猪胸膜肺炎放线杆菌可视化LAMP检测方法的建立与应用  

Establishment and application of visual LAMP method for Actinobacillus pleuropneumoniae detection

作　　者：朱海鹏 郑旭 王慧 高洁 司舒晗 杜涛峰[1,2] 穆杨 ZHU Hai-peng;ZHENG Xu;WANG Hui;GAO Jie;SI Shu-han;DU Tao-feng;MU Yang(College of Veterinary Medicine,Northwest Agricultural and Forestry University,Yangling 712100,China;Shaanxi Scientific Observing and Experimental Station of Veterinary Pharmacology and Diagnostic Technology,Ministry of Agriculture,Yangling 712100,China)

机构地区：[1]西北农林科技大学动物医学学院,陕西杨凌712100 [2]农业部兽用药物与诊断技术陕西科学观测试验站,陕西杨凌712100

出　　处：《中国兽医科学》2022年第7期837-845,共9页Chinese Veterinary Science

基　　金：国家自然科学基金项目(31972675);瑞东农牧(山东)有限责任公司横向课题项目(TG2020082);大学生创新训练项目(S202110712252)。

摘　　要：为建立检测猪胸膜肺炎放线杆菌(APP)的可视化环介导等温扩增(LAMP)方法,对比猪APP主要流行毒株的ApxⅣA基因序列,选择高度保守区域设计LAMP引物,以钙黄绿素(Calcein)为指示剂,对反应体系和反应条件优化后建立了一种快速检测猪APP的可视化LAMP方法。采用建立的方法在63℃水浴锅中扩增40 min后,85℃作用10 min即可完成检测。用该方法检测大肠杆菌、绿脓杆菌、多杀性巴氏杆菌、金黄色葡萄球菌、猪霍乱沙门菌、猪链球菌、猪肺炎支原体、副猪嗜血杆菌等,均不发生交叉反应。以10倍稀释的APP基因组DNA为模板,评价方法的敏感性,结果最低检出量为1.67×10^(-5)ng/μL,比普通PCR方法的最低检出量1.67×10^(-2)ng/μL高1000倍;在反应体系中加入钙黄绿素,反应结束后阳性反应管颜色从橘黄色变为黄绿色且在紫外光下有绿色荧光。采用建立的方法检测312份临床样品,共检出阳性样品46份,而采用普通PCR方法仅检出阳性样品10份。该方法的建立为快速诊断猪传染性胸膜肺炎提供了技术支持。To establish a visual loop-mediated isothermal amplification(LAMP)method for detecting Actinobacillus pleuropneumoniae(APP)in pigs,LAMP primers were designed by comparing ApxⅣA gene sequences of the main epidemic strains of porcine APP.Using calcein as an indicator,a visual LAPM method for rapid detection of porcine APP was established after the reaction system and reaction conditions were optimized.The amplification can be completed within 40 min in a 63℃water bath and then stopped at 85℃for 10 min.Escherichia coli,Pseudomonas aeruginosa,Pasteurella multocida,Staphylococcus aureus,Salmonella cholerae,Streptococcus suis,Mycoplasma pneumoniae,and Haemophilus parasuis were detected by this method,and no cross-reaction occurred.Using 10-fold diluted APP genomic DNA as templates,the sensitivity of the established method was evaluated.The results showed that the minimum detection amount was 1.67×10^(-5)ng/μL,which was three orders of magnitude higher than the minimum detection amount of the conventional PCR method.When calcein was added to the reaction system,the color of the positive reaction tube changed from orange to yellow-green and showed green fluorescence under the ultraviolet lamp.The established visual porcine AAP-LAMP method was used to detect 312 clinical samples,and 46 samples were positive,while only 10 positive samples were detected by the conventional PCR method.The establishment of this method provides a practical method for the rapid diagnosis of porcine contagious pleuropneumonia.

关 键 词：猪胸膜肺炎放线杆菌 可视化环介导等温扩增 ApxⅣA基因 钙黄绿素 

分 类 号：S852.619[农业科学—基础兽医学]