A method for detecting foodborne pathogenic bacteria

Abstract

The present invention belongs to the technical field of food safety detection, and in particular to a method for detecting foodborne pathogens. The method places the characteristic gene of the sample to be tested in a test tube, and then uses gelatin to fix the CRISPR / Cas12a detection system on the lid of the test tube. After the characteristic gene of the sample to be tested in the test tube is amplified by RPA at 37°C, the temperature is raised to 43°C to melt the gelatin, so that the amplified product reacts with the CRISPR / Cas12a detection system in the test tube, and the test result is determined by fluorescence detection after the reaction is completed. The present invention has low cost, simple operation, strong specificity, high accuracy, good stability, does not require large-scale instruments and equipment, has the advantage of visualization, and greatly reduces the risk of aerosol contamination caused during the detection process, which is more conducive to the application in food safety detection technology.

Description

Technical Field

[0001] The invention belongs to the technical field of food safety detection, and specifically relates to a method for detecting foodborne pathogenic bacteria. Background Art

[0002] Foodborne pathogens can cause foodborne diseases and seriously endanger human health. For example, Listeria monocytogenes ( Listeria monocytogenes Listeria monocytogenes (LM) is the most pathogenic foodborne pathogen in the genus Listeria and can cause zoonotic diseases. It is widely present in nature. The bacteria can still grow and reproduce in an environment of 4°C. It is one of the main pathogens of refrigerated food that threaten human health. Its incidence rate is low, but the mortality rate after infection is extremely high. The main clinical manifestations are fever, cramps, chills, back pain, headache, diarrhea, vomiting, etc. Severe cases can cause meningitis, osteomyelitis, myocarditis, spontaneous abortion, sepsis and other diseases, which have a serious impact on food safety, human health and social economy.

[0003] Therefore, establishing a rapid and accurate detection method is an effective preventive measure to reduce the risk of infection. At present, the detection methods of foodborne pathogens include traditional culture methods, immunological methods and molecular biology methods. The traditional culture method is to use chromogenic culture medium to culture the target bacteria. This method relies on colony morphology to distinguish the bacterial type, which has low accuracy, cumbersome operation and long time. The immunological method relies on antibodies to identify the target bacteria. Such methods are costly and prone to non-specific reactions. The molecular biological method determines the type of pathogen by detecting the specific genes of pathogens. It has high accuracy, but most technologies rely on expensive detection instruments, or have high requirements for operators and detection environments. There is a lack of a fast means to achieve on-site detection. With the continuous advancement of CRISPR / Cas detection technology, it has become possible to combine some isothermal amplification methods to detect pathogens on-site. Among them, recombinase polymerase amplification technology (RPA) is often selected as a means of amplification before CRISPR / Cas detection due to its high sensitivity and efficiency, in order to achieve the purpose of rapid detection. However, if the CRISPR / Cas12a system is added directly after the RPA amplification is completed, it is very easy to cause aerosol contamination (such as the patent CN 115747353 A); if the CRISPR / Cas12a system is added immediately after the RPA system is prepared, it will affect the amplification of the RPA system. For example, patent CN117512215 A uses paraffin to isolate the RPA and CRISPR / Cas12a systems. Liquid paraffin needs to be added to the upper layer of the CRISPR / Cas12a reaction system while hot, and then solidified. Finally, it is heated at 55°C for 3 minutes to melt the solid paraffin into liquid, and the two-stage reaction system is mixed to observe the results. This requires high temperature control during the operation, and high-temperature heating will also affect the enzyme activity in subsequent reactions, resulting in decreased sensitivity. Summary of the invention

[0004] The purpose of the present invention is to solve the deficiencies of the prior art and provide a method for detecting foodborne pathogens, which specifically adopts the following technical solutions:

[0005] A method for detecting foodborne pathogenic bacteria, the detection method comprising the following steps:

[0006] The characteristic gene of the sample to be tested is placed in a test tube, and then the CRISPR / Cas12a detection system is fixed on the lid of the test tube using gelatin. After the characteristic gene of the sample to be tested in the test tube is amplified by RPA at 37°C, the temperature is raised to 43°C to melt the gelatin, so that the amplified product reacts with the CRISPR / Cas12a detection system in the test tube, and the test result is determined by fluorescence detection after the reaction is completed;

[0007] The amount of gelatin used is 0.325mg-0.65mg.

