Objective An RT-qPCR method to accurately detect gyrovirus 3 (GyV3) was established.
Methods A pair of specific primers and an FAM-labeled fluorophobe were designed according to the conserved region of GyV3 VP2 in Genbank database. Reaction conditions of a rapid TaqMan RT-qPCR assay were optimized, and specificity, sensitivity, repeatability, and simulation tests conducted to verify the applicability of the methodology.
Results The assay exhibited a high sensitivity with a minimum detection limit of 1.585 copies·μL−1, a specificity free of cross-reactivity with other prevalent avian pathogens, and a reproducibility with less than 1% coefficients of variation on intra- and inter-batch determinations. On a simulated sample with an added recombinant plasmid to the chicken liver tissue DNA, the assay positively identified GyV3 with same result as did the SYBR Green Ⅰ PCR.
Conclusion The newly developed TaqMan RT-qPCR assay showed high sensitivity, specificity, repeatability, and rapid turn-around time in detecting GyV3. It was considered appropriate for clinical diagnosis and epidemiological investigation on the virus.
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