Rapid detection of Potato Y potyvirus in potato farms of Kermanshah using RT-PCR amplification of the P1-protease gene and its cloning.
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.
Mirmomeni
M H
MH
Cell and Molecular Unit, Department of Biology, Razi University, Kermanshah, Iran.
Sharifi
A
A
Sisakhtnezhad
S
S
D001483:Base Sequence; D003001:Cloning, Molecular; D017931:DNA Primers; D007492:Iran; D017800:Potyvirus / Q000201:enzymology / Q000235:genetics / Q000302:isolation & purification*; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D012697:Serine Endopeptidases / Q000235:genetics*; D011198:Solanum tuberosum / Q000821:virology*; D014764:Viral Proteins / Q000235:genetics*
D017931:DNA Primers; C409826:P1 protein, potyvirus; D014764:Viral Proteins; D012697:Serine Endopeptidases