[0008] The present invention uses gelatin to fix the CRISPR / Cas12a detection system on the lid of the detection tube. Then, after the gene amplification reaction of the sample to be tested in the detection tube is completed, the lid does not need to be opened additionally, and the temperature is directly raised to the second stage reaction. As the temperature rises, the gelatin melts, and the CRISPR / Cas12a detection system is directly dripped into the tube for reaction. A 365nm ultraviolet lamp is used to observe whether the product after the RPA-CRISPR / Cas12a system reaction emits fluorescence to determine whether the sample contains target bacteria. The characteristics of gelatin that it maintains a solid state on the lid during the RPA reaction stage (37°C) and can be converted into a liquid state during the CRISPR reaction stage (43°C) are utilized to achieve the two-step reactions of RPA and CRISPR in the same tube. At the same time, gelatin can effectively enhance the fluorescence signal of the reaction system, making it more conducive to on-site detection.

[0009] As a further preferred embodiment, the above detection method specifically comprises the following steps:

[0010] The sample to be tested is placed in LB medium for bacterial culture, and the whole genome DNA of the sample to be tested is extracted;

[0011] The whole genome DNA is placed in a test tube, and the CRISPR / Cas12a detection system is fixed on the lid of the test tube with gelatin, and the lid is closed. The whole genome DNA is used as a template in the test tube to perform an RPA amplification reaction at 37° C. After the reaction is completed, an amplification product is obtained;

[0012] After the RPA amplification reaction is completed, the temperature in the detection tube is raised to 43°C to melt the gelatin. The CRISPR / Cas12a detection system on the lid of the detection tube is dripped into the tube to react with the amplification product. After the reaction is completed, the detection result is determined by fluorescence detection.

[0013] As a further preferred embodiment, the above-mentioned foodborne pathogenic bacteria include Listeria monocytogenes.

[0014] As a further preferred embodiment, the heating and boiling method is used to extract the whole genome DNA of the sample to be tested.

[0015] As a further preferred embodiment, the reaction system of the above RPA amplification reaction specifically includes:

[0016] 7.35μL buffer solution A, 0.5μL upstream primer, 0.5μL downstream primer, 0.5μL whole genome DNA of the sample to be tested, 3.03μL ultrapure water, 0.63μL buffer solution B; the concentrations of the upstream primer and the downstream primer are both 10μM. In the above process, the RPA amplification process can use a commercial kit. Among them, buffer solution A and buffer solution B are the buffer solutions required for amplification in the universal RPA kit, containing enzymes, Mg 2+ The necessary components for amplification are as follows, and the RPA amplification system is 12.51 μL.

[0017] As a further preferred embodiment, the nucleic acid sequences of the upstream primer and the downstream primer are shown as SEQ ID NO.1 and SEQ ID NO.2 respectively.

[0018] The sequence of SEQ ID NO.1 is as follows:

[0019] F1: 5'-TCGATCACTCTGGAGGATACGTTGCTCAATT-3';

[0020] The sequence of SEQ ID NO.2 is as follows:

[0021] R1: 5'-TATGTTGAACAATTTCGTTACCTTCAGGATC-3'.

[0022] As a further preferred embodiment, the CRISPR / Cas12a detection system is 13 μL, specifically comprising:

[0023] Includes: 1.95μL buffer solution, 1.95μL crRNA, 1.3μL Cas12a, 1.3μL fluorescent reporter probe, 6.5μL gelatin solution.

[0024] As a further preferred embodiment, the nucleic acid sequence of the above crRNA is shown in SEQ ID NO.3.

[0025] The sequence of SEQ ID NO.3 is as follows:

[0026] crRNA: 5'-UAAUUUCUACUAAGUGUAGAUUUGGGAUGAAGUAAAUUAUG-3'.

[0027] As a further preferred embodiment, the fluorescent reporter probe is an oligonucleotide sequence labeled with both a fluorescent group and a quencher group.

[0028] As a further preferred embodiment, the gelatin includes porcine gelatin and bovine gelatin; and the concentration of the gelatin solution is 0.1 g / mL.

