Effects of Sample Matrix, RNA Extraction Method, Carrier RNA, and Storage Conditions on RT-qPCR Detection of Foot-and-Mouth Disease Virus

Reliable molecular detection of foot-and-mouth disease virus (FMDV) depends on RT-qPCR performance and pre-analytical factors, including sample matrix, RNA extraction method, carrier RNA supplementation, and storage conditions. This study compared silica membrane- and magnetic bead-based RNA extraction workflows for detection of FMDV serotypes O, A, and SAT2 in serum, PBS-based swab suspension, and tissue homogenates at three virus dilutions, with and without carrier RNA. FMDV RNA stability in serum stored at room temperature or −20 °C for up to 60 days and following five freeze–thaw cycles was also evaluated using serotype O. Extraction performance was matrix-dependent. In serum, silica-based workflow yielded significantly lower Ct values than magnetic bead-based extraction. In tissue homogenates, magnetic bead extraction yielded significantly lower Ct values with carrier RNA, whereas carrier RNA impaired silica-based extraction. No significant differences were observed for PBS-based swab suspension samples. FMDV RNA detectability remained stable at −20 °C for 60 days, whereas room-temperature storage caused progressive signal loss, which was reduced by carrier RNA. Five freeze–thaw cycles did not progressively affect RNA detectability. These findings demonstrate that the effects of extraction workflow and carrier RNA supplementation on FMDV RT-qPCR detectability differed according to the sample matrix and experimental conditions, highlighting the importance of matrix-specific optimization of pre-analytical procedures.