羊多杀性巴氏杆菌与布鲁氏菌双重套式PCR检测方法的建立及应用  

Establishment and Application of Double Nested PCR for Detecting Pasteurella multocida and Brucellain Sheep and Goats

作　　者：吴昊天 满初日嘎[1] 刘昂 何美荣 侯思梦 李崇瑞 杜丽[1] 陈巧玲 陈思 王凤阳[1] 李昌 金宁一 WU Hao-tian;Manchuriga;LIU Ang;HE Mei-rong;HOU Si-meng;LI Chong-rui;DU Li;CHEN Qiao-ling;CHEN Si;WANG Feng-yang;LI Chang;JIN Ning-yi(School of Animal Science and Technology,Hainan University,Key Laboratory of Tropical Animal Breeding and Disease Research of Hainan Province,Haikou Key Laboratory of Animal Genetic Engineering,Haikou,Hainan,570228,China;Institute of Military Veterinary Medicine,Academy of Military Medical Sciences,Changchun,Jilin,130122,China)

机构地区：[1]海南大学动物科技学院,海南省热带动物繁育与疫病研究重点实验室,海口市动物基因工程重点实验室,海南海口570228 [2]军事医学研究院军事兽医研究所,吉林长春130122

出　　处：《动物医学进展》2022年第11期12-17,共6页Progress In Veterinary Medicine

基　　金：海南省自然科学基金项目(321RC477);海南省院士创新平台科研专项(YSPTZX202013);国家肉羊产业技术体系项目(财政部农业部CARS38)。

摘　　要：为了建立一种高灵敏度和强特异性的羊多杀性巴氏杆菌与布鲁氏菌的双重检测方法,根据多杀性巴氏杆菌kmt1基因序列和布鲁氏菌BCSP31基因序列分别设计了内外两对引物,构建了双重套式PCR检测方法。结果表明,该方法可以特异性扩增kmt1和BCSP31片段,并能从多种组织样本的DNA中准确检测到多杀性巴氏杆菌与布鲁氏菌的核酸片段。检测沙门氏菌、猪链球菌、大肠埃希氏菌、金黄色葡萄球菌、铜绿假单胞菌、鲍曼不动杆菌、海藻糖巴氏杆菌和斯氏假单胞菌均为阴性;对于kmt1的检测下限为70.9 copies/μL,对于BCSP31检测下限为89.5 copies/μL;用于检测动物的组织样本符合率为100%。建立的方法有良好的敏感性、特异性和适应性,可以用于动物检疫诊断。In order to establish accurate and rapid detection method of Pasteurella multocidaand Brucellain sheep and goats,two pairs of internal primers and two pairs of external primers were designed according to the gene sequence of Pasteurella multocida kmt1and the sequence of Brucella BCSP31.The results showed that the method can specifically amplify the kmt1and BCSP31,and can accurately detect Pasteurella multocida and Brucella from the DNA of various tissue samples.The detection of Salmonella,Streptococcus suis,Escherichia coli,Staphylococcus aureus,Pseudomonas aeruginosa,Acinetobacter baumannii,Pasteurella trehalose and Pseudomonas sternii were all negative;the lower limit of detection for kmt1was 70.9copies/μL,the lower limit of detection for BCSP31was 89.5copies/μL;the coincidence rate of detecting samples form infected animals is 100%.The results showed that the method established in this experiment has good sensitivity,specificity and adaptability,and can be used for animal quarantine or disease diagnosis.

关 键 词：多杀性巴氏杆菌 布鲁氏菌 套式PCR 

分 类 号：S852.614[农业科学—基础兽医学] S858.26[农业科学—兽医学]