蜜蜂球囊菌荧光PCR快速检测方法的建立及应用  

Establishment and Application of Real-time PCR for Detection of Ascosphaera apis

作　　者：宋战昀[1] 王振国[1] 冯新[2] 王伟利[1] 罗雁非[1] 刘金华[1] 姜永莉[1] 

机构地区：[1]吉林出入境检验检疫局,吉林长春130062 [2]吉林大学畜牧兽医学院,吉林长春130062

出　　处：《动物医学进展》2010年第12期49-53,共5页Progress In Veterinary Medicine

基　　金：国家质检总局科学基金资助项目(2005IK052)

摘　　要：以蜜蜂球囊菌ITS1、ITS2和5.8 S rDNA保守序列(U68313)为参考,设计一对特异性引物和一条TaqMan探针,建立了一种快速检测蜜蜂球囊菌的荧光PCR方法。该方法灵敏度达1 ng/μL的阳性DNA比常规PCR高10倍。检测的特异性高,与蜜蜂幼虫芽胞杆菌、蜂房蜜蜂球菌、蜜蜂败血杆菌无交叉反应,同时可避免常规PCR因电泳造成的污染。应用该方法对蜜蜂及蜂制品的实际样品和模拟样品进行检测,结果显示所建立的荧光PCR检测方法在4 h内即可报告结果,与传统病原菌分离培养、常规PCR相比较,该方法具有快速、灵敏、特异、重复性好等优点,可以用于蜜蜂及其制品中蜜蜂球囊菌的快速检测和进出境检疫。A real-time PCR assay was developed for detection of bee chalkbrood disease.According to the nuclear rDNA region containing the internal transcribed spacer regions and 5.8 S rDNA（ITS1,5.8S,ITS2） in GenBank,a pair of primers and TaqMan probe were designed.The assay could detect 1 ng/μL of the standards,which is more than 10-fold sensitive than conventional PCR.No cross-reaction was detected with Paenibacillus larvae,Melissococcus pluton and Bacillus septicaemiae,and the assay can avoid the contamination by PCR electrophoresis.Compared to isolation and culture of the pathogen and conventional PCR,the advantages of this method are rapid response,high sensitivity,good selectivity and good repeatability.Moreover,it can be used for detection of Ascosphaera apis from bees and bee products in the quarantine.

关 键 词：蜜蜂白垩病 蜜蜂球囊菌 TAQMAN探针 荧光PCR 

分 类 号：S895.137[农业科学—特种经济动物饲养]