Development of simplex and multiplex RT-qPCR assays for the detection of three cryptic viruses of black-grass (Alopecurus myosuroides).

Simplex and multiplex RT-qPCR assays were developed for Alopecurus myosuroides partitivirus 1 (AMPV1), Alopecurus myosuroides partitivirus 2 (AMPV2) and Alopecurus myosuroides varicosavirus 1 (AMVV1), and compared to the existing conventional PCR assays. All assays had a high specificity and their sensitivity was increased compared to the conventional RT-PCR assays. As viral quantification is an important element in comparative experiments, the effect of high- and low-temperature drying treatments, prior to RNA extraction and analysis, was studied and optimised. AMVV1 detection was reduced by both drying treatments, but particularly by the high-temperature. AMPV1 and AMPV2 detection on the other hand was not impeded by the drying treatments, and enables standardisation of plant tissue prior to extraction, in particular for quantitative analysis.