Huang Wenlong, Chen Meiyi, Wang Yiwen, Li Li, Niu Tianmin, Guo Xin, Wang Jiaxuan, He Kaifeng, Wei Zhengkai, Liu Quan
College of Animal Science and Technology, Foshan University, Foshan 528225, Guangdong Province, PR China.
College of Veterinary Medicine, Southwest University, Chongqing 400715, PR China.
J Gen Virol. 2025 May;106(5). doi: 10.1099/jgv.0.002094.
Alongshan virus (ALSV) is a recently discovered tick-borne zoonotic virus. Currently, there is no rapid and accurate clinical method for ALSV detection. This study aimed to develop a loop-mediated isothermal amplification (LAMP) assay for precise ALSV infection detection. Specific primers were designed based on the S1 segment of the ALSV NE-TH4 strain's genome (GenBank accession no. ON408067.1). The reaction time, temperature and concentration of the neutral red staining solution in the LAMP assay were optimized. Thorough evaluations of specificity, sensitivity and repeatability led to the development of a visually interpretable LAMP assay. The optimal amplification time was 50 min. The minimum detection limit for cDNA was as low as 0.005 pg μl, and sensitivity for standards was 1.68×10 copies per μl, surpassing that of PCR and real-time PCR. No cross-reactivity was observed with Jingmen tick virus, Bole tick virus 4 and Beiji nairovirus. These results indicate that the LAMP assay is more sensitive and accurate than PCR and real-time PCR. The developed LAMP assay allows for on-site detection, reduces testing costs and provides rapid and accurate results. Thus, it lays a solid foundation for the prevention and control of emerging tick-borne ALSV.
阿龙山病毒（ALSV）是一种最近发现的蜱传人畜共患病毒。目前，尚无用于检测ALSV的快速准确的临床方法。本研究旨在开发一种环介导等温扩增（LAMP）检测法，用于精确检测ALSV感染。基于ALSV NE-TH4株基因组的S1片段（GenBank登录号：ON408067.1）设计了特异性引物。对LAMP检测中的反应时间、温度和中性红染色溶液浓度进行了优化。通过对特异性、敏感性和重复性的全面评估，开发出了一种可目视判读的LAMP检测法。最佳扩增时间为50分钟。cDNA的最低检测限低至0.005 pg/μl，标准品的敏感性为每微升1.68×10个拷贝，超过了PCR和实时PCR。未观察到与荆门蜱病毒、博乐蜱病毒4和贝吉内罗病毒的交叉反应。这些结果表明，LAMP检测法比PCR和实时PCR更灵敏、准确。所开发的LAMP检测法可进行现场检测，降低检测成本，并提供快速准确的结果。因此，它为预防和控制新出现的蜱传ALSV奠定了坚实基础。
