Real-time reverse transcription recombinase polymerase amplification for rapid detection of murine hepatitis virus

Murine hepatitis virus (MHV) is a highly infectious murine coronavirus that has a high potential for causing harm to host animals. This study aimed to develop a real-time reverse transcription recombinase polymerase amplification (RTRPA) method for rapid detection of MHV in laboratory mice. Methods: Specific primers and probes for RT-RPA assay were designed targeting the conserved region in the M gene of the MHV reference strain (accession no. FJ6647223) according to the TwistDx manual instructions. The specificity, sensitivity, and reproducibility of the RT-RPA method were evaluated and compared with those of the standard RT-qPCR method. The clinical applicability of this assay was evaluated using 68 field samples. Results: Amplification using the newly developed RT-RPA assay was completed within 20min at 37 degrees C, white that using the RT-qPCR method required nearly 60min. The RT-RPA method exhibited an obvious timesaving advantage. Both RT-RPA and RT-PCR methods had the same limit of detection, which was 4.45 x10(1) copies/mu L. The specificity was indicated by a tack of cross-reaction with MHV, pneumonia virus of mice, Sendai virus, hantavirus, minute virus of mice, and reovirus type III. The MHV detection rate of RT-RPA assays was 13.63% (9/66) and RT-qPCR assays was 15.15% (10/66). Cohen's "kappa" (kappa) analysis results exhibited a very good agreement between two methods with the value of kappa >= 0.750(since kappa=0.939) and p <0.0005 (since p =0.000). Conclusion: The RT-RPA assay offers an alternative toot for simple, rapid, and reliable detection of MHV in laboratory mice and has significant potential for application in laboratories.