Study Of Spoilage Microorganisms In Food For Medical Purpose And Its Detection Method Based On Digital PCR

Food spoilage microorganisms are the general term for microorganisms that can cause physical or chemical changes in food and cause food to lose its original quality.In recent years,with the rapid development of the medical food industry,there has been an increasing demand for its testing.In this study,we first screened and identified spoilage microorganisms in medical foods through traditional culture methods,and analyzed the microbial diversity of three special medical foods and two enteral nutrients during storage at different temperatures based on high-throughput sequencing technology.Finally,digital PCR technology was used to establish a high-efficiency and sensitive detection method for Enterococcus faecalis and Enterococcus hirae in medical foods.(1)Using a combination of traditional culture and 16S rRNA sequencing methods,21 different commercially available medical foods were separated and identified for spoilage microorganisms.The results show that Bacillus is the dominant bacterial group in the spoilage of medical food.Lactobacillus,Pediococcus,Staphylococcus and Enterococcus have also been detected;(2)Analyze the bacterial microbial diversity of medical foods at different storage times and temperatures.The storage temperature and storage time have a greater impact on the diversity of bacteria in enteral nutrition.There are different abundances of Bacillus and Lactobacillus in all samples;(3)Design specific primers based on 16S rRNA gene of Enterococcus faecalis,and select a set of best primer probes to optimize temperature and reaction system.Enterococcus faecalis.The real-time quantitative PCR method was used to detect plasmid standards of different copy numbers,and the detection limit was only 1.22×10~3 copies/?L.Based on real-time quantitative PCR reaction conditions,a digital PCR detection method was established to detect different concentrations of DNA and simulated contaminated samples.The literature shows that the digital PCR method can be an order of magnitude better than the real-time fluorescent quantitative PCR method,and it does not require standard products and standard curves,which is more convenient;(4)According to the gyrb gene of Enterococcus hirae,three sets of specific primers and probes are designed.Using real-time quantitative PCR method to detect plasmid standards of different copy numbers,the detection limit can only reach 3.89×10~3copies/?L;for different concentrations of DNA and simulated contamination samples,the accurate quantitative detection limit is 10~3 CFU/g.Based on the real-time quantitative PCR reaction conditions,a droplet digital PCR detection method was established.The accurate quantitative concentration can reach 3.89×10~2 copies/?L,the detection limit of simulated contamination samples is 10~2 CFU/g.