3种血清型沙门氏菌多重PCR检测方法的建立

Establishment of multiplex PCR methods for determination of 3 kinds of Salmonella serotypes

目的建立多重聚合酶链式反应(multiplex polymerase chain reaction,mPCR)法检测3种常见血清型沙门氏菌的分析方法。方法以肠炎沙门氏菌Hat基因、鼠伤寒沙门氏菌Stm-4495基因、乙型副伤寒沙门氏菌sdfI基因设计合成特异性引物,提取沙门氏菌基因组DNA为扩增模板,验证引物特异性,优化多重PCR退火温度及引物浓度,检测方法特异性及检出限,并利用该方法对人工污染冷鲜鸡肉进行检测。结果3对引物特异性强且无交叉影响;25μL多重反应体系中,引物STM、HAT、SDF最佳终浓度分别为:0.5、0.5、0.4μmol/L,最佳退火温度为60℃;11株阴性对照菌无目标条带检出,方法特异性良好,以3种血清型沙门氏菌纯培养物DNA混合物为模板,检出限低至DNA质量浓度1 pg/μL;人工污染鸡肉经过12 h增菌,能同时检测出3种血清型沙门氏菌的检测限为:肠炎沙门氏菌(4.0±1.0)CFU/g、鼠伤寒沙门氏菌(8.0±0.9)CFU/g、乙型副伤寒沙门氏菌(8.0±0.6)CFU/g。结论本方法特异性强、检测限低,对食品中3种常见血清型沙门氏菌快速检测具有重要意义。

Objective To establish a method for determination of for 3 common serotypes of Salmonella by multiplex polymerase chain reaction(mPCR).Methods S.enteritidis Hat gene,S.typhimurium Stm-4495 gene,and S.paratyphi B sdfI gene were used as target gene to design specific primers.Salmonella genomic DNA was extracted as an amplification template to verify the specificity of primers.The primer concentration and annealing temperature were optimized and the specificity and limit of detection of method were determined to develop detection method of mPCR.Results Three pairs of primers could amplify target genes with great specificity and no-cross effect.The optimal final concentrations of primers STM,HAT,SDF were 0.5,0.5,0.4μmol/L,respectively,and the optimum annealing temperature was 60℃.Eleven negative control bacteria were detected without target bands.The detection limit of 3 serotypes of Salmonella was as low as 1 pg/μL.The detection limits of 3 serotypes S.Enteritidis,S.Typhimurium and S.Paratyphi B were(4.0±1.0)CFU/g,(8.0±0.9)CFU/g and(8.0±0.6)CFU/g respectively after 12 h enrichment in contaminated chilled chicken.Conclusion This method has high specificity and low detection limit and is of great significance for rapid detection of 3 common serotypes of Salmonella.