Establishment And Preliminary Application Of Detection Methods Based On CRISPR/Cas12a For Foodborne Psychrotrophic Bacteria

Psychrotrophic bacteria are microorganisms whose optimal growth temperature is about 15°C or below,maximum growth temperature is about 20°C,and minimum growth temperature is about 0°C or below.Among them,Yersinia enterocolitica and Listeria monocytogenes are are important foodborne bacteria,often found in frozen foods.Some Yersinia enterocolitica are pathogenic and cause Yersiniosis,which can lead to intestinal infections and mesenteric lymphadenitis,and can be life-threatening when severe.Listeria monocytogenes is also a common foodborne pathogen,causing mild to severe gastroenteritis,encephalitis and sepsis,and hsa a high mortality rate.The above-mentioned foodborne psychrotrophic bacteria pose a serious challenge to global public health,making the detection of psychrotrophic bacteria contamination in cold chain foods a top priority in food safety prevention and control.Compared to other bacteria,psychrotrophic bacteria can grow at low temperatures and contaminate food ingredients,so rapid,low-cost and sensitive field detection techniques are needed in the production and distribution of refrigerated foods.The existing assays for psychrotrophic bacteria have their advantages,but most tend to be slow,require large equipment and do not provide immediate results.In recent years,the CRISPR/Cas system,consisting of Clustered regularly interspaced short palindromic repeats（CRISPR）and CRISPR associated proteins（Cas）,has been gradually applied to food testing.And the CRISPR/Cas12a protein has become a hot topic of research because of its non-specific ss DNA cleavage activity triggered by cr RNA targeting to its complementary DNA sequence.This light-emitting technology with convenient detection equipment can realize the on-site detection of factory production or market sales,which is convenient for enterprises or regulatory authorities to understand the contamination status of psychrotrophic bacteria in food in time to ensure the hygiene and safety of food.In this study,a rapid and sensitive visual nucleic acid detection method was established using the CRISPR/Cas12a system combined with Recombinase Polymerase Amplification（RPA）technology for in on-site detection of food-borne psychrotrophic bacteria.The details are as follows:1.Quantitative Real-time PCR（qPCR）methods for Yersinia enterocolitica and Listeria monocytogenes were established separately.Under the spiked condition,the qPCR standard curve equation for Yersinia enterocolitica was Y=-3.390X+37.00,with a correlation coefficient（R²）of 0.9986 and a minimum detection limit of 10²CFU/g.The qPCR standard curve equation for the detection of Listeria monocytogenes was established as Y=-3.651X+42.31,with a correlation coefficient（R²）of 0.9796and a minimum detection limit of 10³CFU/g.2.A CRISPR/Cas12a-RPA assay platform pathogenic Yersinia enterocolitica was established.The method targets the highly conserved gene ail and combines the visualization of RPA with the Cas12a system.After optimizing a series of conditions such as the RPA reaction temperature,the primer concentration of 480 n M,reaction temperature of 37°C and reaction time of 15 min were finally determined for this RPA,and the method can complete the detection of target genes in about 45 min.The established assay is highly specific with a minimum detection limit of 10^-6ng/μL for the genome and 1.7 CFU/g for artificially contaminated pork.the method requires only a blue light transilluminator for visualization and is a fast and convenient method for rapid field detection.3.A CRISPR/Cas12a system with a micro-amplification（CRISPR/Cas12a-MA）assay platform was established for Listeria monocytogenes.The method allows amplification and detection of Listeria monocytogenes hly target genes in the same vessel,avoiding possible aerosol contamination.Moreover,by optimizing the RPA amplification volume to 5μL,the amplification reaction system can be efficiently utilized while significantly reducing the system cost.The minimum detection limit of the method was 4.7×10^-9ng/μL for the genome and 4.4 CFU/g for the manually spiked pork,and achieve the detection within 25 min by naked eye observation or microplate reader.The results showed that CRISPR/Cas12a-MA can meet the demand for on-site detection of Listeria monocytogenes in food.4.Investigate the contamination of various types of meat on the market with foodborne psychrotrophic bacteria.In the 47 meat samples tested,the detection rate of pathogenic Yersinia enterocolitica in small colitis was 4.26%,and the detection rate of Listeria monocytogenes was 6.38%.Among them:in 22 pre-prepared meat products,the total detection rate of pathogenic Yersinia enterocolitica and Listeria monocytogenes was 4.54%;in 25 frozen meat products,the total detection rate of pathogenic Yersinia enterocolitica was 4%and the total detection rate of Listeria monocytogenes was 8%.Foodborne psychrotrophic bacteria was detected in all meat products circulating in the market,and it is recommended to strengthen its monitoring and management.In terms of method application,both the qPCR method and the Cas12a-based method showed good accuracy when comparing and analyzing the natural samples.The Cas12a-based assay platform required portable equipment,short detection time and high sensitivity,which was superior to the traditional qPCR method.In summary,two assays for pathogenic Yersinia enterocolitica and two assays for Listeria monocytogenes were established in this study.Both methods showed reliability in sample analysis.And the Cas12a-based method has the advantages of higher sensitivity,more rapid and visualization compared to qPCR.These methods provide support and assurance for the prevention and control of foodborne psychrotrophic bacteria.