蜜蜂白垩病PCR快速检测方法的建立及初步应用  

Development and Application of Polymerase Chain Reaction for Detection of Bee Chalkbrood Disease

作　　者：宋战昀[1] 冯新[2] 刘金华[1] 赵柏林[3] 石建平[1] 王振国[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预防兽医学国家重点学科,吉林长春130062 [3]吉林农业大学,吉林长春130118

出　　处：《蜜蜂杂志》2009年第10期8-11,共4页Journal of Bee

基　　金：国家质检总局科学基金资助项目(2005IK052)

摘　　要：根据Genebank网站公布的蜜蜂球囊菌ITS1,5.8s,ITS2rDNA保守序列(U68313)设计出一对特异性引物ASCU和ASCD,可特异性的扩增蜜蜂球囊菌ITS1,5.8srRNA,ITS2序列上大小为471bp的片段。优化了PCR最佳反应条件和体系;特异性试验结果表明与蜜蜂幼虫芽孢杆菌、蜂房蜜蜂球菌、蜜蜂败血杆菌无交叉反应,具有良好的特异性。敏感性试验结果表明敏感性可达7CFU/mL的蜜蜂球囊菌。通过将定量的蜜蜂球囊菌添加到蜂蜜、花粉、蜂胶和蜂王浆等蜂产品中进行模拟检疫,结果显示所建立的PCR检测方法适用于上述蜂产品中蜜蜂球囊菌的检疫。该方法与病原菌分离培养等传统检疫方法相比较,具有快速、灵敏、特异等优点,可用于蜜蜂及蜂产品中白垩病的进出口检疫及快速诊断。A polymerase chain reaction was developed to detect bee chalkbrood disease. According to the nuclear rDNA region containing the internal transcribed spacer regions and 5.8S rDNA （ITS1, 5.8S, ITS2） in Genebank , a pair of PCR primers named ASCU and ASCD was designed. The PCR product was 471bp Only Ascosphaera apis strains were am- plified the expected fragment. No cross-reaction was de- tected with Paenibacillus larvae, Melissococcus pluton and Bacillus septicacmiae. The results showed that this pair of primers had very high specificity. Sensitivity experiments were also performed and proved that the detection limit of this method was 7 CFU/mL. Then applied the method to detect Ascosphaera＇ apis in artificial inoculated honey, pollen , propolis , royal jelly etc bee products, the PCR method could detect Ascosphaera apis from simulation samples. The results indicated that the PCR method was more rapid, specific and sensitive than the bacteriological examination, and can be used for detection of Ascosphaera ap/s from bee and bee products in the quarantine.

关 键 词：蜜蜂白垩病 蜜蜂球囊菌 聚合酶链式反应 检测 

分 类 号：S895.137[农业科学—特种经济动物饲养]