Optimization of Multiplex PCR for detection of important potato viruses
2010
Khelghati bana, Fatemeh
Healthy potato seed tubers are most important in terms of potato production.Viruses and virus like pathogens are of the most important ones among potato pathogens, that could reduce potato yield substantially. The major viruses affecting potato production are potato virus Y (PVY), potato virus X (PVX), potato virus S (PVS), potato virus M (PVM), potato virus A (PVA) and potato leaf roll virus (PLRV). According to the aims of Seed and Plant Certification and Registertion Institute in improving national seed and plant quality , optimaztion of sensitive, specific and low cost economic methods to detect pathogens in different seed and plant verieties including potato seed tubers are developing. Applying these methods is specially important in regards to the basic and prebasic seeds due to the probability of the presence of low virus infection level. Among the nucleic acid _based detection methods , multiplex _PCR assay appeals to scientists because of sensitivity , specificity and capacity to detect more than one target simultaniousely. In this project potato tubers were first incubated under favorit conditions to sprout and then, the sprouts were homogenized using exteraction buffer to prepare test samples. Samples have been tested using DAS-ELISA method for potato viruses.The infected tubers were cultivated and the total RNA were extracted from infected plants. Finally, the multiplex RT- PCR was optimized using specific primers for PLRV, PVS, PVY, PVX coat protein genes as well as for the 18 s r RNA gene as an internal control
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