四重荧光定量PCR法鉴定肠炎、鼠伤寒以及伤寒沙门氏菌

目的建立四重荧光定量PCR体系鉴定肠炎沙门氏菌、鼠伤寒沙门氏菌以及伤寒沙门氏菌。方法针对沙门氏菌属特异性ompC基因、肠炎沙门氏菌sdf基因、鼠伤寒沙门氏菌STM4495和伤寒沙门氏菌STY2021序列设计引物和TaqMan探针,建立多重荧光定量PCR体系,进行特异性与敏感性研究。结果 28株不同血清型的沙门氏菌均扩增出ompC基因,其他13株非沙门氏菌均未出现ompC的非特异性扩增。sdf、STM4495、STY2021的探针和引物分别特异性扩增出肠炎沙门氏菌、鼠伤寒沙门氏菌以及伤寒沙门氏菌,而25株其他血清型沙门氏菌以及13株非沙门氏菌均未见扩增曲线。敏感性试验显示,该体系的最低检测限分别为48 pg/mL(ompC)、560 pg/mL(sdf)、530 pg/mL(STM4495)、35 pg/mL(STY2021)。结论该方法特异好、灵敏高、能够快速检测沙门氏菌并鉴定肠炎沙门氏菌、鼠伤寒沙门氏菌以及伤寒沙门氏菌。

Identification of Salmonella enteritidis, Salmonella typhimurium and Salmonella typhi by quadruple fluorescence quantitative PCR

Objective To establish quadruple fluorescence quantitative PCR for identification of Salmonella enteritidis, Salmonella typhimurium and Salmonella typhi. Methods Primers and TaqMan probes were designed for Salmonella species-specific ompC gene, Salmonella enteritis sdf gene, Salmonella thyphimurium STM4495 and Salmonella typhi STY2021 sequences, and the multiplex fluorescence quantitative PCR system was established to investigate the specificity and sensitivity. Results The ompC gene was amplified from 28 different serotypes of Salmonella, and the other 13 non-Salmonella did not show non-specific amplification of ompC. The probes and primers of sdf, STM4495 and STY2021 were used to specifically amplify Salmonella enteritis, Salmonella typhi and Salmonella typhi respectively, while no amplification curve was observed for 25 other serotype Salmonella and 13 non-Salmonella strains. The limits of detection of this method were 48 pg/mL (ompC), 560 pg/mL (sdf), 530 pg/mL (STM449), and 35 pg/mL (STY2021). Conclusion The method is specific, sensitive, and can quickly detect Salmonella and identify Salmonella enteritidis, Salmonella typhimurium and Salmonella typhi.