细菌bla_(NDM-1)基因PCR检测方法的建立及应用  

Establishment and Application of PCR for Detection of Bacterial bla_(NDM-1) Gene

作　　者：温肖会[1] 翟少伦[1] 丘婷[2] 吕殿红[1] 贾春玲[1] 袁洁[1] 黄忠[1] 周秀蓉[1] 魏文康[1] 

机构地区：[1]广东省农业科学院动物卫生研究所,广东广州510640 [2]华南农业大学兽医学院,广东广州510642

出　　处：《动物医学进展》2013年第12期145-149,共5页Progress In Veterinary Medicine

基　　金：广东省促进科技服务业发展计划项目(2012B040302010);广州市行业工程技术研究中心建设项目(穗科信字[2012]224-26号);所长基金(2010SZJJ-005)

摘　　要：为了调查养殖场和农贸市场中的耐药细菌是否携带blaNDM-1基因,根据GenBank已公布的blaNDM-1基因序列设计PCR引物,优化PCR反应体系和反应条件,建立了blaNDM-1基因的PCR检测方法,以人工合成的blaNDM-1基因作为阳性质控品,以大肠埃希菌、肺炎克雷伯菌、阴沟肠杆菌和铜绿假单胞菌等4株标准菌株为阴性对照,验证耐药细菌blaNDM-1基因PCR检测方法的特异性、敏感性、重复性,并对229株大肠埃希菌分离株和31株链球菌分离株进行检测。结果表明,blaNDM-1基因PCR检测方法的特异性、敏感性、重复性试验均成立,该方法扩增的目的基因片段为241bp,能检出的最小基因组DNA浓度为9.18×10-7μg/μL;229株大肠埃希菌分离株和31株链球菌分离株blaNDM-1基因PCR检测结果均没有出现目的条带,表明从广东省部分地区分离到的细菌分离株中没有携带blaNDM-1基因。该方法可作为耐药细菌blaNDM-1基因的早期检测、快速筛查手段在兽医临床耐药细菌检测中应用。To investigate whether the blaNDM-1 gene was carried by drug-resistant bacteria in the farms and farmer′s markets,the PCR primers were designed according to the sequence of blaNDM-1 gene from GenBank and the PCR system and reaction conditions were optimized.The specificity,sensitivity,repeatability of PCR detection method for blaNDM-1 gene was verified by positive control of synthetic blaNDM-1 gene and negative control of 4 reference strains including of E.coli,Klebsiella pneumoniae,Enterobacter cloacae and Pseudomonas aeruginosa.229 isolates of E.coli and 31 isolates of Streptococcus were detected by PCR method.The results showed that the specificity,sensitivity,repeatability of PCR method for blaNDM-1 gene was established and the target gene was about 241bp.The minimal detectable DNA concentration was 9.18×10-7 μg/μL.There was no target fragment on agarose gel for 229 isolates of E.coli and 31 isolates of Streptococcus.It showed that blaNDM-1 gene was not carried by bacterial isolates from some area of Guangdong province.The PCR method can be used on monitoring of drug-resistant bacteria as an early diagnosis and rapid screening method for the drug-resistant bacteria carrying blaNDM-1 gene in veterinary clinic.

关 键 词：blaNDM-1基因 耐药细菌 聚合酶链反应 

分 类 号：S852.6[农业科学—基础兽医学] R915[农业科学—兽医学]