[0029] As a further preferred embodiment, the above fluorescence detection includes using a 365 nm ultraviolet light source flashlight to illuminate and observe the fluorescence signal with the naked eye or using a fluorescence signal detection instrument to detect.

[0030] The present invention combines RPA with the CRISPR / Cas12a detection system, and uses 365nm ultraviolet light to observe whether the product after the reaction of the RPA-CRISPR / Cas12a system emits fluorescence to determine whether the sample contains the target bacteria. The upstream and downstream primers in the reaction system of the RPA amplification reaction are characterized in that they can specifically amplify the target fragment, and the fragment can be recognized by crRNA, thereby activating the cleavage activity of Cas12a, so that the fluorescent reporter probe labeled with both the fluorescent group and the quenching group is cut to emit fluorescence.

[0031] The beneficial effects of the present invention are:

[0032] (1) The present invention uses gelatin to fix the CRISPR / Cas12a detection system on the lid, so that the CRISPR / Cas12a detection system can be separated from the RPA amplification system. After the RPA amplification reaction is completed, there is no need to open the lid, and the temperature is raised to directly carry out the second step reaction (the CRISPR / Cas12a detection system is dripped into the tube and mixed with the RPA amplification reaction product), thereby avoiding the problem of contamination caused by opening the lid in the prior art and greatly reducing the risk of aerosol contamination during the detection process.

[0033] (2) The present invention has low cost, simple operation, strong specificity, high accuracy, good stability, and does not require large-scale instruments and equipment. It only requires a small heating device (such as a small metal bath) and a 365nm ultraviolet excitation flashlight. It has the advantage of visualization and is more conducive to grassroots use. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1 Shown is a visualization of the detection results of RPA-CRISPR / Cas12a detecting a standard strain of Listeria monocytogenes;

[0035] Figure 2 Shown is the effect of different amounts of gelatin added on the fluorescence intensity of the RPA-CRISPR / Cas12a detection system;

[0036] Figure 3 Shown is the test result of salmon samples containing Listeria monocytogenes at different enrichment times;

[0037] Figure 4 Shown is a graph showing the specific results of RPA-CRISPR / Cas12a detection of Listeria monocytogenes;

[0038] Figure 5 Shown is the detection limit result graph of Listeria monocytogenes detected by RPA-CRISPR / Cas12a;

[0039] Figure 6 Shown is a comparison of the detection effects of gelatin and paraffin-based RPA-CRISPR / Cas12a detection schemes. DETAILED DESCRIPTION

[0040] The following will be combined with the embodiments and drawings to clearly and completely describe the concept, specific structure and technical effects of the present invention, so as to fully understand the purpose, scheme and effect of the present invention. It should be noted that the embodiments and features in the embodiments of this application can be combined with each other without conflict.

[0041] In the following embodiments, the RPA amplification process can use a commercial kit; wherein buffer solution A and buffer solution B are buffer solutions required for amplification in a general RPA kit, including enzyme, Mg 2+ The RPA amplification system is 12.51 μL. The negative control in the following examples is ultrapure water.

[0042] Example 1

[0043] One-pipeline RPA-CRISPR / Cas12a for detection of Listeria monocytogenes

[0044] Use complete enrichment medium to culture Listeria monocytogenes overnight, take 1 mL of bacterial solution and centrifuge (8000 rpm, 10 min), discard the supernatant, add 200 μL of ultrapure water, heat for 10 min (95 ℃), centrifuge (8000 rpm, 10 min), take the supernatant, and obtain the template DNA to be tested.

[0045] Each experimental group contains 12.5μL of RPA system, including: 7.35μL buffer solution A, 0.5μL (10μM) upstream primer, 0.5μL (10μM) downstream primer, 0.5μL whole genome DNA of the sample to be tested, 3.03μL ultrapure water, 0.63μL buffer solution B, and CRISPR / Cas12a system, including: 1.95μL buffer solution, 1.95μL crRNA, 1.3μL Cas12a, 1.3μL fluorescent reporter probe, 6.5μL 10% gelatin. After the RPA reaction is completed, it is directly heated to 43°C, and the CRISPR / Cas12a system is dripped into the test tube for detection, and finally the test results are observed by ultraviolet light.

