Rapid detection primer, probe and kit for Listeria monocytogenes in food and application of rapid detection primer, probe and kit

Abstract

The invention provides an RPA primer, a probe, a kit and a detection method for detecting listeria monocytogenes in food. According to the invention, primers are designed and optimized for the hlyA gene of Listeria monocytogenes. The designed primer can effectively detect the listeria monocytogenes from different food matrixes and has excellent specificity, sensitivity and inclusiveness, and the primer and the detection method can be used for rapid detection of the listeria monocytogenes in food.

Description

Technical Field

[0001] The invention belongs to the field of rapid microbial detection, and in particular relates to rapid detection of Listeria monocytogenes in food. Background Art

[0002] Listeria monocytogenes (Listeria monocytogenes), also known as Listeria monocytogenes, is the only zoonotic pathogen in the genus Listeria. It is a Gram-positive facultative anaerobic intracellular parasite that is commonly found in food. Humans, livestock, and poultry can all be infected and cause listeriosis. Under the action of biofilm, Listeria monocytogenes can grow and survive under extreme conditions, and is resistant to high salt, acid, and alkali. Because of its strong resistance to environmental and physical and chemical factors, it can be widely present in nature. Because Listeria monocytogenes can grow and reproduce at 4°C, it is also one of the main pathogens that threaten human health in cold chain foods. Food is an important source of infection. Therefore, it is possible for food to be contaminated with Listeria monocytogenes to varying degrees during pre-processing, transportation, and storage. When people consume contaminated food, they may develop corresponding listeriosis and experience a series of clinical reactions such as gastroenteritis, meningitis, sepsis, fetal abortion, monocytosis, etc. It may cause illness in all age groups, among which newborns, the elderly, pregnant women, and those with weakened immune systems have the highest incidence rates.

