Submitted to: Phytopathology
Publication Type: Abstract Only
Publication Acceptance Date: 3/11/2004
Publication Date: 6/1/2004
Citation: Schneider, W.L., Stone, A.L., Sherman, D.J., Damsteegt, V.D. 2004. Detection and quantification of plum pox potyvirus double-stranded RNA with real-time PCR. Phytopathology. 94:S93.
Technical Abstract: Double-stranded RNA (dsRNA) is commonly associated with RNA virus replication. In order to study the replication of plum pox potyvirus (PPV) a two step reverse transcription-real time PCR assay was developed. The reverse transcription step uses plus and minus strand specific primers to synthesize cDNA specific to the plus and minus strands. The strand specific cDNAs are used in real-time PCR reactions. The relative quantities of plus and minus strands are determined by comparing the cycle threshold (CT) value from the plus strand reaction to the minus strand reaction. Plus and minus strand PPV transcripts were used to test and quantify the system. The plus and minus strand assay CT values were equal when the transcripts were equimolar. When one or the other transcript was in excess the CT values for the plus and minus assays differed significantly, with the CT value for the high concentration transcript lower. The assay was also tested on purified PPV dsRNA and total RNA from infected plants. As in the transcript assay, the minus and plus strand CT values for the purified dsRNA were equal. However, in the infected plant assay the CT value for the plus strand assay was 9-10 cycles lower than the minus strand assay CT value, suggesting that the ratio of plus to minus strands was roughly 1000 to one.