• Development of a real-time multiplex RT-PCR to differentiate between Deformed Wing Virus (DWV) subtypes.

FERNANDA GONZALEZ; MORAGAS M; FERRUFINO CECILIA; RODRIGUEZ GRACIELA; MARIA JOSE DUS SANTOS

Congreso; 48th International Apicultural Congress Apimondia 2023; 2023

Deformed wing virus (DWV) is a highly prevalent pathogen that can be transmitted by the Varroa destructor mite. DWV can circulate in different variants (DWV-A, DWV-B, DWV-C). The presence of the virus has been reported in Argentina, but the DWV-A and DWV-B variants have recently been characterized by sequencing. Therefore, it is necessary to have tools that allow the rapid and reliable identification of the circulating variant in a certain region under study. The objective of this work was to design a multiplex RT-qPCR to differentiate the DWV-A from DWV-B/C variants. For this, a unique set of primers (forward/reverse) was designed that amplifies a 180 bpfragment within the helicase gene and that is conserved among all DWV variants described. Variant differentiation was further assessed by using two labeled probes (22 bp), one specific for the DWV-A variant and the other for DWV-B/C. Positive controls used for DWV-A included a positive bee sample and a plasmid containing the target sequence, while the DWV-B/C positive control was a synthetic gene (ultramer). Appropriate assays were performed to determine the dynamic range, detection limit, and precision of the technique, according to MIQE guidelines for real-time PCR experiments. Efficiency calculated with a standard curve was prepared using known concentrations of each control (2.3 x 107 to 101 gene copies/µl) was………..To assess the specificity of the technique, positive and negative samples for DWV previously identified in our laboratory were used. All qPCR reactions were performed in a final volume of 25 µl. For this multiplex design, a concentration of 800 nM was used for each primer and 200 nM for each specific probe. The qPCR thermal profile consisted of 40 cycles of 15 seconds at 95 °C, 30 seconds at 55 °C, and 1 minute at 60 °C. A standard curve was prepared using known concentrations of each control (2.3 x 107 to 101 gene copies/µl) to determine the detection limit and define the dynamic range. The RT-qPCR developed is a sensitive and fast technique that allows the identification of the DWV-A and DWV-B/C variants. In our region, the DWV-A variant predominates and the DWV-B variant was recently identified; thus, the RT-qPCR would allow monitoring colonies and evaluating DWV variants in different ecoregions of the country.