Li Zhan-Hong, Zhang Zhen-Xing, Zhu Pei, Li Zhuo-Ran, Liu Peng, Yang Qi, Yang Qiu-Yan, Yang Zhen, Song Jian-Ling
Yunnan Tropical and Subtropical Animal Virus Diseases Laboratory, Yunnan Animal Science and Veterinary Institute, Kunming, 650224, China.
Key Laboratory of Transboundary Animal Diseases Prevention and Control (Co-construction by Ministry and Province), Kunming, 650224, China.
BMC Vet Res. 2025 Aug 29;21(1):529. doi: 10.1186/s12917-025-04970-y.
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.
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 min at 64 °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 < 0.01), and the nucleic acid of EV-G could be tested from the fecal samples of porcine at different infection stages.
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.
肠道病毒G（EV-G）在全球范围内的猪中广泛流行，许多国家都报道了EV-G与其他致腹泻病原体的共同感染，对养猪业构成威胁。目前有一些可用于检测EV-G的方法；然而，用于检测EV-G的逆转录环介导等温扩增（RT-LAMP）方法尚未开发出来。本研究的目的是建立一种用于检测EV-G的高灵敏度且可视化的RT-LAMP检测方法。
开发并评估了一种用于可视化检测猪肠道病毒G（EV-G）的比色逆转录环介导等温扩增（RT-LAMP）检测方法。该检测方法可在64℃下45分钟内完成，使用比色LAMP预混液可肉眼观察结果。RT-LAMP检测方法的检测限（LOD）为46.8拷贝的EV-G RNA，与先前描述的实时逆转录聚合酶链反应（qRT-PCR）方法相当，比传统RT-PCR灵敏度高100倍。该检测方法特异性扩增EV-G的RNA，与其他猪病原体无交叉扩增。在临床评估中，所建立的RT-LAMP检测方法与先前报道的qRT-PCR的Kappa系数为0.862（P<0.01），并且可以从不同感染阶段的猪粪便样本中检测出EV-G核酸。
在本研究中，我们成功建立了一种用于检测EV-G的可视化、灵敏、特异、快速且便捷的RT-LAMP方法，并成功应用于临床样本中EV-G的检测。