[0003] At present, the World Health Organization (WHO) lists Listeria monocytogenes as one of the four major foodborne pathogens. With the development of the economy and the faster pace of life, the consumption ratio of cold chain food is increasing. Since Listeria monocytogenes can still survive in low temperature environments and is very difficult to be completely eliminated in the production environment, the risk of Listeria monocytogenes in cold chain foods is increasing. Therefore, the monitoring of Listeria monocytogenes in cold chain foods is becoming increasingly important. The current detection methods mainly include traditional identification methods, molecular detection methods, and immunological detection methods. Among them, the traditional identification method is time-consuming and difficult to meet the needs of rapid biological risk monitoring in food. The downstream detection speed of molecular detection methods and immunological detection methods is very fast, but it is still difficult to break through the technical bottleneck of long detection time. Research on the detection of Listeria monocytogenes using various methods has also been carried out at home and abroad. In 2023, Guo Rui (Guo Rui et al., Establishment of a method for rapid detection of foodborne Listeria monocytogenes based on fluorescent probe recombinase-mediated isothermal amplification technology, Food Safety Guide. 2023, (32), 101-104+108.), et al. established a method for rapid detection of Listeria monocytogenes based on fluorescent probe recombinant enzyme mediated isothermal nucleic acid amplification (RAA) technology. Specific primers and probes were designed for the conserved sequence of the hlyA gene of Listeria monocytogenes. The established fluorescent RAA method can complete the detection of sample nucleic acid at 39°C within 20 minutes. The minimum detection limit of bacterial liquid nucleic acid is 10 3CFU / mL, and no cross-reaction with other strain nucleic acids. Chen Dawei (Chen Dawei et al., Establishment of a rapid detection method for Listeria monocytogenes based on RPA / CRISPR-Cas 12a technology, Meat Research. 2023, 37(09), 46-51.) et al. established a rapid detection method for Listeria monocytogenes based on RPA / CRISPR-Cas12a technology, selected the Listeria monocytogenes lysin virulence gene hly as the detection target gene, and used a visual fluorescent reporter system to determine the amplification results. At 39°C, the detection of Listeria monocytogenes can be completed within 30 minutes, and the detection limit of artificial simulated contaminated food salmon fillets can reach 10CFU / mL. Wang Fengjiao (Wang Fengjiao et al., Study on the detection method of multiple recombinase polymerase amplification for three common foodborne pathogens, Journal of Food Safety and Quality. 2021, 12(20), 8121-8127.) et al. established a rapid detection method for multiple recombinase polymerase amplification (RPA) of Salmonella, Listeria monocytogenes and Bacillus cereus in food. The Salmonella invA gene, Listeria monocytogenes hlyA gene and Bacillus cereus 16S RNA sequence were selected as target genes for amplification. The amplification of the three target genes was completed at 35°C in 20 minutes. The sensitivity of Salmonella, Listeria monocytogenes and Bacillus cereus was 2.70×10 5 , 1.30×10 5 , 1.44×10 4 CFU / mL, which can be used for the detection of artificially contaminated samples and actual samples. Huang Xinxin (Huang Xinxin et al., Detection of Listeria monocytogenes in food by fluorescent recombinase-mediated isothermal amplification, Bulletin of Microbiology. 2021, 48(12), 4989-5000.) et al. established a fluorescent recombinase-mediated isothermal amplification method to detect Listeria monocytogenes, and designed specific primers and probes based on the conserved region of the hlyA gene. The established fluorescent RAA method was applied to the detection of food matrices and actual samples. The optimal reaction temperature of the rapid detection method is 42°C, and the detection reaction is completed within 30 minutes. The sensitivity of pure bacteria reaches 3×10 2 CFU / mL. He Jie (He Jie et al., Establishment of a recombinase polymerase amplification detection method for Listeria monocytogenes in food. Chinese Journal of Food Hygiene 2021, 33(03), 274-278.) et al. established a recombinase polymerase amplification (RPA) detection system for Listeria monocytogenes in food, and designed specific strong primers and probes for the specific gene hlyA. The established RPA method only takes 30 minutes to react at 37°C, and can specifically detect Listeria monocytogenes, with the minimum template concentration as low as 0.5ng / μL. Artificial bacterial infection experiments show that the detection sensitivity can reach 10 5CFU / reaction. Du (Du, XJ et al., Recombinase Polymerase Amplification Combined with Lateral Flow Strip for Listeria monocytogenes Detection in Food. J Food Sci 2018, 83 (4), 1041-1047.) et al. established a rapid detection technology for Listeria monocytogenes based on RPA-LF (RPA-lateral flow strip). For the sensitivity test of the standard strain, the detection experiment can be completed at 39°C in 15 minutes, with a sensitivity of 300 fgDNA or 15 CFU / reaction. After 6 hours of bacterial enrichment, the sensitivity can reach 1.5 CFU / reaction. Wang (Wang, L et al., Rapid and Specific Detection of Listeria monocytogenes With an Isothermal Amplification and Lateral Flow Strip Combined Method That Eliminates False-Positive Signals From Primer-Dimers. Front Microbiol 2019, 10, 2959.) et al. established a rapid detection technology for Listeria monocytogenes based on RPA-LF (RPA-lateral flow strip). At 37°C and 42°C, 100 fg / 50 μL can be detected in 25 minutes. 10 CFU of positive strains were inoculated in 25 g of sample, and it could be detected after 6 hours of bacterial growth. Xiao (Xiao, Y et al., A rapid and inexpensive nucleic acid detection platform for Listeria monocytogenes based on the CRISPR / Cas12a system. Talanta 2023, 259, 124558.) et al. established a rapid detection technology for Listeria monocytogenes based on CRISPR / Cas12a, which can complete the rapid detection of Listeria monocytogenes within 25 minutes, and the detection sensitivity can reach 4.4 CFU / g.Sarah Azinheiro (Azinheiro, S. et al., Application of Recombinase Polymerase Amplification with Lateral Flow for a Naked-Eye Detection of Listeria monocytogenes on Food Processing Surfaces. Foods 2020.9(9).) et al. established an RPA-LF (RPA-lateral flow test strip) rapid test method to detect Listeria monocytogenes in food production environments. After 24 hours of bacterial growth, the sensitivity can reach LOD using bacterial count technology. s0 The value is 4.2 CFU / cm 2 LOD 95 The value is 18.2 CFU / cm 2. Chen (Chen, K. et al., A Rapid and Sensitive Europium Nanoparticle-Based Lateral Flow Immunoassay Combined with Recombinase Polymerase Amplification for Simultaneous Detection of Three Food-Borne Pathogens. Int J Environ Res Public Health 2021, 18(9).) et al. established a rapid detection technology based on multiple RPA-LF (RPA-lateral flow test strip), which can simultaneously detect Vibrio parahaemolyticus, O157:H7, and Listeria monocytogenes. The detection sensitivities are 9.0 CFU / mL for Vibrio parahaemolyticus, 7.0 CFU / mL for Vibrio parahaemolyticus, and 4 CFU / mL for Listeria monocytogenes. Jin (Jin, B. et al., Simultaneous Detection of Five Foodborne Pathogens Using a Mini Automatic Nucleic Acid Extractor Combined with Recombinase Polymerase Amplification and Lateral Flow Immunoassay. Microorganisms 2022, 10 (7).) et al. established a rapid detection method for these species of pathogens based on multiple RPA-LF (RPA-lateral flow test strip) technology, targeting the nuc gene of Staphylococcus aureus, the toxR gene of Vibrio parahaemolyticus, the rfbE gene of Escherichia coli O157:H7, the hlyA gene of Listeria monocytogenes, and the fimY gene of Salmonella as target fragments. The RPA test showed the best performance at 37°C for 15 minutes. The detection limit of the optimized RPA-LFIA can reach 10 1 CFU / mL. There was no cross-reaction among the forty-eight strains. In addition, the average recovery in the spiked food samples was 90.5-104.5%.

