Effects of Sample Matrix, RNA Extraction Method, Carrier RNA, and Storage Conditions on RT-qPCR Detection of Foot-and-Mouth Disease Virus.
- 1Institute of Veterinary Medicine of Serbia, Janisa Janulisa 14, 11000 Belgrade, Serbia.
- 2Faculty of Veterinary Medicine, University of Thessaly, Trikalon 224, 43131 Karditsa, Greece.
Abstract
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.
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BibTeX
@article{milicevic2026effects,
title = {{Effects of Sample Matrix, RNA Extraction Method, Carrier RNA, and Storage Conditions on RT-qPCR Detection of Foot-and-Mouth Disease Virus.}},
author = {Vesna Milićević and Bojan Milovanović and Zorana Zurovac Sapundžić and Sofija Šolaja and Dimitrije Glišić and Đorđe Aksić and Ljubiša Veljović and George Valiakos},
journal = {Veterinary sciences},
year = {2026},
month = oct,
doi = {10.3390/vetsci13101096},
url = {https://europepmc.org/article/MED/42944190},
note = {CC BY},
}
RIS
TY - JOUR
TI - Effects of Sample Matrix, RNA Extraction Method, Carrier RNA, and Storage Conditions on RT-qPCR Detection of Foot-and-Mouth Disease Virus.
AU - Vesna Milićević
AU - Bojan Milovanović
AU - Zorana Zurovac Sapundžić
AU - Sofija Šolaja
AU - Dimitrije Glišić
AU - Đorđe Aksić
AU - Ljubiša Veljović
AU - George Valiakos
PY - 2026
DA - 2026/10/15
JO - Veterinary sciences
DO - 10.3390/vetsci13101096
UR - https://europepmc.org/article/MED/42944190
AB - 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.
SN - 2306-7381
ER -