Establishment And Application Of Three Foodborne Pathogens Detection Methods Based On Multiplex PCR And SERS Technology

The rapid development of large-scale aquaculture not only solves the needs of human meat,eggs and milk,but also brings serious environmental pollution.The production of a large number of livestock manure pollutants makes the natural environment on which human beings depend to survive threatened.A large number of pathogenic microorganisms contained in livestock and poultry manure can cause the spread of human and livestock infectious diseases through various ways.Salmonella,E.coli,and Staphylococcus aureus are the three main foodborne pathogens.In recent years,there have been many reports of food poisoning caused by the three pathogens.Most of the infections are related to the consumption of contaminated milk,ground beef,water and dairy products.Foodborne diseases are increasingly becoming an important public health problem due to its high incidence and harm to society.Therefore,rapid and accurate monitoring of food-borne pathogens is the control and prevention of human food-borne pathogens one of the most effective ways.In this study,detection methods for E.coli,Salmonella,and Staphylococcus aureus based on multiplex polymerase chain reaction(PCR)and surface-enhanced Raman scattering(SERS)were established by optimizing the detection protocol.These detection methods improve the detection efficiency of food-borne pathogens and play a guiding role in the monitoring of food-borne pathogens.The main research contents are as follows:1.Establishment of detection method for three foodborne pathogens based on multiplex PCRThe specific primers for E.coli,Salmonella and Staphylococcus aureus were designed by targeting the gene sequences of uid A,hil A and clf A,to verify that there was no cross reaction between the primers.The amplification conditions of the optimized multiplex PCR system were annealing temperature 56?,primer concentration 2?mol/L and Taq mix enzyme dosage 12.5?L.Multiplex PCR specificity tests verified that all the positive strains amplified the target band,while none of the negative strains amplified the fragment.The multiplex PCR sensitivity test had a detection limit of 10~4CFU/m L for triple PCR reactions and a minimum detectable DNA amount of 50 pg/?L.2.Establishment of detection method for three foodborne pathogens based on SERSThe silver sol prepared by the optimized sodium citrate reduction method was used as the reinforcing substrate.The ultraviolet-visible-near infrared spectrophotometer detected that the absorption peak of silver colloid was around 410 nm.The transmission electron microscope observed that the silver nanoparticles were spherical,uniform in size,and about 40 nm in particle size.Under the electron microscope,the silver nanoparticles were uniformly wrapped on the surface of the bacteria,which significantly enhanced the detection signal.The acquisition parameters were optimized under a laser with a wavelength of 785 nm.The optimal parameters were laser power of 100 m W,spectrum acquisition time of 60 s,cycle times of 1 time and slit aperture of 200?M.Raman attribution analysis of three bacteria showed that there were 10 distinct Raman peaks in Salmonella ATCC 9150,9 Raman peaks in CMCC 44102 and ATCC 6538.The first two principal components of PCA analysis contained most of the information of spectral data,and the cumulative contribution rate reached 93.8%.The minimum detection limit of E.coli and Staphylococcus aureus by SERS was 10~4CFU/ml,and that of Salmonella was 10~3CFU/ml.3.Application of multiplex PCR and SERS in detection of livestock manure and polluted waterThe reliability of the multiplex PCR method was verified by the detection of the Artificially Polluted samples and the comparison between the genomic extraction of feces and the DNA extraction of enrichment solution.The established multiplex PCR method was used to detect 42 animal and poultry manure collected from the farm.Among pig feces samples,E.coli was detected in 22 swine stool samples and 8 Salmonella were detected.and in 20 chicken stool samples,only E.coli was detected.Multiplex PCR method detected a total of 18 sewage samples,all of which detected E.coli,of which 8 were detected by Salmonella.Staphylococcus aureus was not detected in all samples.The SERS method was established to collect Raman signals.60 samples were detected for E.coli,19for Salmonella and none for Staphylococcus aureus.The detection rate of Salmonella by multiplex PCR was 26.7%.The detection rate of Salmonella by SERS was 31.6%.Conclusion:(1)The multiplex PCR and SERS technology for the specific detection of Salmonella,E.coli and Staphylococcus aureus were successfully established.In clinical applications,it can detect Escherichia coli and Salmonella in feces and sewage in livestock farms.(2)Compared with multiplex PCR,the SERS method established in this study improves the detection limit of Salmonella with higher sensitivity.In clinical applications,the detection rate of Salmonella by SERS technology is higher than that of multiplex PCR method,and SERS only requires the cultured bacteria to perform Raman scanning and result comparison in the liquid pool,which relatively shortens the detection time.