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.
Download Full PDF Version (Non-Commercial Use)