利用16S rDNA建立种特异性PCR快速检测鸭疫里默氏菌  

Rapid identification of Riemerella anatipestifer on the basis of specific PCR amplifying 16S rDNA

作　　者：曲丰发[1] 蔡畅[1] 郑献进[1] 张大丙[1] 

机构地区：[1]中国农业大学动物医学院农业部预防兽医学重点开放实验室,北京100094

出　　处：《微生物学报》2006年第1期13-17,共5页Acta Microbiologica Sinica

摘　　要：鸭疫里默氏菌感染是危害养鸭业的主要疾病，用表型指标鉴定鸭疫里默氏菌存在不足，因此有必要建立检测该菌的种特异性PCR法。利用已登录的鸭疫里默氏菌、大肠杆菌、沙门氏菌、多杀性巴氏杆菌的16S rDNA基因序列，设计了一对鸭疫里默氏菌16S rDNA基因的特异性引物190f和843r，分别以基因组DNA和菌落提取液为模板，从1-19型鸭疫里默氏菌参考菌株和代表亚型、变异株和可能新型的国内分离株共26株细菌中均扩增出大小为654bp的特异性片段，而扩增鸭大肠杆菌、鸭沙门氏菌和禽多杀性巴氏杆菌等感染鸭的常见细菌的结果均呈阴性。分别将鸭疫里默氏菌基因组DNA和菌落提取液进行10倍梯度稀释，基因组DNA的最小检出量为50pg，菌落最小检出量为15CFU／mL。结果说明，该PCR法具有较好的特异性和敏感性，可用于快速鉴定鸭疫里默氏菌。Riemerella anatipestifer （RA） infection is the main disease causing severe losses in duck production. Because RA is characterized more by the absence than by the presence of specific phenotypic properties and different scholar had the different results of biochemical detection, it can＇t always be identified quickly and correctly only by the phenotypic properties or biochemical characteristics. The research object was to develop a species specific PCR method for RA detection. Because of the conserved structure of rRNA and appropriate size of 16S rRNA, a multiple alignment of 16S rDNA （gene coding 16S rRNA） was processed among RA, Escherichia coli, Pasteurella multocida, Salmonella enteritidis and Salmonella gallinarum, which are the main bacteria causing duck diseases. A pair of species specific primers named 190f and 843r were selected from the variable regions of 16S rDNA depending on the result of multiple alignment. Using BLAST on NCBI website for a sequence similarity search, the results showed that this pair of primers had very high specificity, except for having a lower sequence similarity with some species of Flavobacterium and Chryseobacterium. A PCR assay was performed and the template was extracted using the bacteria gcnomic DNA extraction kit and boiling method respectively. A chelate resin named Chelex 100 was used in the boiling method at the same time. Under the annealing temperature of 60℃, all the 26 RA strains, including 19 representative strains of serotypes 1 - 19 and 7 domestic isolated strains, showed the same 654bp fragment after PCR, while there was no amplification with isolates of other bacterial species. Also a series of sensitivity experiments were performed and proved that the detection limit of this method was 50pg genomic DNA, 1.5 × 10^6 CFU/mL and 15 CFU/mL, when the template was prepared with genomic DNA extraction kit, only boiling method and boiling method with Chelex 100 respectively. 12 clinical cases which were probably infected with RA were chosen to identify the

关 键 词：鸭疫里默氏菌 16S RDNA 种特异性PCR 

分 类 号：S854.43[农业科学—临床兽医学]