Rapid detection primer, probe and kit for listeria monocytogenes in environment 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 an environment. The primer and the probe show good inclusiveness and exclusiveness. Meanwhile, the invention also provides a rapid detection method of the pseudomonas aeruginosa based on the detection primer and the probe. According to the method, the listeria monocytogenes in the environment can be rapidly detected within 15 minutes by adopting a lateral flow test strip.

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 an environment. 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 to cause Listeria monocytogenes contamination to varying degrees during food pre-processing, transportation, and storage. Eating contaminated food may cause corresponding listeriosis, and 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 immunocompromised people have the highest incidence rate. At present, the World Health Organization (WHO) lists Listeria monocytogenes as one of the four major foodborne pathogens.

[0003] With the development of 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 environment and is very difficult to be completely eliminated in the production environment, the risk of Listeria monocytogenes in cold chain food is increasing. Therefore, it is necessary to develop a technical method for rapid detection of Listeria monocytogenes in the environment. Summary of the invention

[0004] The present invention designs and verifies specific primers and probes based on the RPA detection principle, and establishes a lateral flow test strip detection technology for Listeria monocytogenes. The technology has low detection cost and fast detection speed. Without the assistance of large-scale equipment, only simple training of detection personnel is required to complete the detection of Listeria monocytogenes in the environment.

[0005] The present invention provides an RPA composition for detecting Listeria monocytogenes in an environment, wherein the RPA composition comprises upstream and downstream primers and a probe, wherein the upstream primer sequence is 5'-GGATAAGTAAGGCTTTCAAGCTCACTATCCG-3',

[0006] The downstream primer sequence is 5'-CGATGGCAACCCTGGATGACGACGCTCCAC[THF]TGTATAGCTGGTCGGTCGG-3', wherein [THF] is a tetrahydrofuran residue, and the probe sequence is 5'-CGAACAAAGCAGACCTAATAGCAATGTTGAA-3'. Preferably, the 5' end of the upstream primer is modified with biotin.

[0007] The present invention also provides a kit for detecting Listeria monocytogenes in an environment, wherein the kit comprises the RPA composition.

[0008] The present invention also provides a method for detecting Listeria monocytogenes in an environment, which comprises the following steps:

[0009] (1) After applying the environment with a sterile cotton swab, place it in the culture medium for shaking culture;

[0010] (2) collecting the bacterial cells by centrifugation, and performing RPA detection using the RPA composition after lysis;

[0011] (3) Take the amplified product and conduct lateral flow test strip detection, and judge the test result based on the color development.

[0012] Preferably, the shaking culture time is 24-48 hours. Further preferably, the lysis is boiling at 100° C. for 5 minutes.

[0013] The present invention has the following advantages:

[0014] 1. The primers and probes screened by the present invention have good exclusivity and have no cross-reaction with all the tested strains;

[0015] 2. The designed primers and probes have good inclusiveness, and different clones of Listeria monocytogenes can be effectively detected;

[0016] 3. The sensitivity of the designed primer probe is 6.5 X 10 2 CFU / mL, and the detection can be completed in 15 minutes. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 : Specificity test of primers and probes under different reaction times.

[0018] 1. Proteus vulgaris; 2. Listeria welch; 3. Listeria stuartii; 4. Salmonella arizona; 5. Lactobacillus casei; 6. Listeria gestatium; 7. Listeria ivanovii; 8. Blank; 9. Staphylococcus aureus; 10. Bacillus cereus; 11. Pseudomonas aeruginosa; 12. Listeria innocua; 13. Vibrio parahaemolyticus; 14. Bacillus megaterium

[0019] Figure 2 : Inclusive testing of primers and probes

[0020] 1-8 are 8 clones separated from the standard strain; 9-30 are strains of Listeria monocytogenes separated from the sample; 31 is a positive control; 32 is a blank control.

[0021] Figure 3 : Exclusivity test of primers and probes

[0022] 1. Bacillus megaterium (CICC 10324); 2. Vibrio parahaemolyticus (ATCC17802);

[0023] 3. Salmonella Alexandria (ATCC13314); 4. Bacillus cereus (ATCC10876); 5. Listeria grisei (CICC21670); 6. Pseudomonas aeruginosa (ATCC27853); 7. Common Proteobacterium (CMCC49027); 8. Proteobacterium (ATCC 12453); 9. Listeria stuartii (CICC21671); 10. Listeria innocua (ATCC33090); 11. Listeria welshigeni (CICC21672); 12. Lactobacillus casei (CGMCC1.2435); 13. Listeria ivanovii (CICC21663); 14. Staphylococcus aureus (ATCC25923);

