Rapid Detection of Potato Y potyvirus in Potato Farms of Kermanshah using RT-PCR Amplification of the P1- Protease Gene and Its Cloning

Abstract

In this study, the RT-PCR method was used to detect the Y virus in potato tubers and leaves. Samples suspicious of virus infection with symptoms of virus infection were gathered from farms in Kermanshah and placed in plastic bags and kept at -80 degrees C temperature in order to maintain the RNA of the virus until extraction. The extraction and purification of RNA were carried out using Tri-Reagent kit. One of the virus genes is the P1 protease gene which codes a proteinase enzyme. This enzyme plays a role in breaking the initial polyprotein. For amplification of this gene three primer, including primer-1, primer-2 and primer-3, were designed and used. Using primer 1 and reverse transcriptase enzyme, cDNA was synthesized and then PCR was performed using the primers 1, 2 and 3. The PCR products were examined by agarose gel electrophoresis (1%). Consequently, two pieces of DNA (400 and 800 bp) were yielded which were identical to the genome DNA sequencing. Thus, the proposed technique is a convenient method for quick and accurate detection of viruses and, therefore, the application of this method for detecting Potato Y virus in potato farms is recommended.