禽大肠杆菌、肠炎、鼠伤寒、鸡白痢及鸡伤寒沙门菌多重PCR方法的建立及应用

Multiplex PCR assay for detection of Avian pathogenic E. coli,Salmonella enteritidis, Salmonella typhimurium, Salmonella gallinarum and Salmonella pullorum

【背景】大肠杆菌病和沙门菌病是最常见的家禽细菌性疾病,给养禽业造成严重经济损失。另外,禽大肠杆菌和沙门菌也是重要的人畜共患病原菌,可通过禽类及其产品传播给人类,对人类健康造成严重威胁。加强禽大肠杆菌和沙门菌的快速鉴别检测,对养禽业和公共卫生都具有重要意义。【目的】建立禽大肠杆菌、肠炎沙门菌、鼠伤寒沙门菌、鸡白痢沙门菌和鸡伤寒沙门菌的多重PCR检测方法。【方法】通过比较分析确定禽致病性大肠杆菌、肠炎沙门菌、鼠伤寒沙门菌、鸡白痢沙门菌和鸡伤寒沙门菌的特异靶标基因,设计5对特异性引物,通过条件优化建立多重PCR方法,分析该多重PCR方法的特异性、敏感性及可靠性。【结果】该方法能特异性地鉴定禽致病性大肠杆菌、肠炎沙门菌、鼠伤寒沙门菌、鸡白痢沙门菌和鸡伤寒沙门菌,每个PCR反应的最低检出限分别为10~3 CFU细菌和100 pg基因组DNA。临床分离菌株检测显示,多重PCR与传统血清学方法结果一致。【结论】建立的多重PCR方法能够快速鉴别禽致病性大肠杆菌和不同血清型沙门菌,对禽大肠杆菌病和沙门菌病的流行病学调查及临床检测具有重要意义。

[Background]Colibacillosis and salmonellosis are the most common bacterial diseases in poultry,causing economically devastating to poultry industries.Salmonella and avian pathogenic E.coli(APEC)are important zoonotic pathogens,also with potential threat to human health.Thus,it is necessary to strengthen the rapid detection and differential diagnosis of these bacterial diseases.[Objective]We developed multiplex PCR to efficiently detect APEC,Salmonella enteritidis,Salmonella typhimurium,Salmonella gallinarum and Salmonella pullorum.[Methods]The special genes of APEC,Salmonella enteritidis,Salmonella typhimurium,Salmonella gallinarum and Salmonella pullorum were screened and selected for primers designing.A multiplex PCR method was developed by optimization of conditions.Then,the sensitivity and usage for detection of clinical isolates were determined.[Results]A multiplex PCR was established for accurately and effectively detection of APEC,Salmonella enteritidis,Salmonella typhimurium,Salmonella gallinarum and Salmonella pullorum.The sensitivity of the multiplex PCR was 10~3 bacterial colony forming units(CFUs)and 100 pg genomic DNA.The results of multiplex PCR for detection of clinical isolates agreed well with those of traditional serological assays.[Conclusion]Multiplex PCR developed in this study could efficiently detect APEC,Salmonella enteritidis,Salmonella typhimurium,Salmonella gallinarum and Salmonella pullorum.