Real-time PCR method for the detection of figwort mosaic virus (FMV) to complement the FMV 34S promoter-specific PCR assay used for screening of genetically modified plants.

摘要

In this study a new real-time PCR assay for the detection of figwort mosaic virus (FMV) DNA is described. This assay targets a 113-bp-long sequence of the FMV open reading frame Vll, a non-conserved coding region among the caulimoviruses. Detection of FMV DNA is useful to complement screening for the FMV 34S promoter (P-FMV), a genetic element present in several genetically modified (GM) plants. The specificity of the assay was assessed against closely related plant viruses, plant species of agronomic importance or susceptible to infection by FMV, and various GM plants containing the P-FMV sequence. No cross-reactivity of the assay against the tested nontarget organisms was observed. The limit of detection of the FMV real-time PCR assay was determined at approximately 5 copies per reaction. The robustness of the method was tested using different real-time PCR instruments and PCR master mixes with no negative effects on the PCR efficiency and linearity being observed. When amplifying the FMV target DNA at a concentration level close to the detection limit, no negative influence on the PCR performance was observed in the presence of background genomic DNA from various plant species. The precision data of the in-house validation were in line with generally accepted performance requirements. In summary, the method appears fit for the purpose of a specific identification test for the presence of genomic FMV DNA, while preventing false-positive results during P-FMV screening.