[0046] The reaction procedure is as follows:

[0047] RPA amplification was performed at 37°C for 15 minutes, then directly heated to 43°C, and the CRISPR / Cas12a system was dripped into the test tube to detect the RPA amplification product. The reaction lasted for 45 minutes, and finally the test results were observed by ultraviolet light. Figure 1 shown by Figure 1 It can be seen that this method can detect three strains of Listeria monocytogenes from different sources (ATCC 13932, BNCC 185987, BNCC375908), and the negative group of the corresponding experiment has no fluorescent signal.

[0048] Example 2

[0049] Investigate the effect of gelatin addition on fluorescence signal intensity

[0050] Use complete enrichment medium to culture Listeria monocytogenes overnight, take 1 mL of bacterial solution and centrifuge (8000 rpm, 10 min), discard the supernatant, add 200 μL of ultrapure water, heat for 10 min (95 ℃), centrifuge (8000 rpm, 10 min), take the supernatant, and obtain the template DNA to be tested.

[0051] (1) Without gelatin: Each experimental group contained 12.5 μL of RPA system, including: 7.35 μL buffer solution A, 0.5 μL (10 μM) upstream primer, 0.5 μL (10 μM) downstream primer, 0.5 μL whole genome DNA of the sample to be tested, 3.03 μL ultrapure water, 0.63 μL buffer solution B, RPA reaction at 37 °C for 15 min, open the lid and add CRISPR / Cas12a system, including: 1.95 μL buffer solution, 1.95 μL crRNA, 1.3 μL Cas12a, 1.3 μL fluorescent reporter probe, 6.5 μL ultrapure water, heat to 43 °C for 45 min, and finally observe the test results by ultraviolet light.

[0052] (2) Adding gelatin: Each experimental group contained 12.5 μL of RPA system, including: 7.35 μL buffer solution A, 0.5 μL (10 μM) upstream primer, 0.5 μL (10 μM) downstream primer, 0.5 μL whole genome DNA of the sample to be tested, 3.03 μL ultrapure water, 0.63 μL buffer solution B, and CRISPR / Cas12a system, including: 1.95 μL buffer solution, 1.95 μL crRNA, 1.3 μL Cas12a, 1.3 μL fluorescent reporter probe, 6.5 μL Gelatin (added at concentrations of 5%, 10%, and 20%; corresponding to added masses of 0.325 mg, 0.65 mg, and 1.3 mg) was reacted with RPA at 37°C for 15 min, then directly heated to 43°C. The CRISPR / Cas12a system was dripped into the detection tube to detect the RPA amplification product, reacted for 45 min, and finally observed the detection results by ultraviolet light.

[0053] The above results of the effect of gelatin on the fluorescence signal intensity are as follows Figure 2 shown by Figure 2 It can be seen that adding an appropriate amount of gelatin (0.325 mg and 0.65 mg) can effectively improve the fluorescence signal intensity.

[0054] Example 3

[0055] Detection of Listeria monocytogenes in salmon samples

[0056] Take salmon as the sample to be tested (other foods can refer to this example process). Purchase salmon from the market, take 5 portions of 25g salmon, sterilize under ultraviolet light for 2 hours, add about 50 CFU of Listeria monocytogenes to each sample, and then culture the sample in 225 mL LB1 culture medium, where 1 acridonic acid (5.0 mg / vial) and 1 acriflavine (3.0 mg / vial) are added to each 225 mL culture medium. Starting from the 6th hour, take 1 mL of bacterial solution every 1 hour, centrifuge the bacterial solution (8000 rpm, 10 min), discard the supernatant, add 200 μL ultrapure water, heat for 10 minutes (95 ℃), centrifuge (8000 rpm, 10 minutes), take the supernatant, and obtain the template DNA to be tested. Then perform a one-tube RPA-CRISPR / Cas12a test.

[0057] The specific scheme of the one-tube RPA-CRISPR / Cas12a method is as follows:

[0058] The RPA-CRISPR / Cas12a detection reaction system includes: 12.5 μL of RPA system, including: 7.35 μL buffer solution A, 0.5 μL (10 μM) upstream primer, 0.5 μL (10 μM) downstream primer, 0.5 μL whole genome DNA of the sample to be tested, 3.03 μL ultrapure water, 0.63 μL buffer solution B, and CRISPR / Cas12a system, including: 1.95 μL buffer solution, 1.95 μL crRNA, 1.3 μL Cas12a, 1.3 μL fluorescent reporter probe, 6.5 μL 10% gelatin.