[0024] 15. Bifidobacterium infantis (CICC6069) 16. Blank control

[0025] Figure 4 , primer and probe sensitivity testing

[0026] 1. 6.5X10 6 CFU / mL; 2, 6.5X10 5 CFU / mL; 3, 6.5X10 4 CFU / mL; 4, 6.5X10 3 CFU / mL; 5, 6.5X10 2 CFU / mL; 6, 65CFU / mL; 7, 6.5CFU / mL; 8, 6.5CFU / 10mL

[0027] Figure 5 2. Monitoring of Listeria monocytogenes in the production environment of enterprises

[0028] 1-18, Environmental samples taken from enterprises; 19, Blank control; 20, Positive control DETAILED DESCRIPTION

[0029] 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.

[0030] Example 1: DNA extraction

[0031] 1.1 DNA extraction methods for samples without food matrix

[0032] Use standard strains to culture on Baird-Parker lines, pick characteristic colonies, dilute in phosphate buffer, take 1mL of bacterial solution, centrifuge at 12000g for 10min to collect bacteria, and remove the 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 the supernatant as RPA amplification template, and store at -20℃ for later use.

[0033] 1.2 DNA extraction method of Listeria monocytogenes in the environment

[0034] Use a sterile cotton swab to smear the countertop and other areas that are in direct contact with food. Place the smeared cotton swab in 10mL of PBS solution and shake for 1 minute to fully suspend the bacteria on the cotton swab in the PBS solution. Centrifuge the PBS solution at 8000rpm for 10min to precipitate the bacteria. Discard the supernatant, add 100μL of pathogenic bacteria nucleic acid extraction and lysis solution (Yizhi Technology, YZV0802) to the precipitate, and boil at 100℃ for 5min. Centrifuge at 1000g for 3min, take the supernatant as the RPA amplification template, and store at -20℃ for later use.

[0035] Example 2: Design of primers and probes

[0036] The template sequences used in designing primers were all from the National Center for Biotechnology Information (NCBI) database, and primer express software was used to carry out primer design.

[0037] For act A (JN703930.1), more than 10 pairs of primers and probes were synthesized. After verification, a total of 4 pairs of primers and probes could successfully identify Listeria monocytogenes. After repeated verification, the pair of primers and probes with the best working performance were selected for subsequent experiments. The upstream sequence of the primers screened was 5'-GGATAAGTAAGGCTTTCAAGCTCACTATCCG-3',

[0038] The probe sequence is 5'-CGATGGCAACCCTGGATGACGACGCTCCAC[THF]TGTATAGCTG GTCGGTCGG-3', wherein [THF] is a tetrahydrofuran residue, and the downstream sequence of the primer is 5'-CGAACAAAGCAGACCTAATAGCAATGTTGAA-3'.

[0039] Example 3: Reaction system and reaction conditions

[0040] The RPA amplification reaction was carried out using the fast constant temperature amplification reagent (NFO) (YZL0110 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, ddH 2 O 12.5μL, magnesium acetate solution 2.5μL, including Mg 2+ The final concentration is 14mM. After adding the system, mix well, centrifuge, and react at 36℃ for 15-60mins. After the reaction is completed, take 10μL and add 190μL ddH 2 O in a centrifuge tube, mix well, take 100 μL and drop it into the sample well of a colloidal gold test strip (YZL0111 Yizhi Technology), and read the test result after 5 minutes.

[0041] In order to further improve the sensitivity of the test strip, the amplification reaction time was extended from 15 min to 30 min. The experimental results are as follows Figure 1 As shown, increasing the reaction time will destroy the specificity of the reaction, causing the primers and probes to cross-react with Listeria monocytogenes, Staphylococcus aureus, and Pseudomonas aeruginosa. Therefore, the present invention finally determined that the reaction time of 15 minutes was the optimal reaction condition.

[0042] Example 4: Inclusion Test

[0043] According to the "GB4789.45-2023 National Food Safety Standard Microbiological Test Method Validation General Rules", the designed primers and probes were tested for inclusion, and the standard strain Listeria monocytogenes (ATCC19115) and Listeria monocytogenes isolated from food were selected for inclusion testing. Among them, 8 clones of the standard strain were selected for inclusion verification experiments, and 22 clones of Listeria monocytogenes isolated from food were selected for inclusion verification experiments. When conducting the inclusion test, the inoculation level was about 100 times the minimum detection level of the Listeria monocytogenes detection method in the "GB 4789.30-2016 National Food Safety Standard Food Microbiological Test Listeria monocytogenes Test". The results are as follows Figure 2 As shown, the results show that the primers and probes designed by the present invention have good inclusiveness, and different clones of Listeria monocytogenes can be effectively detected.