[0004] In addition, CN105671197A discloses a method for detecting foodborne pathogenic bacteria Listeria monocytogenes, using the hlyA gene as a target to design primers and probes, combining RPA and lateral flow (LFD) test strip technology for detection, and the detection minimum line can reach 10pg / μL, and the test results can be obtained in about 20 minutes. CN108034737A discloses RPA primers and probes and a detection method for detecting Listeria monocytogenes, using the actA gene as a target to design primers and probes, and the detection lower limit can reach 18cfu / reaction, and has good specificity. CN108148894A discloses a method and application of a recombinase polymerase isothermal amplification combined with a test strip to detect Listeria monocytogenes, which selects the hlyA gene as a detection target gene, designs a primer pair specific to Listeria monocytogenes, uses Listeria monocytogenes genomic DNA as a template, performs specific isothermal amplification, and applies a colloidal gold test strip to obtain the test results. The method only takes 10 minutes for constant temperature amplification at 39°C, and the colloidal gold test strip obtains the test result within 5 minutes, and its detection line is 300fg. CN110878338A discloses RPA primers, RPA kits and their applications for detecting Listeria monocytogenes. Primers are designed based on the hlyA gene of Listeria monocytogenes, and the reaction time is only 40 minutes.

[0005] However, the detection matrix in the food field is complex, and the sugar, salt, and oil components in food have a great influence on the sensitivity of PCR detection (Schrader, C. et al., PCR inhibitors-occurrence, properties and removal. Journal of Applied Microbiology, 113 (5), 1014-1026.). Therefore, evaluating the influence of different food matrices on reaction sensitivity is an important technical component for developing rapid detection methods for food matrices. Most of the above documents use standard strains or artificially constructed contaminated samples for detection, and none of them discloses the effective detection of Listeria monocytogenes in complex food matrices, nor does it provide a rapid detection technology solution with excellent specificity, sensitivity and tolerance. Summary of the invention