[0059] The reaction procedure is as follows:

[0060] RPA amplification was performed at 37°C for 15 min, then directly heated to 43°C, and the CRISPR / Cas12a system was dripped into the detection tube to detect the RPA amplification product, reacted for 45 min, and finally observed the detection results by ultraviolet light. The fluorescence signal can be read by a fluorescence reader.

[0061] The above results are Figure 3 shown by Figure 3 It can be seen that this method can quickly detect whether the contaminated salmon sample contains Listeria monocytogenes 7 hours after the sample has been cultured, and is suitable for the rapid detection of Listeria monocytogenes.

[0062] Example 4

[0063] Specificity verification of the one-tube RPA-CRISPR / Cas12a system for detecting Listeria monocytogenes

[0064] The whole enrichment medium was used to culture monocytogenes, enteropathogenic Escherichia coli, enteroinvasive Escherichia coli, Shiga toxin-producing Escherichia coli, enteroaggregative Escherichia coli, Yersinia enterocolitica, Staphylococcus wortii, Burkholderia cereus, Bacillus thuringiensis, Burkholderia gladiolus, Bacillus cereus, Staphylococcus aureus, Vibrio parahaemolyticus, Salmonella and other pathogenic bacteria overnight. Take 1m bacterial solution and centrifuge (8000 rpm, 10 min), discard the supernatant, add 200μL ultrapure water, heat for 10min (95 ℃), centrifuge (8000 rpm, 10 min), take the supernatant, and obtain the template DNA to be tested. Then perform a one-tube RPA-CRISPR / Cas12a detection. The specific scheme of one-tube RPA-CRISPR / Cas12a detection is as follows:

[0065] The one-tube RPA-CRISPR / Cas12a detection system includes: 12.5μL RPA system, including: 7.35μL buffer solution A, 0.5μL (10μM) upstream primer, 0.5μL (10μM) downstream primer, 0.5μL whole genome DNA of different samples to be tested (Listeria monocytogenes, enteropathogenic Escherichia coli, enteroinvasive Escherichia coli, Shiga toxin-producing Escherichia coli, etc.), 3.03μL ultrapure water, 0.63μL buffer solution B, and CRISPR / Cas12a system, including: 1.95μL buffer solution, 1.95μL crRNA, 1.3μL Cas12a, 1.3μL fluorescent reporter probe, 6.5μL 10% gelatin.

[0066] The reaction procedure is:

[0067] RPA amplification was performed at 37°C for 15 minutes, then directly heated to 43°C, and the CRISPR / Cas12a system was dripped into the detection tube to detect the RPA amplification product, reacted for 45 minutes, and finally observed the detection results by ultraviolet light. The fluorescence signal can be read by a fluorescence reader.

[0068] Search for Listeria monocytogenes in NCBI oeLh Gene sequence, Vibrio parahaemolyticus td Gene sequence and Bacillus cereus cesB The gene sequence was then delivered to Shanghai Sangon Biotechnology Co., Ltd. to synthesize the positive template of the above gene, and the synthesized positive template was subjected to one-tube RPA-CRISPR / Cas12a detection. The specific scheme of one-tube RPA-CRISPR / Cas12a detection is as follows:

[0069] The one-tube RPA-CRISPR / Cas12a detection system includes: 12.5μL RPA system, including: 7.35μL A buffer solution, 0.5μL (10μM) upstream primer, 0.5μL (10μM) downstream primer, 0.5μL different gene positive templates, 3.03μL ultrapure water, 0.63μL B buffer solution, and CRISPR / Cas12a system, including: 1.95μL buffer solution, 1.95μL crRNA, 1.3μL Cas12a, 1.3μL fluorescent reporter probe, 6.5μL 10% gelatin.

