Quantitative determination of cucumber mosaic virus genome RNAs in virions by real-time reverse transcription-polymerase chain reaction

Abstract

A real-time RT-PCR procedure using the green fluorescent dye SYBR Green I was developed for determining the absolute and relative copies of cucumber mosaic virus (CMV) genomic RNAs contained in purified virions. Primers specific to each CMV ORF were designed and selected. Sequences were then amplified with length varying from 61 to 153 bp. Using dilution series of CMV genome RNAs prepared by in vitro transcription as the standard samples, a good linear correlation was observed between their threshold cycle (Ct) values and the logarithms of the initial template amounts. The copies of genomic RNA 1, RNA 2, RNA 3 and the subgenomic RNA 4 in CMV virions were quantified by this method, and the ratios were about 1.00:1.17:3.58:5.81. These results were confirmed by Lab-on-a-chip and northern blot hybridization assays. Our work is the first report concerning the relative amounts of different RNA fragments in CMV virions as a virus with tripartite genome.