Development And Application Of The CRISPR/Cas13a And RPA-based Methods For Detection Of Listeria Monocytogenes

Listeria monocytogenes is an important food-borne zoonotic pathogen.It has the highest fatality rate among food infection pathogens,averaging up to 30%and is of great significance in food safety.With the development of economy,people have diverse sources of food.Listeria monocytogenes has a strong adaptability to the environment and can grow slowly at 4°C.The biofilm formed is conducive to its permanent pollution.Once humans become infected,they are prone to clinical symptoms such as sepsis,meningitis,and miscarriage,which seriously affect human health.At the same time,related food recalls and environmental sanitation also cause certain economic losses.Since food animals are likely to be contaminated during production,slaughter,and sales,this provides convenient conditions for the colonization,growth and spread of Listeria monocytogenes.The current detection methods for Listeria monocytogenes in food are mainly bacterial culture,biochemical reactions,and serological typing,which are not conducive to the detection of large quantities of samples,and are also time-consuming and labor-intensive.With the rapid development of molecular biology,PCR-based nucleic acid amplification techniques are becoming gradually widely used,which can quickly detect multiple batches of samples,greatly shorten the detection time,and have a higher relative accuracy rate.However,there are still some uncertain factors,such as false positive that interfere with the accuracy of detection.Therefore,more accurate and rapid detection methods are needed in food safety practice to overcome these shortcomings.In this study,a series of PCR-based nucleic acid amplification techniques were developed to detect Listeria monocytogenes in the animal-derived food chain,with the aim of establishing a more accurate and stable detection method than the existing rapid detection methods.The feasibility of the method was validated by using an optimal detection system for Listeria monocytogenes in simulated food samples.The distribution of Listeria Monocytogenes in the production,slaughter and sale of samples was analyzed.And the feasibility of the method was verified by comparison with national standards in the context of obtaining the epidemiological background of foodborne Listeria monocytogenes.A method was developed for the quantitative detection of Listeria monocytogenes by real-time quantitative PCR(q PCR).Using the iap gene of Listeria monocytogenes as the target,the recombinant plasmid was constructed and the standard curve was drawn.The reaction conditions and reaction system of q PCR were optimized,and the sensitivity and specificity of the method were analyzed.The results showed that the CT value of the drawn standard curve has a good linear relationship with the logarithmic value of the plasmid template,and the sensitivity can reach4.68×10² copies/?L;the spiked milk and spiked beef samples were selected for simulation analysis,and the sensitivity can reach 5.34×10¹ CFU/m L and 5.34×10²CFU/g,respectively.In the case of pre-enrichment,raw milk,faeces,raw beef,slaughter knives,cutting boards,frozen fish and frozen pork collected in the processes of breeding,slaughter and sales,etc.was 10.23%(9/88).This method is suitable for rapid batch detection of Listeria monocytogenes,and has the characteristics of high efficiency,accuracy and quantitative analysis.In view of the limitation of the detection of Listeria monocytogenes on site or at the grassroots unit,we then developed a method for the detection of Listeria monocytogenes based on Recombinase Polymerase Amplification(RPA),which optimized the reaction conditions and reaction system.At the same time,the sensitivity,specificity and repeatability of the method were analyzed.The sensitivity can reach 1.5 ng/?L,and it only takes less than 1 h from amplification to result observation.The sensitivity of the method for spiked samples and actual samples is slightly lower than that of q PCR method.But the detection time is shorter than q PCR detection time,and the operation is convenient,as well as the reaction site is not limited to the laboratory and is more meaningful for on-site detection.In view of the fact that PCR methods are prone to false positives and the sensitivity of existing methods cannot meet the needs of food safety testing.A CRISPR/Cas13a-based nucleic acid detection method for Listeria monocytogenes was established in the experiment,combined with RPA isothermal amplification and fluorescent probes,which based on the establishment of the first two rapid detection methods and the advantages of the latest CRISPR precise gene editing.The Cas13a protein was expressed and purified successfully,and cr RNA was designed and purified for the target of Listeria monocytogenes.The Cas13a protein was verified to have detection activity after binding to target.Furthermore,a SHERLOCK-LM detection method was developed.In a 50?L system,a visual detection result can be obtained within 5 minutes by adding a sample at one time.The method is rapid,quantitative,and highly sensitive,with a sensitivity of 1.5×10^-3ng/?L.the method was also compared for the spiked enriched sample and the spiked unenriched sample.The small difference in detection between the two methods suggests that the method is also suitable for food samples without pre-enrichment.The detection results of actual samples are consistent with the q PCR,which further improved the sensitivity and stability of the method,making it highly practical.