A kit for simultaneous detection of Listeria monocytogenes and drug-resistant gene fosX

Abstract

A method for simultaneous detection of Listeria monocytogenes and drug resistance genes fosX The kit belongs to the field of bacterial molecular diagnosis technology. The present invention solves the problem of traditional detection of Listeria monocytogenes and its drug resistance genes in the prior art. fosX The method has the technical problems of low efficiency and high equipment dependence. This paper provides a method for the simultaneous detection of Listeria monocytogenes and its drug-resistant genes. fosX The kit comprises: an EmDEA specific primer pair and an RNA probe, as well as a nucleic acid amplification enzyme system and a signal amplification enzyme system. fosX The kit has strong specificity, repeatability, stability, and reliability, with a sensitivity as low as 500 copies / μL. The kit provided by the present invention has a simple operation process and can be carried out in rural areas, remote areas, or any other scenarios requiring immediate testing, thus having broad application prospects.

Description

Technical Field

[0001] The present invention belongs to the technical field of bacterial molecular diagnosis, and in particular relates to a kit for simultaneously detecting Listeria monocytogenes and the drug-resistant gene fosX. Background Art

[0002] Listeria monocytogenes is a major foodborne pathogen that can be transmitted to humans and animals. Infection with Listeria monocytogenes can cause serious illnesses such as meningitis, sepsis, myocarditis, and miscarriage in pregnant women, posing a significant threat to human health. The presence of Listeria monocytogenes in food poses a significant challenge to food safety. In recent years, with the accelerated pace of life and the increasing popularity of fast food and ready-to-eat foods, the potential harm of Listeria monocytogenes has become increasingly prominent.

[0003] Traditional methods for detecting Listeria monocytogenes mainly rely on isolation and identification methods, including three steps: bacterial growth, isolation, and identification. However, this method is time-consuming, cumbersome, and prone to contamination during operation. It cannot meet the technical requirements of online detection during food production and rapid detection before food is put on the market and consumed. In addition, Listeria monocytogenes is resistant to fosfomycin, which is mainly caused by fosX Gene-mediated, but existing molecular detection methods mostly target broad-spectrum drug-resistant genes (such as β-lactamase genes) and lack certain specificity. Currently, there is no method for simultaneously detecting pathogens and their drug-resistant genes, and there are problems of step-by-step operation and low detection efficiency.

[0004] Currently, the primary technology for high-sensitivity molecular detection is fluorescent quantitative PCR. After more than two decades of development, this technology has become highly mature and widely used in various molecular testing scenarios. However, this technology requires expensive and inconvenient instruments to perform, and the test time typically exceeds 40 minutes. These issues have significantly limited the application of fluorescent quantitative PCR in the field of point-of-care testing (POCT).

[0005] In order to address the shortcomings of fluorescent quantitative PCR in the field of rapid nucleic acid detection, various technologies for rapid nucleic acid amplification under isothermal conditions have become the focus of research in recent years. The main technologies include the following: transcription-mediated amplification and its derivative technologies, but due to the constraints of their reaction mechanisms, their overall detection time is long and their sensitivity is also limited, making it impossible to achieve high-sensitivity detection within a short period of time; loop-mediated isothermal amplification is fast and sensitive, but its anti-contamination ability is weak, resulting in false positive test results, thus limiting its application in the direction of rapid nucleic acid detection; there are many studies related to recombinase polymerase amplification technology (RPA technology) or its derivative technologies, but its complex operation and weak fluorescence signal make it difficult to meet the needs of ultra-fast and high-sensitivity detection, and it is currently rarely used in clinical testing.

[0006] Therefore, the technical personnel in this field are eager to develop a rapid, sensitive and specific detection method to achieve the simultaneous detection of Listeria monocytogenes and its drug resistance genes. fosX Synchronous detection solves the technical problems of low efficiency and high equipment dependence of traditional detection methods. Summary of the Invention

[0007] The present invention solves the problem of traditional detection of Listeria monocytogenes and its drug resistance genes in the prior art. fosX The method has the technical problems of low efficiency and high equipment dependence. A kit for simultaneously detecting Listeria monocytogenes and the drug-resistant gene fosX is provided.

[0008] One of the purposes of the present invention is to provide a method for simultaneously detecting Listeria monocytogenes and its drug-resistant genes fosX A kit comprising: an EmDEA-specific primer pair and an RNA probe;

[0009] The EmDEA-specific primer pair includes VirR-F4, VirR-R4, fosX-F2, and fosX-R3, wherein the nucleotide sequence of VirR-F4 is shown in SEQ ID NO.4, and the nucleotide sequence of VirR-R4 is shown in SEQ ID NO.16; the nucleotide sequence of fosX-F2 is shown in SEQ ID NO.20, and the nucleotide sequence of fosX-R3 is shown in SEQ ID NO.33;

[0010] The RNA probe includes VirR-RNA3 and fosX-RNA4, the nucleotide sequence of VirR-RNA3 is shown in SEQ ID NO.9, and the nucleotide sequence of fosX-RNA4 is shown in SEQ ID NO.28.