[0006] Based on the principle of fluorescent probe-RPA detection technology, the present invention designs specific primers and probes for Listeria monocytogenes, and verifies the inclusiveness, exclusivity and sensitivity of the primers and probes according to relevant standard requirements; in order to break through the technical bottleneck of too long pre-enrichment time, the present invention optimizes the pre-enrichment culture conditions of Listeria monocytogenes, and uses shaking culture to shorten the pre-enrichment time to 8 hours; according to the relevant requirements of GB29921-2021 National Food Safety Standard "Limits of Pathogenic Bacteria in Pre-packaged Foods", frozen drinks (ice cream, popsicles, popsicles), dairy products (cheese), meat products (sauced pork elbows, sausages, refrigerated chicken breasts), ready-to-eat fresh-cut fruits and vegetables (strawberries, fresh-cut vegetables) and ready-to-eat aquatic products (seafood rice balls, salmon, Arctic scallops, sweet shrimps) are selected as food matrices to artificially contaminate samples. The contaminated samples were tested using the GB 4789.30-2016 national food safety standard "Food Microbiology Test for Listeria Monocytogenes" and the rapid test method established by the present invention. The detection limit LOD of the rapid test method of the present invention was 50 The detection limit of Listeria monocytogenes is 2-6 CFU / mL (different food matrices have a slight effect on the detection limit of the method). Overall, the rapid detection method established by the present invention can complete the rapid detection of Listeria monocytogenes within 20 minutes after 8 hours of pre-enrichment, and the detection limit is no more than 2.5 times that of the national standard method GB4789.30, which fully complies with the relevant requirements of GB4789.45-2023 "General Rules for Validation of National Standard Microbiological Test Methods for Food Safety".

[0007] In order to realize the detection of Listeria monocytogenes in food, the present invention provides an RPA primer for detecting Listeria monocytogenes in food, wherein the upstream primer sequence is 5'-AACTGGAGCGAAAACAATAAAAGYAAGCTAG-3', and the downstream primer sequence is 5'-CCGGTCATCAATTACCGTTCTCCACCATTCC-3'. Preferably, the 5' end of the upstream primer is modified with biotin.

[0008] The present invention also provides an RPA probe for detecting Listeria monocytogenes in food, wherein the sequence of the probe is

[0009] 5'-CGTCCATCTATTTGCCAGGTAACGCRAGAAA[FAM-dt]A[THF][BHQ1-dt]

[0010] AATGTTTACGCYAAAG[C3 Spacer]-3';

[0011] Among them, FAM-dt is a fluorescent group labeled with FAM; THF is a tetrahydrofuran residue; BHQ1-dt is a fluorescence quenching group; C3 Spacer is a C3 blocking group, wherein Y is C / T, and R is A / G.

[0012] The present invention also provides an RPA kit for detecting Listeria monocytogenes in food, which comprises the aforementioned RPA primers for detecting Listeria monocytogenes, and more preferably, further comprises the aforementioned RPA probe.

[0013] The present invention also provides an RPA composition for detecting Listeria monocytogenes in food, wherein the composition comprises the RPA primers and the RPA probes.

[0014] At the same time, the present invention also provides a method for detecting Listeria monocytogenes in food, which comprises the following steps:

[0015] (1) extracting genomic DNA of the sample to be tested;

[0016] (2) preparing an RPA reaction system, which comprises the aforementioned RPA composition for detecting Listeria monocytogenes in food and the genomic DNA of the sample to be tested extracted in step (1);

[0017] (3) performing an amplification reaction on the reaction system configured in step (2) at a constant temperature;

[0018] (4) Determine whether the genomic DNA of the sample to be tested contains Listeria monocytogenes based on the amplification reaction results.

[0019] Preferably, the amplification reaction result is determined based on the fluorescence signal value.

[0020] Preferably, the RPA probe is added to the method for fluorescence signal detection. More preferably, a pre-enrichment step is performed before step (1), and the enrichment step is performed by shaking culture. More preferably, the shaking culture time is more than 8 hours, preferably 8 hours.

[0021] Furthermore, the food is selected from frozen drinks, dairy products, meat products, ready-to-eat fresh-cut fruits and vegetables or ready-to-eat seafood.

[0022] At the same time, the present invention also provides an RPA reaction system, wherein the reaction system is:

[0023] RPA reaction buffer: 29.4 μL

[0024] The upstream primer (10 μM) 2 μL

[0025] The downstream primer (10 μM) 2 μL

[0026] The probe (10 μM) 0.6 μL

[0027] ddH2O 12.5μL

[0028] Mg 2+ 2.5μL

[0029] Template: 1 μL.