[0070] The reaction procedure is:

[0071] RPA amplification was performed at 37°C for 15 minutes, then directly heated to 43°C, and the CRISPR / Cas12a system was dripped into the detection tube to detect the RPA amplification product, reacted for 45 minutes, and finally observed the detection results by ultraviolet light. The fluorescence signal can be read by a fluorescence reader.

[0072] The results are as follows Figure 4 As shown, Figure 4 1-19 shown in A in the figure correspond to the test results of Listeria monocytogenes ATCC 13932, Listeria monocytogenes clinical strain 1 (isolated from milk), Listeria monocytogenes clinical strain 2 (isolated from milk powder), Listeria monocytogenes clinical strain 3 (isolated from fish), Listeria monocytogenes clinical strain 4 (isolated from shrimp), enteropathogenic Escherichia coli, enteroinvasive Escherichia coli, Shiga toxin-producing Escherichia coli, enteroaggregative Escherichia coli, Yersinia enterocolitica, Staphylococcus vortex, Burkholderia cereus, Bacillus thuringiensis, Burkholderia gladiolus, Bacillus cereus, Staphylococcus aureus, Vibrio parahaemolyticus, Salmonella and negative control respectively; Figure 4 The ad shown in B in the figure correspond to the genes hlyA, tdh , cesB and negative control test results, and by Figure 4 It is known that the characteristic genes for non-monocytogenes Listeria td , cesB The test results were all negative, with no fluorescent signal, Listeria monocytogenes and gene oeLh The positive template detection result has a strong fluorescent signal, which can indicate that the detection result of the method established by the present invention is accurate and reliable.

[0073] Example 5

[0074] Validation of the detection limit of a one-tube RPA-CRISPR / Cas12a system for detecting Listeria monocytogenes

[0075] The concentration of the DNA template of Listeria monocytogenes was measured using Qubit 4 and then serially diluted 5-fold to 5.15×10 -4 ng / μL. Then, a one-tube RPA-CRISPR / Cas12a test was performed. The specific protocol for the one-tube RPA-CRISPR / Cas12a test is as follows:

[0076] The one-tube RPA-CRISPR / Cas12a detection system includes: 12.5μL RPA system, including: 7.35μL A buffer solution, 0.5μL (10μM) upstream primer, 0.5μL (10μM) downstream primer, 0.5μL whole genome DNA of the sample to be tested with different concentrations, 3.03μL ultrapure water, 0.63μL B buffer solution, and CRISPR / Cas12a system, including: 1.95μL buffer solution, 1.95μL crRNA, 1.3μL Cas12a, 1.3μL reporter probe, 6.5μL 10% gelatin.

[0077] The reaction procedure is:

[0078] RPA amplification was performed at 37°C for 15 minutes, then directly heated to 43°C, and the CRISPR / Cas12a system was dripped into the detection tube to detect the RPA amplification product, reacted for 45 minutes, and finally observed the detection results by ultraviolet light. The fluorescence signal can be read by a fluorescence reader.

[0079] Take 1mL of Listeria monocytogenes cultured overnight in the whole enrichment medium and perform 10-fold gradient dilutions, and count them by plate counting method. Take 100μL of the first dilution gradient dilution bacterial solution, centrifuge the bacterial solution (8000 rpm, 10min), discard the supernatant, add 100μL ultrapure water, heat for 10min (95℃-100℃), centrifuge (8000 rpm, 10 min), take the supernatant, and obtain the template DNA to be tested. The extracted DNA is then used for one-tube RPA-CRISPR / Cas12a detection. The specific scheme of one-tube RPA-CRISPR / Cas12a detection is as follows:

[0080] The one-tube RPA-CRISPR / Cas12a detection system includes: 12.5μL RPA system, including: 7.35μL A buffer solution, 0.5μL (10μM) upstream primer, 0.5μL (10μM) downstream primer, 0.5μL whole genome DNA of the sample to be tested with different concentrations, 3.03μL ultrapure water, 0.63μL B buffer solution, and CRISPR / Cas12a system, including: 1.95μL buffer solution, 1.95μL crRNA, 1.3μL Cas12a, 1.3μL reporter probe, 6.5μL 10% gelatin.