[0011] In a preferred embodiment of the present invention, the 5' end of the RNA probe is labeled with a reporter group, and the 3' end of the RNA probe is labeled with a quencher group; the reporter group is any one of FAM, HEX, TET, Cyanine dye-, ROX or Texas Red, and the quencher group is any one of TAMRA, BHQ1, BHQ2, BHQ3, Iowa Black or BBQ.

[0012] In a preferred embodiment of the present invention, the reaction system of the EmDEA specific primer pair is: the DNA sample to be tested , Forward F / reverse R primers , RNA probes , activating liquid , freeze-drying protective agent.

[0013] In a preferred embodiment of the present invention, the activation solution is composed of: NTP nucleoside triphosphate and buffer.

[0014] In a preferred embodiment of the present invention, the lyoprotectant includes a nucleic acid amplification enzyme system and a signal amplification enzyme system; the nucleic acid amplification enzyme system is composed of: recombinase, single-strand binding protein, polymerase and ATP energy regeneration enzyme; the signal amplification enzyme system is composed of: positioning enzyme and cutting enzyme.

[0015] A second object of the present invention is to provide a method for using the above-mentioned kit for non-diagnostic purposes, the method comprising the following steps:

[0016] S1: Collect the sample to be tested, add high-efficiency lysis buffer, and react at 95°C for 10 minutes to obtain the DNA of the sample to be tested;

[0017] S2: Add the sample DNA obtained in S1 to the reaction system of the EmDEA specific primer pair, perform the amplification reaction using a handheld metal bath and a handheld fluorescence detector, and determine the test result based on the fluorescence signal Tt value.

[0018] In a preferred embodiment of the present invention, the amplification reaction is carried out at 42° C. for 30 cycles and for a reaction time of 10-30 min.

[0019] In a preferred embodiment of the present invention, the high-efficiency lysis solution is any one of pH=8 Tris, NaCl, EDTA, SDS or PVP-40, and a broad-spectrum protease.

[0020] In a preferred embodiment of the present invention, the criterion for determining the fluorescence signal Tt value in S2 is:

[0021] If no fluorescent signal Tt value appears during the amplification cycle, the test result is negative, indicating that the sample is not infected with Listeria monocytogenes or does not contain Listeria monocytogenes resistance genes. fosX ;

[0022] If the fluorescent signal Tt value appears during the amplification cycle, the test result is positive, indicating that the sample is infected with Listeria monocytogenes or contains Listeria monocytogenes resistance genes. fosX .

[0023] The third object of the present invention is to provide the above-mentioned kit for detecting Listeria monocytogenes and its drug-resistant genes fosX The application does not involve the diagnosis and treatment of diseases.

[0024] Beneficial effects of the present invention: The present invention provides a method for simultaneously detecting Listeria monocytogenes and its drug-resistant genes fosX A kit comprising: an EmDEA-specific primer pair and an RNA probe; the EmDEA-specific primer pair comprises VirR-F4, VirR-R4, fosX-F2, and fosX-R3, the VirR-F4 nucleotide sequence is shown in SEQ ID NO.4, and the VirR-R4 nucleotide sequence is shown in SEQ ID NO.16; the fosX-F2 nucleotide sequence is shown in SEQ ID NO.20, and the fosX-R3 nucleotide sequence is shown in SEQ ID NO.33; the RNA probe comprises VirR-RNA3 and fosX-RNA4, the VirR-RNA3 nucleotide sequence is shown in SEQ ID NO.9, and the fosX-RNA4 nucleotide sequence is shown in SEQ ID NO.28.

[0025] The present invention simplifies the DNA extraction step by adding a lysis solution and heat treating the DNA for 10 minutes. Based on the enzyme-mediated dual amplification nucleic acid amplification technology, the target nucleic acid can be amplified to 10 by integrating the nucleic acid amplification enzyme system and the signal amplification enzyme system under a constant temperature of 42°C for 10-30 minutes. 9 times, and generate more than 10,000 fluorescent signals from one nucleic acid amplification product under a constant temperature of 42°C, ultimately achieving the goal of detecting Listeria monocytogenes and its drug-resistant genes in the sample to be tested. fosX The purpose of efficient and rapid detection.