[0030] Preferably, Mg in the system 2+ The final concentration is 2 to 15 mM, more preferably 14 mM. 2+ It can be a magnesium chloride solution or a magnesium acetate solution, preferably a magnesium acetate solution.

[0031] The positive and progressive effects of the present invention are:

[0032] 1. The primers and probes designed by the present invention have excellent specificity and tolerance.

[0033] 2. The results of the present invention show that different food matrices have a slight effect on the sensitivity of the rapid detection method. When designing primers and probes at different sites on the same target, the amplification efficiency is completely different. When designing multiple sets of primers and performing primer sensitivity tests, the present invention selects primers and probes with excellent sensitivity, specificity and tolerance, and tests them with standard strains. The LOD 50 2.2x10 4 CFU / mL.

[0034] 3. The validation of the rapid test method established in the present invention is in full compliance with the relevant requirements of GB4789.45-2023 "General Rules for Validation of National Standard Microbiological Test Methods for Food Safety", and the selected food matrix is ​​relatively complete.

[0035] 4. The rapid test method established by the present invention can complete the whole process from sample collection to test completion within 9 hours (enrichment + testing). The RLOD of the rapid test method and the national standard test method is no higher than 2.5, which fully complies with the relevant requirements of GB4789.45-2023 "General Rules for Validation of National Standard Microbiological Test Methods for Food Safety". BRIEF DESCRIPTION OF THE DRAWINGS

[0036] Figure 1A and Figure 1B : Inclusive testing of RPA primers and probes for Listeria monocytogenes detection: Figure 1A The first set of primers and probes were inclusive; Figure 1B The second set of primers and probes are inclusive.

[0037] Figure 2A and Figure 2B : Exclusivity testing of RPA primers and probes for Listeria monocytogenes detection: Figure 2AExclusivity testing of the first set of primers and probes; Figure 2B Exclusivity test of the second set of primers and probes.

[0038] Figure 3A and Figure 3B :Sensitivity testing of RPA primers and probes for detection of Listeria monocytogenes: Figure 3A Exclusivity testing of the first set of primers and probes; Figure 3B The second set of primers and probes are tested for exclusivity. Figure 3A :(1)5x10 6 CFU / mL; (2) 5x10 5 CFU / mL; (3) 5x10 4 CFU / mL; Figure 3B :(1)5x10 6 CFU / mL; (2) 5x10 5 CFU / mL; DETAILED DESCRIPTION

[0039] The present invention will be further described in detail below in conjunction with the accompanying drawings and embodiments. It is to be understood that the specific embodiments described herein are only used to explain the present invention, rather than to limit the present invention. It should also be noted that, for ease of description, only the parts related to the present invention are shown in the accompanying drawings, rather than all.

[0040] Example 1: DNA extraction

[0041] 1.1 DNA extraction methods for samples without food matrix

[0042] Use standard strains to streak culture on Listeria monocytogenes chromogenic medium, pick characteristic colonies, dilute in phosphate buffer, take 1mL of bacterial solution, centrifuge at 12000g for 10min to collect bacteria, and remove supernatant. Add 100μL of pathogenic bacteria nucleic acid extraction and lysis solution (Yizhi Technology, YZV0802) to the precipitate, boil at 100℃ for 5min. Centrifuge at 1000g for 3min, take supernatant, use as RPA amplification template, and store at -20℃ for later use.

[0043] 1.2 DNA extraction methods for Listeria monocytogenes in food matrices

[0044] Weigh 25g of sample into a conical flask containing 225mL of LB1 enrichment solution and culture at 180rpm for 4-8h. After enrichment, take 1mL of enrichment solution and centrifuge at 500rpm for 1min to precipitate food impurities and take the supernatant. Centrifuge at 12000rpm for 10min, take the precipitate to obtain bacteria. After discarding the supernatant, add lysis solution (Yizhi Technology, YZV0802) to the precipitate and boil at 100℃ for 5min. Centrifuge at 1000g for 3min, take the supernatant as RPA amplification template and store at -20℃ for later use.

