Home > Archive>Volume 42, Issue 2, 2023 >32-37. DOI:10.13300/j.cnki.hnlkxb.2023.02.005
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Establishment of visual LAMP method for detecting Mannheimia haemolytica in cattle
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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

Clc Number:

S852.61

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    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.

    Fig.1 Temperature （A） ， Mg2+ concentration（B）， dNTPs concentration（C） and loop primers concentration（D） optimization of visual LAMP
    Fig.2 Specificity of visual LAMP
    Fig.3 Sensitivity of visual LAMP to detect bacterium
    Fig.4 Sensitivity of visual LAMP to detect lktC plasmid
    Fig.5 Sensitivity of PCR to detect A6-ZMD bacterium
    Fig.6 Sensitivity of PCR to detect lktC
    Fig.7 Comparison of visual LAMP and PCR to detect negative mice tissues
    Fig.8 Comparison of visual LAMP and PCR to detect positive mice tissues
    Table 1 Primers used in this study
    Reference
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刘宁宁,易萍,郭爱珍,胡长敏,陈颖钰. Establishment of visual LAMP method for detecting Mannheimia haemolytica in cattle[J]. Jorunal of Huazhong Agricultural University,2023,42(2):32-37.

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  • Received:October 11,2022
  • Online: March 31,2023
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