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| 联合环介导等温扩增和横向流动试纸条可视化检测副猪嗜血杆菌 |
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广敏1,靳洪振1,彭康尧1,李华明1,项维1,陈瑞格1,雷连成1,2*,张付贤1*
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| (1.长江大学 动物科学学院, 湖北 荆州 434023;2.吉林大学 动物医学学院, 长春 130062) |
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| 摘要: |
| 为快速鉴定临床上的副猪嗜血杆菌(Haemophilus parasuis，HPS)感染，本研究采用环介导等温扩增(LAMP)技术结合横向流动试纸条(LFD)测定法进行核酸扩增和结果的可视化判定。选择副猪嗜血杆菌inf B基因中保守序列设计5组环介导等温扩增引物，筛选最适引物组；用生物素和6-FAM标记内引物用于LAMP扩增反应，结合LFD建立了副猪嗜血杆菌LAMP-LFD快速检测方法，并测试该方法的特异性、灵敏度、重复性和临床应用效果。结果表明:1)优化后的LAMP在66 ℃下运行30 min，LFD可以在5 min内特异性、直观地检测到副猪嗜血杆菌；2)特异性强，与其他5种细菌无交叉反应；3)敏感度高，其最低检测浓度为1.285×10-12 ng/μL，较普通PCR方法灵敏度显著提高；4)采用该方法对52份临床样品进行检测，副猪嗜血杆菌阳性率为19.2%(10/52)，与LAMP检测结果的符合率为100%。综上，本研究建立的LAMP-LFD方法可特异、灵敏、准确地应用于副猪嗜血杆菌的可视化检测，而且操作简单、仪器设备要求及检测成本低、耗时短，适用于基层养殖场副猪嗜血杆菌感染的临场、即时检测，具有较高的推广价值。 |
| 关键词: 副猪嗜血杆菌 环介导等温扩增技术 横向流动试纸条 可视化 快速检测 |
| DOI：10.11841/j.issn.1007-4333.2023.01.15 |
| 投稿时间：2022-05-24 |
| 基金项目:国家自然科学资金项目(31520103917) |
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| Combined loop-mediated isothermal amplification and transverse flow test paper for visual detection of Haemophilus parasuis |
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GUANG Min1,JIN Hongzhen1,PENG Kangyao1,LI Huaming1,XIANG Wei1,CHEN Ruige1,LEI Liancheng1,2*,ZHANG Fuxian1*
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| (1.College of Animal Science, Yangtze University, Jingzhou 434023, China;2.College of Veterinary Medicine, Jilin University, Changchun 130062, China) |
| Abstract: |
| In order to rapidly identify the clinical Haemophilus parasuis (HPS)infection, loop-mediated isothermal amplification(LAMP)technology was used in this study to amplify nucleic acid and visualize the results by lateral flow test strip(LFD). Five groups of loop-mediated isothermal amplification primers were designed based on the conserved sequence of inf B gene of Haemophilus parasuis, and the optimal primer group was selected. A rapid detection method for Haemophilus parasuis LAMP-LFD was established by using biotin and 6-FAM-labeled internal primers for LAMP amplification reaction combined with LFD. The specificity, sensitivity, repeatability and clinical application effect of this method were tested. The results showed that: 1)The optimized LAMP was operated at 66 ℃ for 30 min, and LFD could specifically and intuitively detect Haemophilus parasuis within 5 min; 2)It has strong specificity and no cross-reaction with other five bacteria; 3)The sensitivity was high, and the lowest detection concentration was 1. 285×10-12 ng/μL, which was significantly higher than that of the conventional PCR method; 4)This method was used to detect 52 clinical samples. The positive rate of Haemophilus parasuis was 19. 2%(10/52), and the coincidence rate with LAMP test results was 100%. In summary, the LAMP-LFD method established in this study can be applied to the visual detection of Haemophilus parasuis specifically, sensitively and accurately. Moreover, the LAMP-LFD method has the advantages of simple operation, low equipment requirements, low detection cost and short time consumption, which is suitable for real-time detection and has high promotional value. |
| Key words: Haemophilus parasuis loop-mediated isothermal amplification lateral flow dipstick visualization rapid detection |
