Establishment And Application Of TaqMan Real-time Quantitative PCR For Detection Of Two Food-borne Pathogens

Clostridium perfringens is a zoonotic pathogen widely distributed in natural environments.The bacteria can often cause food poisoning,necrotizing enteritis,enterotoxemia and other diseases.It has a serious impact on human health development and the development of livestock and poultry industry.Pseudomonas aeruginosa is also an opportunistic foodborne pathogen prevalent in the natural environment.Capable of causing disease in a variety of hosts including plants,nematodes,insects,and mammals.The spread and death rates from chronic infections caused by the bacteria are also on the rise.Therefore,the bacteria also pose a serious threat to human and animal health.The detection of these two bacteria in the "Inspection Method of the National Standard of Food Safety for Drinking Natural mineral Water"(GB 8538-2016)of our country was stipulated in the relevant regulations.At present,the detection methods of these two bacteria include isolation culture identification,conventional PCR detection and ring mediated isothermal amplification,etc.But these methods are generally time-consuming and have high requirements for operation.Taqman probe real-time quantitative PCR detection method has the advantages of high specificity,high sensitivity,high throughput and fast,and has been widely used in various fields.In this study,based on the known sequences of C.perfringens and P.aeruginosa NCBI,specific target gene sequences were screened,and TaqMan real-time fluorescent quantitative PCR primers and probes were designed to optimize the reaction system and reaction conditions.Combined with the filter membrane method in the National Standard for Inspection of Natural Mineral Water for Drinking for Food Safety(GB 8538-2016),the TaqMan real-time quantitative PCR detection methods for C.perfringens and P.aeruginosa were established,and the TaqMan double real-time quantitative PCR detection methods were used to detect direct drinking water samples and tap water samples.The main research results are as follows:(1)Through literature review and analysis of the conserved and specific sequences of C.perfringens and P.aeruginosa in NCBI database,the highly conserved plc gene of C.perfringens(GenBank:MK599266.1)and oprL(GenBank:JQ228528.1)was selected as the target gene.(2)The TaqMan real-time quantitative PCR detection method for C.perfringens and P.aeruginosa was successfully established based on the plc gene and the oprL gene.The method had good specificity and reproducibility.The minimum detection limit was 1×10 copies/μL,and the sensitivity was good.Based on this method,the TaqMan real-time quantitative PCR detection method for C.perfringens and P.aeruginosa simulated contaminated water samples was established.The minimum detection limit was 1 × 102 CFU/mL,which was 3 orders of magnitude more sensitive than the conventional PCR method.(3)The TaqMan real-time fluorescence quantitative PCR method was used to detect C.perfringens.The results showed that Ct value>40 of 90 direct drinking water samples indicated that no C.perfringens was detected.P.aeruginosa was detected in 90 direct drinking water samples and 200 fresh water samples from different seasons by TaqMan real-time quantitative PCR assay.The results showed that no P.aeruginosa was detected in 90 direct drinking water samples with Ct value>40.P.aeruginosa was detected in 3 of the water samples collected in summer and autumn,while no P.aeruginosa was detected in other water samples.(4)Based on the TaqMan real-time quantitative PCR detection method for C.perfringens and P.aeruginosa,the TaqMan dual real-time quantitative PCR detection method was further established.The detection limit of this method was 1×10 copies/μL.Based on this method,a TaqMan dual real-time quantitative PCR method was established for the detection of C.perfringens and P.aeruginosa simulated contaminated water samples.The detection limit was 1×102 CFU/mL.In addition,90 samples of fresh water and 90 samples of direct drinking water were tested.The results showed that Ct values of 3 of the 90 samples were ≤35,indicating that P.aeruginosa was detected.No C.perfringens and P.aeruginosa were detected in 90 direct drinking water samples and other water samples.In summary,the TaqMan real-time quantitative PCR detection method for C.perfringens and P.aeruginosa and the TaqMan dual real-time quantitative PCR detection method established in this study have good specificity,high sensitivity,convenient operation,rapid economy,and time saving.It can provide technical support for the detection of these two bacteria in water and the prevention and control of related diseases.