[0044] Example 5: Exclusivity Test

[0045] Bacterial species closely related to Listeria monocytogenes were selected for exclusive experiments, and 15 non-target bacterial clones were selected for the exclusive experiment. ① Bacillus megaterium (CICC 10324), ② Vibrio parahaemolyticus (ATCC17802), ③ Salmonella Alexandria (ATCC13314), ④ Bacillus cereus (ATCC10876), ⑤ Listeria monocytogenes (CICC21670), ⑥ Pseudomonas aeruginosa (ATCC27853), ⑦ Common Proteobacterium (CMCC49027), ⑧ Proteobacterium (ATCC 12453), ⑨ Listeria monocytogenes (CICC21671), ⑩ Listeria innocua (ATCC33090), Listeria monocytogenes (CICC21672), Lactobacillus casei (CGMCC1.2435), Listeria monocytogenes (CICC21663), Staphylococcus aureus (ATCC25923), Bifidobacterium infantis (CICC6069) was tested for exclusivity. Figure 3 The primers and probes screened have good exclusivity and have no cross-reaction with all the test strains mentioned above.

[0046] Example 6: Sensitivity test

[0047] 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. The bacterial concentration was selected to be 6.5X10 6 CFU / mL; 6.5X10 5 CFU / mL; 6.5X10 4 CFU / mL; 6.5X10 3 CFU / mL; 6.5X10 2 CFU / mL; 65CFU / mL sample solution for sensitivity test. The results are as follows Figure 4 As shown, the sensitivity of the probe of the primer designed by the present invention is 6.5X10 2 CFU / mL.

[0048] Example 7: Detection of Listeria monocytogenes in the environment

[0049] After sensitivity testing, the test sensitivity of the primers and probes designed by the present invention is 6.5X10 2 CFU / mL. Such sensitivity cannot meet the practical requirement of "Listeria monocytogenes must not be detected in the production environment", therefore, when using the primers and probes designed by the present invention to detect Listeria monocytogenes in the environment, it is necessary to enrich the samples. After coating the environment with a sterile cotton swab, cut the cotton swab head into the LB1 culture medium, and after shaking and culturing for 24-48 hours, take 1mL of LB1 culture solution, centrifuge, discard the supernatant, collect the bacteria, add 200uL of lysis solution, boil at 100°C for 5 minutes, and then perform RPA detection. After testing, after enrichment, the detection method developed by the present invention can meet the practical requirement of "Listeria monocytogenes must not be detected in the production environment".

[0050] In order to further verify the practicality of the test method developed by the present invention, we contacted three cold chain food production enterprises to carry out environmental smearing on them, and obtained a total of 18 smear samples. After bacterial enrichment, the 18 smear samples were tested simultaneously with the national standard test and the rapid test technology developed by the present invention. The results are as follows Figure 5 As shown, the rapid test results of the 18 samples obtained were all negative, which was consistent with the test results of the national standard method.

[0051] 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 composition for detecting Listeria monocytogenes in an environment, characterized in that: The RPA composition comprises upstream and downstream primers and a probe, wherein the upstream primer sequence is 5'-GGATAAGTAAGGCTTTCAAGCTCACTATCCG-3', The downstream primer sequence is 5′-CGATGGCAACCCTGGATGACGACGCTCCAC[THF]TGTATAGCTG GTCGGTCGG-3′, wherein [THF] is a tetrahydrofuran residue, and the probe sequence is 5′-CGAACAAAGCAGACCTAATAGCAATGTTGAA -3′.

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

3. A kit for detecting Listeria monocytogenes in an environment, characterized in that: The kit comprises the RPA composition according to claim 1 or 2.

4. A method for detecting Listeria monocytogenes in an environment, comprising the following steps: (1) After applying the environment with a sterile cotton swab, place it in the culture medium for shaking culture; (2) collecting the bacterial cells by centrifugation, and performing RPA detection using the RPA composition according to claim 1 or 2 after lysis; (3) Take the amplified product and conduct lateral flow test strip detection, and judge the test result based on the color development.

5. The method according to claim 4, characterized in that: The shaking culture time is 24-48 hours, and / or the lysis is boiling at 100° C. for 5 minutes.

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