Development and evaluation of single-plex TaqMan real-time quantitative PCR assays for the detection of six tick-borne pathogenic viruses in northeastern China.

BACKGROUND

Northeastern China is a recognized hotspot for tick-borne viral diseases. However, there remains a lack of methods capable of simultaneously detecting these emerging and re-emerging tick-borne viruses.

METHODS

Primer and probe sets were designed using Beacon Designer 8.0, targeting conserved regions of viral genomes from Alongshan virus (ALSV), Tick-borne encephalitis virus (TBEV), Severe fever with thrombocytopenia syndrome virus (SFTSV), Beiji nairovirus (BJNV), Yezo virus (YEZV), and Songling virus (SGLV). Recombinant plasmids were constructed to assess assay sensitivity, while virus-positive cDNA from tick samples was used to verify specificity. The performance of these assays was further validated using field-collected tick samples, with results compared against established reference methods serving as gold standards.

RESULTS

Specific primer and probe sets were designed with amplicon lengths ranging from 83 to 199 bp. Optimization of the reaction components yielded final primer volumes of 0.2-0.5 μL and probe volumes of 0.4-1.0 μL (from 10 μM working stocks). Sensitivity analysis demonstrated a limit of detection (LOD) as low as 10 copies/μL for all six viruses, while specificity testing confirmed no cross-reactivity among the targets. In validation trials, the established TaqMan RT-qPCR assays showed complete concordance with reference methods for ALSV (5/15) and TBEV (4/14). Notably, the TaqMan assays identified additional positive samples for SFTSV (n=1), BJNV (n=3), and YEZV (n=1) that were missed by the reference assays, particularly in samples with low viral loads. Conversely, two samples that tested positive for SGLV using SYBR Green RT-qPCR yielded negative results with the TaqMan RT-qPCR assay.

CONCLUSION

We have successfully developed a suite of highly sensitive and specific single-plex TaqMan RT-qPCR assays for the rapid detection of six key tick-borne viruses in northeastern China. These assays facilitate efficient viral surveillance in tick populations and provide a diagnostic tool with significant potential for clinical applications.