猪肺炎支原体环介导等温扩增检测方法的建立  

Development of Loop-mediated Isothermal Amplification Method for Detection of Swine Mycoplasma hyopneumoniae

作　　者：李鹏[1] 马艳娇[1,2] 于剑 薛晓萌[1] 刘丛[1] 夏伟[1] 

机构地区：[1]黑龙江八一农垦大学动物科技学院,黑龙江大庆163319 [2]讷河市畜牧水产推广中心,黑龙江讷河161300

出　　处：《中国生物制品学杂志》2011年第4期480-483,共4页Chinese Journal of Biologicals

基　　金：大庆市科学技术局项目(SGG2007-048)

摘　　要：目的建立检测猪肺炎支原体(Mycoplasma hyopneumoniae,Mh)的环介导等温扩增(Loop-mediated isothermal am-plification,LAMP)方法。方法根据GenBank中登录的猪肺炎支原体的核苷酸序列,设计4条特异性引物,用外引物进行PCR反应,对内外引物浓度、dNTP+和MgSO4浓度及Bst DNA聚合酶用量等进行优化,建立猪肺炎支原体LAMP检测方法,并对该方法进行特异性和敏感性验证。结果内外引物浓度分别为0.5和0.20 pmol/μl,dNTP+浓度为0.5 mmol/L,MgSO4浓度为3.75 mmol/L,Bst DNA聚合酶用量为8.0和9.6 U时,LAMP反应效果较好;应用该方法检测多杀性巴氏杆菌、猪胸膜肺炎放线杆菌、肺炎双球菌、支气管败血波氏杆菌、副猪嗜血杆菌和链球菌均呈阴性;当猪肺炎支原体的拷贝数低于3时,检测不到该病原体。结论已建立了猪肺炎支原体LAMP检测方法,该方法特异性较好,敏感性较高。Objective To develop a loop-mediated isothermal amplification（LAMP） method for detection of swine Mycoplasma hyopneumoniae.Methods Four specific primers were designed according to the nucleotide sequence of swine M.hyopneumoniae reported in GenBank.The external primers were used for PCR,and the concentrations of internal and external primers,dNTP＋ and magnesium sulfate as well as the units of Bst DNA polymerase added were optimized,based on which a LAMP method for swine M.hyopneumoniae was developed and verified for specificity and sensitivity.Results When the concentrations of internal and external primers were 0.5 and 0.20 pmol/L,while those of dNTP＋ and magnesium sulfate were 0.5 and 3.75 mmol/L respectively,and 8.0 or 9.6 U of Bst DNA polymerase was added,the reaction effect was satisfactory.No Pasteurella multocida,Actinobacillus,Streptococcus pneumonia,Bordetella bronchi,Haemophilus parasuis or Streptococcus were detected by the developed LAMP.The M.hyopneumoniae with a copy number of less than 3 could not be detected by the method.Conclusion A LAMP method for detection of swine M.hyopneumoniae was developed,which showed high specificity and sensitivity.

关 键 词：猪肺炎 支原体 环介导等温扩增 诊断 

分 类 号：S852.62[农业科学—基础兽医学] Q78[农业科学—兽医学]