四川农业大学学报 ›› 2007, Vol. 25 ›› Issue (03): 343-347.doi: 10.16036/j.issn.1000-2650.2007.03.015

• 研究论文 • 上一篇    下一篇

基于鸭疫里默氏杆菌16SrRNA PCR检测方法的建立和应用

杨苗1, 程安春1,2, 汪铭书1,2, 仲崇岳1, 段泽1, 朱德康1,2   

  1. 1. 四川农业大学 动物科技学院, 四川 雅安 625014;
    2. 动物疫病与人类健康四川省重点实验室, 四川 雅安 625014
  • 收稿日期:2006-12-04 出版日期:2007-09-30 发布日期:2017-03-04
  • 基金资助:
    教育部"新世纪优秀人才支持计划"项目(NCET-04-0906);国家"十五"科技攻关重大项目(2004BA901A03);四川省重大基础研究项目(05JY029-109);四川省重点建设学科项目(SZD0418)。

Development and Application of PCR Based on 16SrRNA for the Detection of Riemerella anatipestifer

YANG Miao1, CHENG An-chun1,2, WANG Ming-shu1,2, ZHONG Chong-yue1, DUAN Ze1, ZHU De-kang1,2   

  1. 1. College of Animal Science and Technology, Sichuan Agricultural University, Yaan 625014, Sichuan, China;
    2. The Key Laboratory of Animal Disease and Human Health of Sichuan Province, Yaan 625014, Sichuan, China
  • Received:2006-12-04 Online:2007-09-30 Published:2017-03-04

摘要: 根据GenBank中鸭疫里默氏杆菌(RA)16SrRNA基因序列设计特异性引物,建立了基于鸭疫里默氏杆菌16SrRNA PCR检测方法。以建立的PCR方法对RA、大肠杆菌、沙门氏菌、鸭源金黄色葡萄球菌、两双歧杆菌、短乳酸杆菌、屎肠球菌和短小芽孢杆菌进行检测,结果只有RA DNA能扩增出844 bp的特异条带,其他细菌的DNA不能扩增出任何条带;从凝胶中回收DNA条带,测序结果与GenBank中RA相应序列完全一致。对不同稀释度的RADNA样品进行扩增,PCR对RA DNA最小检出量为22 pg,最少检出RA 80 CFU/mL。以该PCR方法对可疑为RA的临床病料和菌株进行检测和鉴定,结果与细菌分离鉴定吻合率为100%。表明本研究所建立的基于RA 16SrRNA PCR方法具有快速、灵敏、特异的优点,可用于RA感染的检测、分子流行病学调查和分离菌株的快速鉴定。

关键词: 鸭疫里默氏杆菌, 16SrRNA, PCR

Abstract: Primers are designed based on the conserved region of the 16SrRNA nucleotide sequences of Riemerella anatipestifer(RA) in GenBank, and the specific PCR assay for RA detection is developed. Using this assay, we can amplify a differential fragment about 844 bp from RA, but we cannot get the same fragment from Escherichia coli, Salmonella, Staphylococcus aureus, Bifidobucterium pullorum, Lactobacillus aviarius, Enterococcus faecium and Bacillus subtilis. The DNA band was obtained from agarose gel, and the sequence of the PCR product is aligned with the available sequence in GenBank. A series of sensitivity experiments is performed and proves that the detection limit of this assay is 22 pg genomic DNA and 80 CFU/m L. The clinical samples and bacteria isolated from the infected ducks are detected by the PCR assay and the positive coincidence rate comparing RA isolation with PCR is 100 percent. The result shows that the PCR assay is rapid, sensitive and specific which provides a greatly improved tool for diagnosing the infected ducks, doing molecular epidemiology survey of RA and quickly detecting the isolated bacteria.

Key words: Riemerella anatipestifer, 16SrRNA, PCR

中图分类号: 

  • S852.61