4种鸡源致病性沙门氏菌多重PCR检测方法的建立及应用

Development and Application of a Multiplex PCR for Simultaneous Detection of Four Salmonella Species

建立一种多重PCR方法,同时检测肠炎、鼠伤寒、鸡白痢/鸡伤寒沙门氏菌。针对沙门氏菌属特异性invA基因、肠炎沙门氏菌特异性Sdf I序列、鼠伤寒沙门氏菌特异性STM4497基因、鸡白痢/鸡伤寒沙门氏菌flhB基因设计引物,对引物浓度和反应条件进行优化,并对多重PCR特异性和灵敏度进行评估。利用建立的多重PCR方法调查河北地区蛋种鸡生产链中沙门氏菌流行现状。研究建立的多重PCR方法可快速、特异性鉴别肠炎、鼠伤寒、鸡白痢/鸡伤寒沙门氏菌,最低检出限为100 CFU细菌和0.5 ng基因组DNA。沙门氏菌流行病学调查显示,肠炎沙门氏菌是河北地区蛋种鸡生产链中沙门氏菌主要流行血清型。成功建立鉴别肠炎、鼠伤寒、鸡白痢/鸡伤寒沙门氏菌的多重PCR方法,对沙门氏菌流行病学调查及临床快速诊断具有重要意义。

To develop a multiplex PCR method for the simultaneous detection of Salmonella enteritidis,Salmonella typhimurium,Salmonella pullorum/gallinarum,primers were designed based on invA gene of Salmonella genus,Sdf I sequence of Salmonella enteritidis,STM4497 gene of Salmonella typhimurium,and flhB gene of Salmonella pullorum/gallinarum.The primer concentrations and reaction conditions were optimized,and the specificity and sensitivity of multiplex PCR were evaluated.The prevalence of Salmonella in the production chain of laying hens in Hebei area was investigated using this multiplex PCR.The method could be used to identify Salmonella enteritidis,Salmonella typhimruium,Salmonella pullorum/gallinarum rapidly and specifically.The minimum detection limit was 100 CFU bacteria and 0.5 ng genomic DNA.The epidemiological investigation of Salmonella showed that Salmonella enteritidis was the main prevalent serotype of Salmonella in the production chain of laying hens in Hebei area.A multiplex PCR method for detection of Salmonella enteritidis,Salmonella typhimruium,Salmonella pullorum/Salmonella gallinarum was developed,which is of great significance for epidemiological investigation and rapid clinical diagnosis of Salmonella.