甲型副伤寒沙门菌特异性基因筛选及PCR检测体系建立

摘要

以甲型副伤寒沙门菌为检测目标,通过比较基因组和聚合酶链式反应(polymerase chain reaction,PCR)验证方法筛选到4个该血清型的特异性基因,其中以gene_310作为该血清型的检测靶点设计引物PA23;并结合沙门菌属特异性引物139-141,建立一种甲型副伤寒沙门菌的PCR检测方法.优化PCR反应体系,并对该检测体系的特异性、灵敏度、抗干扰能力及人工污染样品检出限等方面进行评价.结果表明,当样品中含有甲型副伤寒沙门菌时,该体系能扩增出2条特异性条带,含有其他血清型的沙门菌仅能扩增出284 bp条带,不含沙门菌无扩增条带产生.灵敏度评价表明,基因组DNA和纯菌菌落检出限分别为32.4 pg/μL和4.3×103 CFU/mL;抗干扰能力实验显示,当鸡肉背景菌群和猪肉背景菌群浓度在106 CFU/mL和4.87×107 CFU/mL时,检出限为6.43×104 CFU/mL.当无菌的鸡肉和猪肉样品中添加NCFU/25 g甲型副伤寒沙门菌时,经10h增菌,检测结果为阳性(0＜N＜10).实验建立甲型副伤寒沙门菌PCR检测方法具有较好的特异性和灵敏度,有很好的应用价值,可在食品安全领域广泛应用.%In this study,four serotype-specific genes of Salmonella Paratyphi A were identified by comparative genomics and PCR.A PCR assay based on the gene_3105 and invA gene was developed and evaluated for the detection of S.Paratyphi A.The electrophoresis pattern showed only two bright specific bands at 284 bp and 384 bp in S.Paratyphi A.The PCR protocol was optimized and the specificity,sensitivity,anti-jamming capability and limit of detection (LOD) for artificially contaminated food were evaluated.The specificity results showed two bright specific bands for S.Paratyphi A,only one specific band at 284 bp for other Salmonella serotypes,no specific bands for non-Salmonella strains.The sensitivity of the PCR assay was 32.4 pg/μL and 4.3 × 103 CFU/mL for genomic DNA and pure colonies,respectively.In the presence of natural background flora enriched from chicken and pork breast samples,the detection limit was 6.43 × 104 CFU/mL.In artificially contaminated chicken and pork,the detection limit was N CFU/25 g after 10 h enrichment (0 ＜ N ＜ 10).In conclusion,the PCR assay for the detection of S.Paratyphi A is specific and sensitive,and has a good application value and can be widely used in the field of food safety.