Development and Application of an RPA‐Based Rapid Point‐of‐Care Testing (POCT) Method for the Detection of Feline Panleukopenia Virus

Abstract

Feline panleukopenia (FP) is a highly prevalent and consequential disease that poses a substantial threat to both adult and juvenile cats across all geographical regions. The causative agent responsible for this disease is the feline panleukopenia virus (FPV). Therefore, it is imperative to develop a facile, efficient, and accurate detection method for FPV. Hence, a recombinase polymerase amplification–lateral flow dipstick assay (RPA–LFDA) method was specifically designed for the detection of FPV. The amplification process was optimized. This investigation focused on evaluating the expansion temperature detection system and revealed an optimal reaction temperature of 39°C. Then, primer combination screening involving nine groups identified F3R2 as the most effective primer set, while dilution ratio experiments determined that a 10‐fold dilution yielded the best amplification products. Our findings demonstrated that the RPA‐LFDA assay had an analytical sensitivity that was capable of detecting as low as 10 target copies per reaction. Furthermore, cross‐reactivity tests demonstrated no interference between feline herpesvirus‐1 (FHV‐1) and feline calicivirus (FCV). To validate our newly developed method against existing techniques in clinical samples from three common sources on the market, we observed superior sensitivity and specificity compared to those of the colloidal gold method (CGM), with a higher positive detection rate using our nucleic acid detection system than CGM. Compared to qPCR as a reference standard, RPA‐LFDA detected 39 out of 44 positive samples (including one false positive), whereas CGM detected 26 out of 44 positive samples. Based on the RPA‐LFDA, the sensitivity was calculated to be 100%, the specificity was 83.33%, the mistake diagnostic rate was 16.67%, the omission diagnostic rate was 0%, and the overall accuracy reached 97.73%. Moreover, the positive coincidence rate was 97.44%, while the negative coincidence rate reached 100%. The agreement κ value was 0.8962. In conclusion, this approach exhibits greater sensitivity than CGM and offers greater convenience and cost‐effectiveness than the qPCR methodology, making it a viable option for the clinical detection of FPV.