›› 2012, Vol. 24 ›› Issue (3): 0-391.

• 论文 •    

鸭疫里默氏杆菌PCR快速检测方法的建立

季权安1,刘燕1, 肖琛闻1,韦强1, 霍翠梅2,鲍国连1,*
  

  1. 1浙江省农业科学院 畜牧兽医研究所,浙江 杭州 310021; 2山东省济宁市畜牧兽医局,山东 济宁 272000
  • 收稿日期:1900-01-01 修回日期:1900-01-01 出版日期:2012-05-24 发布日期:2012-05-24

Establishment of rapid colony PCR method of Riemerellaan alipestifer

JI Quan-an;LIU Yan;XIAO Chen-wen;WEI Qiang;HUO Cui-mei;BAO Gu-|lian;*   

  1. 1Institute of Animal Husbandry and Veterinary Science, Zhejiang Academy of Agricultural Sciences, Hangzhou 310021, China;2 Bureau of Animal Production and Health Ministry of Agriculture in Jining, Shandong Province, Jining 272000,China
  • Received:1900-01-01 Revised:1900-01-01 Online:2012-05-24 Published:2012-05-24

摘要: 鸭疫里默氏杆菌病对养鸭业危害严重,传统及常规检测方法均有不足,因此有必要建立起一种更加快速简捷、敏感实用的菌落PCR法。根据GenBank公布的鸭疫里默氏杆菌ATCC株OMPA基因序列,在基因保守区设计特异性引物,直接挑取单菌落作为模板,从分离鉴定的1,2,10,11型15株菌及1,2,10,11型4株参考菌株中均能扩增出大小为670 bp的特异性核酸片段,而对照的大肠杆菌、多杀性巴氏杆菌的扩增结果均为阴性;将培养菌用TE作10倍梯度稀释,最低检出限量为80个鸭疫里默氏杆菌;建立的菌落PCR与常规PCR法比较,两种方法均能扩增出相同的特异性条带。结果表明,菌落PCR法具有较好的特异性和敏感性,与常规PCR法结果完全一致,且更加快速简捷,在临床应用上具有更好的实用价值。

关键词: 菌落PCR, 常规PCR, 鸭疫里默氏杆菌, OMPA基因

Abstract: Riemerella anatipestifer disease could induce huge damage to the duck industry. It is necessary to establish fast simple and sensitive method due to the backward of present conventional methods. According to published OMPA ATCC strains OMPA gene sequence in GenBank, a pair of genespecific primers in a conservative region was designed by DNAstar software, PCR method was started initially from taking only one of Riemerella anatipestifer colony directly. Fifteen clinical isolated strains include RA1,2,10 and 11 as well as four reference strains include RA1,2,10 and 11 could be amplified the specific 670 bp fragments while E.coli, Pasteurella multocida were negative. Sensitivity test results showed that the PCR detection minimum limit was 80 Riemerellaan atipestifer CFU. The results of newly established colony PCR and conventional PCR method were the same, both could amplified the same band, it proved that the colony PCR method was deserved for further research and application.

Key words: colony PCR, conventional PCR, Riemerella anatipestifer, OMPA