[0045] Example 2: Design of primers and probes

[0046] The template sequences used in the primer design of the present invention are all from the National Center of Biotechnology Information (NCBI) database, primer express software was used to design primers. The gene hlyA (FJ030606.1) in the NCBI database was selected as the target gene for primer and probe design. Primer express software was used to design primers, and the designed primers and probes were screened. Finally, two pairs of primers and probes that can be used for RPA amplification detection of Listeria monocytogenes were obtained. The sequences of the primers and probes are shown in Table 1.

[0047] Table 1 Primer and probe sequences for RPA detection of Listeria monocytogenes

[0048]

[0049] Example 3: Reaction system and reaction conditions

[0050] The RPA amplification reaction was carried out using a fast isothermal amplification reagent (EXO-probe) (YZL0103, Yizhi Technology). According to the configuration requirements of the amplification reagent, the specific reaction system and reaction conditions are as follows: template 1μL, RPA reaction buffer 29.4μL, upstream primer (10μM) 2μL, downstream primer (10μM) 2μL, probe (10μM) 0.6μL, ddH2O 12.5μL, magnesium acetate solution 2.5μL, of which Mg 2+ The final concentration is 14 mM. After adding the system, mix well, centrifuge, react at 39°C for 20 min, and collect FAM fluorescence signals every 30 s.

[0051] Example 4: Inclusion Test

[0052] According to the "GB4789.45-2023 National Food Safety Standard Microbiological Test Method Validation General Rules", the designed primers and probes were subjected to inclusive test experiments. Four strains of Listeria monocytogenes (ATCC19115) and Listeria monocytogenes (CICC21633) were selected, and 22 strains of Listeria monocytogenes isolated from food were selected for inclusive verification experiments. During the inclusive test, the inoculation level was approximately 100 times the minimum detection level of "GB 4789.30-2016 National Food Safety Standard Listeria monocytogenes Test".

[0053] Four strains of Listeria monocytogenes (ATCC19115) and four strains of Listeria monocytogenes (CICC21633) were selected, as well as 22 strains of Listeria monocytogenes isolated from food, for a total of 30 strains for inclusion testing. Figure 1A and Figure 1B As shown, the primers and probes obtained by screening showed good inclusiveness.

[0054] Example 5: Exclusivity Test

[0055] According to the "GB4789.45-2023 National Food Safety Standard Microbiological Test Method Validation General Rules", bacterial species closely related to Listeria monocytogenes were selected for exclusive experiments, and 30 strains of non-target microorganisms were selected for the exclusive experiment.

[0056] The samples were ① Listeria stutzeri (CICC21671), ② Listeria ivanovii (CICC21663), ③ Listeria innocua (ATCC33090), ④ Listeria grayi (CICC21670), ⑤ Listeria welshmanii (CICC21672), ⑥ Bifidobacterium infantis (CICC6069), ⑦ Lactobacillus plantarum (CGMCC1.1856), ⑧ Lactobacillus casei (CGMCC1.2435), ⑨ Pseudomonas aeruginosa (ATCC27853), ⑩ Staphylococcus aureus (ATCC25923), Salmonella Enteritidis (ATCC13076), Streptococcus faecalis (ATCC29212), Vibrio parahaemolyticus (ATCC17802), Pseudomonas putida (CGMCC1.2309), Two strains of Pseudomonas fluorescens (CICC20066) were used to perform exclusive tests on the primers and probes for Listeria monocytogenes obtained by screening. The results are as follows Figure 2A and Figure 2B The primers and probes screened have good exclusivity and have no cross-reaction with all the test strains mentioned above.

