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Dectection of Potato Virus by Multiplex RT-PCR

Author: DaiTingFei
Tutor: WenChengJing；NingHong
School: Sichuan Agricultural University
Course: Of Plant Pathology
Keywords: Potato virus Multiplex RT-PCR detection
CLC: S435.32
Type: Master's thesis
Year: 2011
Downloads: 67
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Abstract


As the world of high-yield crops, potato production and cultivation area in the country in the world. Seed potato in China with foreign seed potato quality gap, the situation is more serious potato disease occurred, causing huge losses. Potato virus disease is an important constraint in potato production. Under field conditions, the potato virus disease usually associated with mixed infection of two or more viruses infect potato plants. Potato virus infection resulting in lower yields, decline in quality, cut caused by potato virus disease in the world each year at least 20%. Large-scale tissue culture virus-free seed potato production control potato virus disease, the excellent production of virus-free seed need fast, accurate and sensitive virus detection technology. Establish a rapid, accurate, sensitive virus detection technology to protect the quality of seed potato and prevent the spread of the virus has important practical significance. Potato virus detection method a lot, basically can be divided into biological experimental methods (indicator plant method), several categories of electron microscopy techniques, immunological methods and molecular biology detection methods. Common potato virus detection methods using potato virus ELISA kits, ELISA for the detection of potato virus detection provides a great convenience, but limited to the host phloem and very low concentrations of phloem viruses, such as potato leaf curl virus, ELISA detection method is still not very sensitive, occasionally there will be false positives occur, and the operation process is very tedious and time-consuming. RT-PCR detection technology is its speed, specificity and high sensitivity, including the identification of the potato plant seed virus disease plays an increasingly large role. The present experiments established the six kinds of unit weight of the potato viruses, the multiplex RT-PCR reaction system, be optimized for each reaction system, while the detection sensitivity was measured. Experiment selected six kinds of potato viruses: potato virus X (PVX), potato virus Y (PVY), potato leafroll virus (PLRV), potato M virus (PVM), potato virus A (PVA), potato virus S ( PVS), are common potato virus have a higher risk of introduction. The experiment material from the submitted sample, and the sample out. Experiments to using extracted potato virus RNA kit method RNAiso Plus reagent method, results showed that the extract both high purity and integrity of RNA, RT-PCR experiments to meet the requirements. According NCBI (?) The reported virus genome sequence designed from conserved regions of sequence specific primers of the potato virus X singlet RT-PCR, first using potato virus X RNA amplification, the amplified fragment size A 740 bp fragment of then recovery of potato virus X single re-amplified products were sequenced, proved by RT-PCR amplification of potato virus X target tape completely correct sequence through NCBI BIAST and the viral sequences have been reported compared. Experiment in the case of the content and type of other components of the reaction system constant by changing the amount of upstream and downstream primers optimized triple reaction composition 20 of the six kinds of viruses, all triple combinations of optimization system is derived and the result detected. By diluting the concentration of each viral cDNA, respectively on the six kinds of viruses in the singlet and triplet RT-PCR detection of the minimum detectable concentration was measured, the results show, singlet detection sensitivity is higher than the triple detection singlet reaction The virus minimum detectable concentration 50-100ng/ul, the minimum detectable concentration of the virus in the triple response to 100-200ng/ul. On this basis, the fourfold RT-PCR reaction system further exploratory studies.

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CLC: > Agricultural Sciences > Plant Protection > Pest and Disease Control > Crop pests and diseases and their prevention > Tuber Crops Pests and Diseases > Diseases and insect pests of potatoes ( potatoes)
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