Dataset Information

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.

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 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).

Conclusion

The 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.

SUBMITTER: Wang X

PROVIDER: S-EPMC9755729 | biostudies-literature | 2022

REPOSITORIES: biostudies-literature

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Publications

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

Wang Xiao X Sui Xin X Ma Yueyu Y Li Ming M Zhang Xu X Fei Dongliang D Ma Mingxiao M

Frontiers in microbiology 20221202

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.<h4>Methods</h4>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 in ...[more]

PMID: 36532497

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Project description:Hepatitis E virus (HEV) is a globally prevalent zoonotic pathogen that is primarily spread through the fecal-oral route, such as by consuming undercooked or contaminated pork. HEV infection leads to an estimated 3.3 million symptomatic cases of viral hepatitis and 70,000 deaths in human populations each year. Therefore, a rapid and accurate method for detecting HEV in serum or stool samples is essential. In this study, we aimed to develop and evaluate two methods for the rapid and convenient detection of HEV RNA: reverse transcription recombinase-aided amplification with lateral flow dipstick (RT-RAA-LFD) and quantitative real-time reverse transcription recombinase-aided amplification (qRT-RAA). We optimized the reaction conditions and assessed their sensitivity and specificity. The RT-RAA-LFD assay completed its reaction at 39°C within 15 minutes, achieving a 95% limit of detection (LOD) of 247 copies/μL. The qRT-RAA assay, completed at 42°C within 20 minutes, had a 95% LOD of 25 copies/μL. Both assays demonstrated no cross-reactivity with other porcine pathogens and exhibited strong specificity. In testing 245 porcine bile and fecal samples, the RT-RAA-LFD assay showed a kappa value of 0.943 (P < 0.001) with a 97.14% (238/245) coincidence rate compared with quantitative reverse transcription PCR. Similarly, the qRT-RAA assay achieved a kappa value of 0.976 (P < 0.001) with a 98.78% (242/245) coincidence rate. In conclusion, these two RT-RAA assays show promising potential as effective diagnostic tools for broad and efficient screening of swine HEV in veterinary clinics.ImportanceHepatitis E virus (HEV) is a globally widespread zoonotic pathogen that poses a significant public health risk. Swine serve as the primary natural host for zoonotic HEV. This study introduces a rapid and precise method for detecting swine HEV RNA, showcasing its potential as an effective diagnostic tool for comprehensive and efficient screening of swine HEV in veterinary clinics.

Project description:BackgroundBovine rotavirus A (BRVA) is considered to be the most common pathogen of severe diarrhea in cattle worldwide, which could lead to the death of newborn calves and cause the significant economic losses to the cattle industry. As a novel isothermal nucleic acid amplification technique, recombinase polymerase amplification (RPA) has been applied widely for the rapid detection of different important pathogens in human and animals.ResultsAn RT-RPA assay based on the real time fluorescence monitoring (real-time RT-RPA) and an RT-RPA assay combined with a lateral flow strip (LFS RT-RPA) were successfully developed by targeting the VP6 gene of BRVA. The RT-RPA assays allowed the exponential amplification of the target fragment in 20 min. After incubation of the LFS RT-RPA on a metal bath at 40 °C, the results were displayed on the lateral flow strip within 5 min, while real-time RT-RPA allowed the real-time observation of the results in Genie III at 42 °C. Both of the two assays showed high specificity for BRVA without any cross-reaction with the other tested pathogens causing diarrhea in cattle. With the standard RNA of BRVA serving as a template, the limit of detection for real-time RT-RPA and LFS RT-RPA were 1.4 × 102 copies per reaction and 1.4 × 101 copies per reaction, respectively. In the 134 fecal samples collected from cattle with diarrhea, the BRVA positive rate were 45.52% (61/134) and 46.27% (62/134) in real-time RT-RPA and LFS RT-RPA, respectively. Compared to a previously published real-time PCR, the real-time RT-RPA and LFS RT-RPA showed a diagnostic specificity of 100%, diagnostic sensitivity of 98.39% and 100%, and a kappa coefficient of 0.985 and 1.0, respectively.ConclusionsIn this study, BRVA was successfully detected in cattle fecal samples by the developed real-time RT-RPA and LFS RT-RPA assays. The developed RT-RPA assays had great potential for the rapid detection of BRVA in under-equipped diagnostic laboratory and the point-of-need diagnosis at quarantine stations and farms, which is of great importance to control BRVA-associated diarrhea in cattle herds.

Dataset Information

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

Methods

Results

Conclusion

Publications

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

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