Quantitative Determination of <italic>Cucumber Mosaic Virus</italic> 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.

Gene copy number is differentially regulated in a multipartite virus

“…Although no direct investigation has addressed this question, some data indicating unequal accumulation of different genome segments can be found, for example, in studies on cucumber mosaic virus [38][39][40][41] , tomato aspermy virus 42,43 and brome mosaic virus 44 -all positive-sense ssRNA viruses unrelated to FBNSV. These earlier studies were either purely descriptive, analysing the physical and/or chemical properties of the virus particle 38,39,42,44 , or focused on technical developments for RNA quantification 40,41,44 . Nevertheless, they suggest that the regulation of segment copy number may be a general feature of multipartite viruses.…”

The quantification of tomato microRNAs response to viral infection by stem-loop real-time RT-PCR

“…Their stem-loop RT primers, forward primers and reverse primers were designed according to criteria mentioned by Chen et al, (2005) and Tang et al (2006). For target mRNAs, the design of the conventional gene-specific forward and reverse primers was based on sequences obtained from clones of AGO1 and ARF8, or from public databases (MYB: AY131230 and AY131231, SCL: DQ087265), and was optimized as per our previous work (Feng et al, 2006). All sequences are summarized in Table 1.…”

Detection of five viruses infecting dormant bulbs by TaqMan-based real-time RT-PCR

“…A quantitative real-time RT-PCR (SYBR Green) has also been used to determine the quantities of different RNA segments of the CMV genome (Feng et al 2006). These real-time RT-PCR assays have been shown to be more sensitive than enzyme-linked immunosorbent assay (ELISA) and conventional PCR (Mumford et al 2000(Mumford et al , 2008Feng et al 2006;Pappu et al 2008). These methods require investigation to verify whether they can detect viruses in dormant bulbs reliably.…”

“…Two different real-time RT-PCR assays for the detection of IYSV were developed; one using TaqMan probe (Mumford et al 2008) and the other using SYBR Green (Pappu et al 2008). A quantitative real-time RT-PCR (SYBR Green) has also been used to determine the quantities of different RNA segments of the CMV genome (Feng et al 2006). These real-time RT-PCR assays have been shown to be more sensitive than enzyme-linked immunosorbent assay (ELISA) and conventional PCR (Mumford et al 2000(Mumford et al , 2008Feng et al 2006;Pappu et al 2008).…”