reaction conditions for multiplePCR, the establishment of synchronous detection of potato PVX, PVY, PVS and PLRV theRT-mPCR system. The collection of field potato leaves and potato seed industry productionof micro sample testing shows that the establishment of multiplex RT-PCR detection systemwith a stable, fast and accurate high, and can be synchronized potato four viruses were identified. ELISA assay and compared with a single RT-PCR method, this detection methodcannot only reduce steps and save time, and reduce cross-contamination and improve thedetection sensitivity, after effectual method of potato virus detection.