首页 > 过刊浏览>2023年第42卷第2期 >32-37. DOI:10.13300/j.cnki.hnlkxb.2023.02.005
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牛溶血性曼氏杆菌可视化LAMP检测方法的建立
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作者:
作者单位:

1.华中农业大学动物医学院/农业微生物资源发掘与利用全国重点实验室,武汉 430070;2.湖北省兽医流行病学国际科技合作基地,武汉 430070;3.湖北洪山实验室, 武汉 430070

作者简介:

刘宁宁，E-mail：673005224@qq.com

通讯作者:

陈颖钰，E-mail：chenyingyu@mail.hzau.edu.cn

中图分类号:

S852.61

基金项目:

湖北省重点研发计划项目（2020BBA055）；宁夏回族自治区重点研发计划项目（2021BEF02028）；国家现代农业（肉牛/牦牛）产业技术体系专项（CARS-37）


Establishment of visual LAMP method for detecting Mannheimia haemolytica in cattle
Author:
Affiliation:

1.College of Veterinary Medicine，Huazhong Agricultural University/ State Key Laboratory of Agricultural Microbiology， Wuhan 430070， China;2.Hubei International Scientific and Technological Cooperation Base of Veterinary Epidemiology (Huazhong Agricultural University)， Wuhan 430070， China;3.Hubei Hongshan Laboratory， Wuhan 430070， China

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    摘要:

    为建立一种能够快速、高效检测牛溶血性曼氏杆菌（Mannheimia haemolytica）的可视化LAMP检测法，利用LAMP引物设计软件，以溶血性曼氏杆菌保守基因lktC序列设计内引物、外引物及环引物，对其反应温度、Mg2+浓度、dNTPs浓度等条件进行优化，并检测了方法的敏感性、特异性及实用性。结果显示，该方法在68.2 ℃、6 mmol/L Mg2+、1.6 mmol/L dNTPs条件下达到最佳，40 min内完成检测并可通过肉眼判定结果，对细菌的最低检测限为6.7×10-1 cfu/μL，对lktC重组质粒的最低检测限为7.9×102 copies/μL，敏感性高；与15种病原均不发生交叉反应，特异性好；对背景清晰小鼠肺组织样本检测结果显示该方法实用性良好，且优于普通PCR方法。表明该方法可快速、高效检测牛溶血性曼氏杆菌。

    Abstract:

    In order to establish a quick and efficient visual LAMP （loop-mediated isothermal amplification） molecular method for detecting Mannheimia haemolytica in cattle， specific software tool was used to design LAMP primers including inner primers， external primers and loop primers targeting M. haemolytica conservative gene lktC. The reaction temperature， Mg2+ concentration， dNTPs concentration and other conditions were optimized. Both bacteria and recombinant plasmids were used to evaluate sensitivity， and 15 related pathogens were used to evaluate specificity. The applicability was evaluated using mice lung tissue samples with clear background. The results showed that the developed LAMP assay achieved the best results under the conditions of 68.2 ℃， 6 mmol/L Mg2+， 1.6 mmol/L dNTPs for 40 min and could be judged by naked eyes. The minimum detection limits were 6.7×10-1 CFU/μL for bacteria and 7.9×102 copies/μL for lktC recombinant plasmid， and there was no cross reaction with 15 other pathogens， indicating high sensitivity and specify. The results of lung tissue samples from mice with clear background showed that the method was better than ordinary PCR method. The above results indicate that this method was successfully constructed and could be used for rapid and efficient detection of M. haemolytica in cattle.

    图1 可视化LAMP方法的温度（A），Mg2+浓度（B），dNTPs浓度（C）和环引物浓度（D）优化Fig.1 Temperature （A） ， Mg2+ concentration（B）， dNTPs concentration（C） and loop primers concentration（D） optimization of visual LAMP
    图2 可视化LAMP的特异性检测Fig.2 Specificity of visual LAMP
    图3 可视化LAMP检测A6-ZMD菌液灵敏度Fig.3 Sensitivity of visual LAMP to detect bacterium
    图4 可视化LAMP检测lktC质粒灵敏度Fig.4 Sensitivity of visual LAMP to detect lktC plasmid
    图5 PCR检测A6-ZMD菌液的灵敏度Fig.5 Sensitivity of PCR to detect A6-ZMD bacterium
    图6 PCR检测lktC基因的灵敏度Fig.6 Sensitivity of PCR to detect lktC
    图7 可视化lktC-LAMP方法和普通PCR方法检测阴性小鼠肺组织Fig.7 Comparison of visual LAMP and PCR to detect negative mice tissues
    图8 可视化LAMP方法和普通PCR方法检测阳性小鼠肺组织Fig.8 Comparison of visual LAMP and PCR to detect positive mice tissues
    表 1 本研究中所用引物信息Table 1 Primers used in this study
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刘宁宁,易萍,郭爱珍,胡长敏,陈颖钰.牛溶血性曼氏杆菌可视化LAMP检测方法的建立[J].华中农业大学学报,2023,42(2):32-37

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  • 收稿日期:2022-10-11
  • 在线发布日期: 2023-03-31
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