Abstract

Salmonella sp. specific gene fragment 3335471 was used to establish a PCR-based assay for rapid detection of Salmonella sp. To establish the PCR assay, internal amplification control (IAC) was constructed using the composite primer method; the optimal concentration was determined to be 365 copies/μL. Storage of freeze-dried PCR solutions at –20°C for 3 months had no effect on detection results. Tests of artificially contaminated milk showed that Salmonella choleraesuis could be detected in 8 h when inoculated at 7 colony forming units (CFU)/10 mL. Next, a quantitative real-time PCR (RT-PCR) for detection of Salmonella sp. was developed. The assays showed high specificity for 13 Salmonella strains and 16 non-Salmonella strains. The optimal concentration of the IAC was 425 copies/μL for RT-PCR. Using S. choleraesuis as the target strain, the detection sensitivity of genome was 1.23 fg/μL and the limit of detection was 1–4 CFU/10 mL in artificially contaminated milk with 6 h of non-selective enrichment. This study established highly efficient, sensitive PCR and RT-PCR systems, providing effective approaches for the rapid detection of Salmonella sp. in the food industry.

中文翻译：

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沙门氏菌快速检测的内控扩增PCR检测方法的建立

摘要

沙门氏菌属。特定基因片段3335471用于建立基于 PCR 的检测，用于快速检测沙门氏菌。为了建立 PCR 检测，使用复合引物方法构建内部扩增对照 (IAC)；最佳浓度确定为 365 拷贝/μL。将冻干 PCR 溶液在 –20°C 下储存 3 个月对检测结果没有影响。对人工污染牛奶的检测表明，以 7 个菌落形成单位 (CFU)/10 mL 接种时，8 小时内可检出猪霍乱沙门氏菌 。接下来，用于检测沙门氏菌的定量实时 PCR (RT-PCR) 。已开发。检测结果显示对 13沙门氏菌具有高特异性菌株和 16 个非沙门氏菌菌株。对于 RT-PCR，IAC 的最佳浓度为 425 拷贝/μL。以猪霍乱沙门氏菌为目标菌株，基因组检测灵敏度为1.23 fg/μL，检测限为1-4 CFU/10 mL人工污染牛奶，非选择性富集6 h。本研究建立了高效、灵敏的PCR和RT-PCR体系，为沙门氏菌的快速检测提供了有效途径。在食品行业。