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

Abstract

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 (RT-RPA) method for rapid detection of MHV in laboratory mice.MethodsSpecific 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.ResultsAmplification using the newly developed RT-RPA assay was completed within 20 min at 37°C, while that using the RT-qPCR method required nearly 60 min. The RT-RPA method exhibited an obvious time-saving advantage. Both RT-RPA and RT-PCR methods had the same limit of detection, which was 4.45 × 101 copies/μL. The specificity was indicated by a lack 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” (κ) analysis results exhibited a very good agreement between two methods with the value of κ ≥ 0.750(since κ = 0.939) and p < 0.0005 (since p = 0.000).ConclusionThe RT-RPA assay offers an alternative tool for simple, rapid, and reliable detection of MHV in laboratory mice and has significant potential for application in laboratories.

Development of Enzymatic Recombinase Amplification Assays for the Rapid Visual Detection of HPV16/18

“…However, the risk of forming dimers and hairpins also increases; thus, we set the primer length at 30–35 bp. Besides, 5–9 mismatches in the primers can be tolerated by ERA assays, but the dimers and hairpins should be avoided [ 29 ]. To evaluate the performance of each candidate primer set, not only the quantity of ERA products, but also the start time of the amplification was considered.…”

“…Agarose gel electrophoresis can be applied to analyze the length and amounts of amplicons, but it takes a relatively long reaction time and requires other equipment [ 26 ]. In contrast, the fluorescent probe can monitor and analyze the results in real-time [ 27 - 29 ], while LFD is extremely portable and easy to operate and can read the results within 5min by the naked eye [ 30 - 32 ]. The probes and enzymes applied for the above methods are quite different.…”

“…The exo-probe, which is used for real-time ERA assay, obtains a tetrahydrofuran (THF) between the fluorophore and quencher. The THF can be identified and split by exonuclease III and the separation of fluorophore and quencher will generate fluorescence signals [ 28 , 29 ]. For ERA-LFD assay, endonuclease IV can identify the THF of the nfo-probe and convert the probe to a forward primer carrying a fluorophore at the 5¢ end [ 30 ].…”

Establishment and Application of CRISPR–Cas12a-Based Recombinase Polymerase Amplification and a Lateral Flow Dipstick and Fluorescence for the Detection and Distinction of Deformed Wing Virus Types A and B

“…The entire reaction can be accomplished at a constant temperature (optimally at 37°C–42°C) within a short time (20 min), making RPA a promising on-site detection method [ 15 ]. RPA has been employed for detecting pathogens such as bacteria, fungi, parasites, and viruses [ 16 , 17 , 18 ].…”