Rapid and sensitive SYBR Green-based RT-qPCR assays for the detection and quantification of mink coronavirus

Mink coronavirus (MCoV) infection poses a serious threat to animal health, resulting in substantial economic losses in mink farming and impeding the sustainable development of the fur industry. Thus, developing both sensitive and specific molecular diagnostics is imperative for effective MCoV surveillance and disease control. In the present study, specific oligonucleotide primers were designed to target the 1b and N genes of MCoV, and two SYBR Green-based RT-qPCR assays - designated as MCoV-1b and MCoV-N, respectively - were established. The assays exhibited extremely high sensitivity, with lower limits of detection of 1.79 × 10 ¹ copies/μL for MCoV-1b and 1.29 × 10 ¹ copies/μL for MCoV-N. No cross-reactivity was observed with other common viruses and melting curve analyses revealed a single sharp peak for each assay, confirming specific amplification of MCoV targets. Reproducibility across three independent experimental runs exhibited low standard deviations and coefficients of variation (CV) of cycle threshold (Ct) values across broad dynamic ranges, indicating stable and consistent performance (CV < 5%). When applied to faecal clinical samples (n = 236), the assays yielded overall positivity rates of 77.97% for the 1b target and 77.54% for the N targets, which are consistent with that from high-throughput sequencing, validating their utility. Collectively, the two RT-qPCR assays developed in this study offer a reliable technical platform for rapid screening, accurate quantification, and epidemiological monitoring of MCoV. The reported molecular diagnostics hold significant practical value in mitigating the risks of MCoV transmission, supporting the sustainable growth of the global mink farming industry and diminishing potential global public health threats.