Page 43 - 《华中农业大学学报（自然科学版）》2023年第2期

第 2 期 刘宁宁 等：牛溶血性曼氏杆菌可视化 LAMP 检测方法的建立 37

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Establishment of visual LAMP method for detecting
Mannheimia haemolytica in cattle

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1
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LIU Ningning ,YI Ping ,GUO Aizhen 1,2,3 ,HU Changmin ,CHEN Yingyu 1,2
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

Abstract In order to establish a quick and efficient visual LAMP （loop-mediated isothermal amplifi⁃
cation） 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. haemolyt⁃
2+
ica conservative gene lktC. The reaction temperature， Mg 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 tis⁃
sue samples with clear background. The results showed that the developed LAMP assay achieved the best
2+
results under the conditions of 68.2 ℃ ， 6 mmol/L Mg ， 1.6 mmol/L dNTPs for 40 min and could be
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judged by naked eyes. The minimum detection limits were 6.7×10 CFU/μL for bacteria and 7.9×10
copies/μL for lktC recombinant plasmid， and there was no cross reaction with 15 other pathogens， indicat⁃
ing 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 suc⁃
cessfully constructed and could be used for rapid and efficient detection of M. haemolytica in cattle.
Keywords loop-mediated isothermal amplification； lktC gene； Mannheimia haemolytica； visualiza⁃
tion detection； comprehensive prevention and control

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