中国兽医科学2025，Vol.55Issue(4)：477-483,7.DOI:10.16656/j.issn.1673-4696.2025.0041
睡眠嗜组织菌可视化LAMP现场检测方法的建立及初步应用
Establishment and preliminary application of a visual loop-mediated isothermal amplification assay for field detection of Histophilus somni
摘要
Abstract
To establish a visual LAMP assay for the detection of Histophilus somni(Hi.somni),a pair of inner primers and a pair of outer primers of LAMP was designed based on the specific sequence of the 16S rRNA gene of Hi.somni,and the visual LAMP detection assay was established by the optimizations of reaction system and amplification procedure,the verifications of specificity,sensitivity and the coincidence rates to conventional PCR assay.The optimization results of reaction procedure showed that the optimal reaction procedure of the established LAMP assay was holding at 58 ℃ for 70 min.The specific test results showed that the assay could detect Hi.somni specifically,and there was no cross-reaction with 14 common bacteria of cattle,sheep and goats,such as Mannheimia haemolytica,Trueperella pyogenes and Pasteurella multocida,indicating the stronger specificity.The sensitivity test results indicated that the minimum detection concentration of the recombinant plasmid pMD19T-His was 4.7 copies/μL,showing the higher sensitivity.The clinical sample test results showed that the positive rates of Hi.somni were 11.49％(10/87)by LAMP assay and 9.20％(8/87)by conventional PCR assay,re-spectively,and that the total coincidence rate,positive coincidence rate and negative coincidence rate between the LAMP assay and the conventional PCR assay were 97.70％(85/87),100％(8/8)and 97.47％(77/79),respectively.The results indicated that the established LAMP assay provides an effective technical support for fast visual field detection of Hi.somni infections in cattle,goats and sheep.关键词
睡眠嗜组织菌/环介等温扩增/现场检测/牛/山羊/绵羊Key words
Histophilus somni/LAMP/field detection/cattle/goat/sheep分类
农业科技引用本文复制引用
李富祥,高华峰,赵文华,宋建领..睡眠嗜组织菌可视化LAMP现场检测方法的建立及初步应用[J].中国兽医科学,2025,55(4):477-483,7.基金项目
云南省奶牛产业技术体系项目(2024KJTX-17) （2024KJTX-17）
云南省重大科技专项计划项目(202102AE090039,202302AE090010) （202102AE090039,202302AE090010）