[0081] The reaction procedure is:

[0082] RPA amplification was performed at 37°C for 15 minutes, then directly heated to 43°C, the CRISPR / Cas12a system was dripped into the test tube, the RPA amplification product was detected, the reaction was carried out for 45 minutes, and finally the test results were observed by ultraviolet light irradiation, and the fluorescence signal could be read by a fluorescence reader.

[0083] Search for Listeria monocytogenes in NCBI oeLh The gene sequence was obtained, and the positive template of the gene was synthesized by Shanghai Sangon Biotechnology Co., Ltd. The synthesized positive template was diluted 10 times to 1zM, and then the diluted positive template was used for one-tube RPA-CRISPR / Cas12a detection. The specific scheme of one-tube RPA-CRISPR / Cas12a detection is as follows:

[0084] The one-tube RPA-CRISPR / Cas12a detection system includes: 12.5μL RPA system, including: 7.35μL buffer solution A, 0.5μL (10μM) upstream primer, 0.5μL (10μM) downstream primer, 0.5μL hlyA gene positive template of different concentrations to be tested, 3.03μL ultrapure water, 0.63μL buffer solution B, and CRISPR / Cas12a system, including: 1.95μL buffer solution, 1.95μL crRNA, 1.3μL Cas12a, 1.3μL reporter probe, 6.5μL 10% gelatin.

[0085] The reaction procedure is:

[0086] RPA amplification was performed at 37°C for 15 minutes, then directly heated to 43°C, and the CRISPR / Cas12a system was dripped into the detection tube to detect the RPA amplification product, reacted for 45 minutes, and finally observed the detection results by ultraviolet light. The fluorescence signal can be read by a fluorescence reader.

[0087] The results are as follows Figure 5 As shown, Figure 5 A and Figure 5 B in the figure is the experimental result diagram of the detection limit of the whole genome of Listeria monocytogenes. Figure 5 A in the figure is the test result picture of ultraviolet light irradiation. Figure 5 B in the figure is the fluorescence signal value result; Figure 5 C and Figure 5 D in the figure is the test result of Listeria monocytogenes enrichment solution with different concentrations. Figure 5 C in the figure is the test result picture of ultraviolet light irradiation. Figure 5 D in is the fluorescence signal value result; Figure 5 E in the equation is different concentrations oeLhThe picture of the test results of the gene under ultraviolet light irradiation; Figure 5 It can be seen that the concentration of the whole genome DNA of Listeria monocytogenes was diluted to 0.01288ng / μL, and the bacterial solution was diluted to 64 CFU and oeLh When the gene is diluted to 10zM, it can still be detected. The method of the present invention has a high sensitivity to Listeria monocytogenes and can be used for the detection thereof.

[0088] Example 6

[0089] Comparison of test results of gelatin and paraffin in one tube test

[0090] Use full bacterial culture medium to culture Listeria monocytogenes overnight, take 1mL of bacterial solution and centrifuge (8000rpm, 10 min), discard the supernatant, add 200μL ultrapure water, heat for 10min (95℃), centrifuge (8000rpm, 10min), take the supernatant, and obtain the template DNA to be tested. Then use gelatin or paraffin to perform one-tube RPA-CRISPR / Cas12a detection. The specific scheme of one-tube RPA-CRISPR / Cas12a detection is as follows:

[0091] Gelatin one-tube detection: Each experimental group contains 12.5μL of RPA system, including: 7.35μL buffer solution A, 0.5μL (10μM) upstream primer, 0.5μL (10μM) downstream primer, 0.5μL whole genome DNA of the sample to be tested, 3.03μL ultrapure water, 0.63μL buffer solution B, and CRISPR / Cas12a system, including: 1.95μL buffer solution, 1.95μL crRNA, 1.3μLCas12a, 1.3μL fluorescent reporter probe, 6.5μL 10% gelatin. After the RPA reaction is completed, it is directly heated to 43°C, and the CRISPR / Cas12a system is dripped into the test tube for detection. The reaction procedure is: perform RPA amplification at 37°C for 15 minutes, then heat directly to 43°C, drip the CRISPR / Cas12a system into the detection tube to detect the RPA amplification product, react for 45 minutes, and finally observe the test results by ultraviolet light. The fluorescence signal can be read by a fluorescence reader.