[0057] Example 6: Sensitivity test

[0058] The standard strain was used as the test object. According to GB4789.45, 24 samples were used for sensitivity test and the sensitivity was calculated. The specific process is as follows: Listeria monocytogenes (ATCC19115) was used to prepare bacterial suspension and the McFarland turbidity of the bacterial suspension was adjusted to 1. The bacterial suspension was diluted 10 times and the colony count of the dilution was performed. 10 samples were taken respectively. -1 ~10 -10 1 mL of diluent was centrifuged at 12000 g for 10 min, the supernatant was removed, and lysis solution (YZV0802, Yizhi Technology) was added to prepare the RPA template, and the RPA experiment was carried out according to the reaction system and reaction conditions of Example 3. After selecting the appropriate concentration of bacterial lysis solution and carrying out 24 test reactions in parallel, the LOD was calculated using formula (1) 50 Calculation of the detection level at which 50% positive results are expected at a certain contamination level.

[0059]

[0060] In order to test the sensitivity of the primers and probes obtained by screening, Listeria monocytogenes (ATCC19115) was picked up and added to phosphate buffer to prepare a bacterial suspension with a McFarland turbidity of 1. The bacterial suspension was serially diluted 10 times, and the dilutions were counted on the plate. Different concentrations of dilutions were used to prepare RPA templates and perform sensitivity tests. The test results are shown in Figure 3A and Figure 3B The first pair of primers and probes, when the bacterial concentration is 5x10 4 CFU / mL, which is close to the sensitivity limit of RPA amplification reaction. The sensitivity of the second pair of primer probes is slightly lower. When the bacterial concentration is 5x10 5 CFU / mL, it is close to the sensitivity limit of the RPA amplification reaction.

[0061] To further determine the LOD of primers and probes 50 The first set of primers and probes selected a bacterial concentration of 2.5x10 4 CFU / mL bacterial lysate was used as the template for RPA amplification. 24 RPA experiments were carried out in parallel, of which 13 were positive. According to formula 1, the LOD 50 The value is 2.2x10 4 CFU / mL. The test bacterial concentration of the second set of primers and probes was 2.5x10 5 CFU / mL, 24 RPA experiments were carried out in parallel, of which 17 were positive. According to formula 1, the LOD 50 The value is 2.84x10 5Therefore, the first set of primers was used in the subsequent examples to conduct sensitivity tests in food matrices.

[0062] Example 7: Sensitivity testing in food matrices

[0063] According to the relevant requirements of GB 29921-2021 National Food Safety Standard "Limits of Pathogenic Bacteria in Prepackaged Foods", frozen drinks, dairy products, meat products, ready-to-eat fresh-cut fruits and vegetables, and ready-to-eat aquatic products need to be tested for Listeria monocytogenes. According to GB 4789.45-2023 "General Rules for Validation of Microbiological Testing Methods in National Food Safety Standards", "If the scope of application of the method to be verified is "food", at least 5 types of food should be selected. If the scope of application is less than 5 types of food, all should be selected, and at least 1 food subcategory should be selected for each type of food". The present invention selects ice cream, popsicles, popsicles (frozen drinks), cheese (dairy products), sauced pork elbows, sausages, refrigerated chicken breasts (meat products), strawberries, fresh-cut vegetables (ready-to-eat fresh-cut fruits and vegetables), salmon, Arctic clams, and sweet shrimps (ready-to-eat aquatic products) as food matrices. After sterilization, Listeria monocytogenes is inoculated at a concentration of 1 to 10 CFU / mL to prepare artificially contaminated samples.

[0064] After the food was sterilized, 25 g of the food sample was weighed into a sterile Erlenmeyer flask containing 225 mL of LB1 enrichment solution, and an appropriate amount of the standard strain of Listeria monocytogenes (ATCC19115) was added. After rotating and culturing at 200 rpm for 2 h, 4 h, 6 h, and 8 h, 1 mL of the enrichment solution was taken, centrifuged at 12000 rpm for 10 min, the supernatant was removed, and 200 μL of lysis solution (YZV0802, Yizhi Technology) was added to the precipitate to prepare the RPA template. The first set of primers and probes were used, and the RPA experiment was carried out according to the reaction system and reaction conditions of Example 3. Twenty detection experiments were carried out in parallel, and control tests were carried out according to GB 4789.30-2016 "National Food Safety Standard Food Microbiological Examination Listeria Monocytogenes Test". Then, according to "GB4789.45-2023 National Food Safety Standard Microbiological Examination Method Validation General Rules", the RLOD (relative detection level) was calculated according to formula (2) to evaluate the rapid screening technology of the present invention.

