Clostridium perfringens is an important zoonotic pathogen with α-toxin gene as major molecular biological detection target.A droplet digital PCR(ddPCR)method was developed using Clper primer and probe with the optimal concentration of primer and probe 0.85 and 0.3 μmol/L respectively and the annealing temperature as 60℃.The DNA of Listeria,Salmonella,Escherichia coli,Enterococcus,Campylobacter and Staphylococcus aureus were assessed by the established method,and all results were negative,indicating that the method had good specificity.Repetitive experiments based on three dilution titers of reference materials of synthesized α-toxin DNA showed that the variation coefficients of both inter-and intra-groups were less than 5%,which indicated good repeatability.The limit of detection and the limit of quantification were determined by detecting seven dilution titers of reference materials,which were 12.39 and 33.97 copies/μL respectively.The established method was verified to be applied in clinic by detecting 68 clinical samples,and had no obvi-ous matrix effect when used for plasmid DNA detection.A reference material certificate(GBW(E)091237)had been issued for the reference material by the State Administration of Market Supervision and Administra-tion after detecting the homogeneity and stability by the ddPCR method and determining the fixed values by 9 laboratories.Overall,the establishment of the ddPCR method for α-toxin gene and the development of DNA reference materials could lay the foundation for standardization of molecular biological detection reagents for C.perfringens.
关键词
产气荚膜梭菌/ddPCR/α-毒素基因/标准物质/特异性/灵敏度/可重复性/定值
Key words
Clostridium perfringens/Droplet digital PCR/α-Toxin gene/Reference material/Specifici-ty/Sensitivity/Repeatability/Fixed value
维普
万方数据