[0092] Paraffin one-tube detection: Each experimental group contains 12.5μL of RPA system, including: 7.35μL buffer solution A, 0.5μL (10μM) upstream primer, 0.5μL (10μM) downstream primer, 0.5μL whole genome DNA of the sample to be tested, 3.03μL ultrapure water, 0.63μL buffer solution B, 6.5μL paraffin (add while hot); CRISPR / Cas12a system, including: 1.95μL buffer solution, 1.95μL crRNA, 1.3μL Cas12a, 1.3μL fluorescent reporter probe. After the RPA reaction is completed, heat to 55℃ to melt the paraffin, so that the RPA system is in contact with the CRISPR / Cas12a system for detection. The reaction procedure is: RPA amplification at 37℃ for 15min, then heated to 55℃ to melt the paraffin, react for 45min, and finally observe the test results by ultraviolet light. The fluorescence signal can be read by a fluorescence reader.

[0093] The results are as follows Figure 6 shown by Figure 6 It can be seen that when gelatin is used to realize one-tube RPA-CRISPR / Cas12a detection, the monocytogenes Listeria oeLh The positive template of the gene can still be detected when diluted to 10zM, but cannot be detected when using paraffin-based detection, indicating that the method of the present invention has a high sensitivity to Listeria monocytogenes and can be used to detect it.

[0094] Although the description of the present invention has been quite detailed and specifically describes several described embodiments, it is not intended to be limited to any of these details or embodiments or any particular embodiment, but should be regarded as providing a broad possible interpretation of these claims in view of the prior art by reference to the appended claims, thereby effectively covering the intended scope of the present invention. In addition, the above description of the present invention is based on the embodiments foreseeable by the inventor, and its purpose is to provide a useful description, and those non-substantial changes to the present invention that have not yet been foreseen may still represent equivalent changes to the present invention.

Claims

1. A method for detecting foodborne pathogens for non-diagnostic purposes, characterized in that: The detection method comprises the following steps: The sample to be tested is placed in a test tube, and then the CRISPR / Cas12a detection system is fixed on the lid of the test tube using gelatin. After the characteristic gene of the sample to be tested in the test tube is amplified by RPA at 37°C, the temperature is raised to 43°C to melt the gelatin, so that the amplified product reacts with the CRISPR / Cas12a detection system in the test tube. After the reaction is completed, the test result is determined by fluorescence detection; The amount of gelatin used is 0.65 mg; the gelatin includes porcine gelatin and bovine gelatin; The foodborne pathogens include Listeria monocytogenes.

2. The detection method according to claim 1, characterized in that: The detection method specifically comprises the following steps: The sample to be tested is placed in LB medium for bacterial culture, and the whole genome DNA of the sample to be tested is extracted; The whole genome DNA is placed in a test tube, and the CRISPR / Cas12a detection system is fixed on the lid of the test tube with gelatin, and the lid is closed. The whole genome DNA is used as a template in the test tube to perform an RPA amplification reaction at 37° C. After the reaction is completed, an amplification product is obtained; After the RPA amplification reaction is completed, the temperature in the detection tube is raised to 43°C to melt the gelatin. The CRISPR / Cas12a detection system on the lid of the detection tube is dripped into the tube to react with the amplification product. After the reaction is completed, the detection result is determined by fluorescence detection.

3. The detection method according to claim 2, characterized in that: The whole genome DNA of the samples to be tested was extracted by heating and boiling method.

4. The detection method according to claim 2, characterized in that: The nucleic acid sequences of the upstream primer and the downstream primer in the RPA amplification reaction are shown as SEQ ID NO.1 and SEQ ID NO.2, respectively.

5. The detection method according to claim 2, characterized in that: The CRISPR / Cas12a detection system is 13 μL and specifically includes: Includes: 1.95μL buffer solution, 1.95μL crRNA, 1.3μL Cas12a, 1.3μL fluorescent reporter probe, 6.5μL gelatin solution.

6. The detection method according to claim 5, characterized in that: The nucleic acid sequence of the crRNA is shown in SEQ ID NO.

3.

7. The detection method according to claim 2, characterized in that: The fluorescence detection includes using a 365 nm ultraviolet light source flashlight to illuminate and observe the fluorescence signal with the naked eye or using a fluorescence signal detection instrument to detect.

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