Colorimetric reverse transcription loop-mediated isothermal amplification assay for visual, sensitive, and specific detection of enterovirus G.

Abstract

BACKGROUND: Enterovirus G (EV-G) is widely prevalent in pigs worldwide, and co-infections of EV-G and other diarrhea-causing pathogens have been reported in many countries, threatening the pig farming industry. There are some methods available for EV-G detection; however, the RT-LAMP method for detecting EV-G has not yet been developed. The aim of thisstudy was to establish a highly sensitive and visual RT-LAMP assay for the detection of EV-G. RESULTS: A colorimetric reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay was developed and evaluated for the visual detection of porcine enterovirus G (EV-G). The assay can be completed in 45&#xa0;min at 64&#xa0;&#xb0;C, and the results can be observed by the unaided eye using the colorimetric LAMP master mix. The limit of detection (LOD) of the RT-LAMP assay was 46.8 copies of EV-G RNA, which is comparable to that of the previously described real-time RT-PCR (qRT-PCR) method and is 100-fold more sensitive than that of conventional RT-PCR. The assay specifically amplified the RNA of EV-Gs, and there was no cross-amplification with other porcine pathogens. In the clinical evaluation, the Kappa coefficient of the established RT-LAMP assay and the previously reported qRT-PCR was was 0.862 (P&#x2009;<&#x2009;0.01), and the nucleic acid of EV-G could be tested from the fecal samples of porcine at different infection stages. CONCLUSIONS: In this study, we successfully established a visual, sensitive, specific, rapid, and convenient RT-LAMP method for the detection of EV-G, which was successfully applied in the detection of EV-Gs in clinical samples.