An Improved Method of Reverse Transcription and Polymerase Chain Reaction (RT-PCR) to Efficiently Detect Potyvirus, Sweet potato feathery mottle virus (SPFMV) RNA in Sweet Potato

Abstract

To improve the detection method for SPFMV in sweet potato leaves, we compared three RNA extraction methods that utilized either guanidine thiocyanate-phenol (ISOGEN™), SDS-phenol or a CF-11 cellulose column. The first method gave the highest yield of total RNA (39.0 μg from 0.1 g leaf sample) with the largest potential to synthesize cDNA by reverse transcription with the shortest extraction time. We then compared three combinations of polymerases for RT-PCR. AMV reverse transcriptase followed by PCR with rTaq DNA polymerase gave the most sensitive detection, in which the amplified DNA band was detected in a sample containing 0.2 pg of total RNA.