[0065]

[0066] The results showed that the RLOD values ​​detected after 2-6 hours of shaking culture were all higher than 2.5, which did not meet the relevant requirements of GB4789.45-2023 "General Principles for Validation of Microbiological Testing Methods for National Food Safety Standards". Shaking culture for 8 hours is the shortest bacterial growth time, so the present invention adopts shaking culture for 8 hours for detection.

[0067] The detection sensitivity of the rapid detection method on different food matrices was tested according to the operation of this embodiment. At the same time, parallel detection experiments were carried out on the samples according to the national standard GB 4789.30-2016 "National Food Safety Standard Food Microbiology Inspection Listeria Monocytogenes Inspection", and the sensitivities of the two detection methods were compared and analyzed. The two detection results are shown in Table 3.

[0068] Table 3 Analysis of sensitivity test results of the rapid screening method for Staphylococcus aureus in food matrices

[0069]

[0070] It can be seen from Table 3 that the rapid test method established in the present invention can complete the whole process from sample collection to detection within 9 hours (enrichment + detection), and the RLOD of the rapid test method and the national standard test method is not higher than 2.5, which fully complies with the relevant requirements of GB4789.45-2023 "General Rules for Validation of National Standard Microbiological Test Methods for Food Safety".

[0071] The above content is a further detailed description of the present invention in combination with a specific preferred embodiment. It cannot be determined that the specific embodiments of the present invention are limited to this. For ordinary technicians in the technical field to which the present invention belongs, several simple deductions or substitutions can be made without departing from the concept of the present invention, which should be regarded as belonging to the protection scope of the present invention determined by the submitted claims.

Claims

1. An RPA primer for detecting Listeria monocytogenes in food, characterized in that: The upstream primer sequence is 5'-AACTGGAGCGAAAACAATAAAAGYAAGCTAG-3'; the downstream primer sequence is 5'-CCGGTCATCAATTACCGTTCTCCACCATTCC-3'.

2. The primer according to claim 1, characterized in that: The 5' end of the upstream primer is modified with biotin.

3. An RPA probe for detecting Listeria monocytogenes in food, characterized in that: The sequence of the probe is 5'-CGTCCATCTATTTGCCAGGTAACGCRAGAAA[FAM-dt]A[THF][BHQ1-dt]AATGTTTACGCYAAAG[C3Spacer]-3'; Among them, FAM-dt is the fluorescent group labeled with FAM; THF is the tetrahydrofuran residue; BHQ1-dt is the fluorescence quenching group; C3Spacer is the C3 blocking group.

4. An RPA kit for detecting Listeria monocytogenes in food, characterized in that: It contains the primer as claimed in claim 1.

5. The kit according to claim 4, characterized in that: The kit further comprises the probe according to claim 3.

6. An RPA composition for detecting Listeria monocytogenes in food, characterized in that: The composition comprises the RPA primer according to claim 1 or 2 and the RPA probe according to claim 3.

7. A method for detecting Listeria monocytogenes in food, comprising the following steps: (1) extracting genomic DNA of the sample to be tested; (2) preparing an RPA reaction system, which comprises the composition of claim 6 and the genomic DNA of the sample to be tested extracted in step (1); (3) performing an amplification reaction on the reaction system configured in step (2) at a constant temperature; (4) Determine whether the genomic DNA of the sample to be tested contains Listeria monocytogenes based on the amplification reaction results.

8. The method according to claim 7, characterized in that: It further includes: Before step (1), a pre-enrichment step is performed, wherein the enrichment step is performed by shaking culture, and the shaking culture time is more than 8 hours.

9. The method according to claim 7 or 8, characterized in that: The food is selected from frozen drinks, dairy products, meat products, ready-to-eat fresh-cut fruits and vegetables or ready-to-eat aquatic products.

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