[0026] The invention provides a method for simultaneously detecting Listeria monocytogenes and its drug-resistant genes fosX The kit has strong specificity, repeatability, stability and reliability, and its sensitivity can be as low as , thereby achieving the early detection of Listeria monocytogenes and its drug-resistant genes in the early stages of infection fosXThe kit provided by the present invention can be used in conjunction with a portable handheld metal bath and a handheld fluorescence detector. It has a simple operation process and low requirements for testing personnel. It can be carried to rural sites, remote areas, or any other scenario where immediate testing is required. It meets the needs of various application scenarios such as food safety monitoring, emergency response, scientific research and education, and public health, and has broad application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 Listeria monocytogenes in Example 1 virR Screening results of EmDEA gene-specific primer pairs and RNA probes;

[0028] Figure 2 Listeria monocytogenes in Example 1 fosX Screening results of EmDEA-specific primer pairs and RNA probes;

[0029] Figure 3 Listeria monocytogenes in Example 3 virR Gene-specific test results graph;

[0030] Figure 4 Listeria monocytogenes in Example 3 fosX Diagram of drug resistance gene specific detection results;

[0031] Figure 5 Listeria monocytogenes in Example 3 virR Gene duplication detection results diagram;

[0032] Figure 6 Listeria monocytogenes in Example 3 fosX Diagram of drug resistance gene duplication detection results;

[0033] Figure 7 Listeria monocytogenes in Example 3 virR Gene sensitivity test result diagram;

[0034] Figure 8 Listeria monocytogenes in Example 3 fosX Diagram of drug resistance gene sensitivity test results;

[0035] Figure 9 The portable detector used in Example 3 was used to detect Listeria monocytogenes on site. virR Gene result map;

[0036] Figure 10 The portable detector used in Example 3 was used to detect Listeria monocytogenes on site. fosX Drug resistance gene results diagram. DETAILED DESCRIPTION

[0037] Those skilled in the art can refer to the content of this document and appropriately improve the process parameters. It is particularly important to note that all similar substitutions and modifications are obvious to those skilled in the art and are considered to be included in the present invention. The methods and applications of the present invention have been described through preferred embodiments. It is obvious that relevant persons can modify or appropriately change and combine the methods and applications described herein without departing from the content and scope of the present invention to implement and apply the technology of the present invention.

[0038] In order to make the purpose, technical solutions and advantages of the present invention more clear, the present invention is further described in detail below in conjunction with specific embodiments. The experimental methods used in the following examples are all conventional methods unless otherwise specified, and the materials, reagents, methods and instruments used are all conventional materials, reagents, methods and instruments in the art unless otherwise specified, and can be obtained from commercial channels by those skilled in the art.

[0039] Example 1: Design and screening of EmDEA-specific primer pairs and RNA probes

[0040] (1) Design of EmDEA-specific primer pairs and RNA probes

[0041] The present invention is based on the Listeria monocytogenes and its drug-resistant gene published by Gen Bank fosX Sequences were aligned with homologous sequences by ClustalX and selected VirR gene (nucleotide sequence shown in SEQ ID NO.37) and drug-resistant gene fosX The specific recognition site fragment (nucleotide sequence shown in SEQ ID NO.38) was used as a template to design EmDEA-specific primer pairs and RNA probes. The EmDEA-specific primer pairs were synthesized by Beijing Liuhe BGI Genomics Co., Ltd., and the RNA primers were synthesized by Suzhou Jingrui Biotechnology Co., Ltd.

[0042] The 5' end of the RNA probe is labeled with a FAM reporter group, and the 3' end of the RNA probe is labeled with a BHQ1 quencher group. The FAM and BHQ1 modified RNA probes are used to display the target gene amplification process to generate a fluorescent signal, which is proportional to the total amount of DNA bound to the probe.

[0043] The sequence information of the designed EmDEA-specific primer pairs and RNA probes is shown in Table 1.

[0044] Table 1

[0045]

[0046]

[0047] (2) EmDEA-specific primer pairs ( VirR ) and RNA probes ( VirR )

[0048] In this example, the basic fluorescence isothermal amplification detection kit (Cat. No. RR032) from Suzhou Jingrui Company was used to conduct the following experiment. The EmDEA-specific primer pairs and RNA probes described in Table 1 were added to the reaction system of the EmDEA-specific primer pairs according to the grouping in Table 2. The specific steps were as follows: the DNA samples to be tested were added to the eight tubes. (Listeria monocytogenes VirR Gene synthesis fragment (plasmid) as the amplification template for the experimental group), Forward F / reverse R primers 、 RNA probes , freeze-dried protective agent, centrifuged instantly, let it stand at room temperature for 2 min, and add activation solution along the wall of the eight-tube , instantaneous centrifugation, oscillation mixing for 10 s, instantaneous centrifugation, amplification reaction using a handheld metal bath and a handheld fluorescence detector, 30 cycles at 42°C, reaction time is 30 min, and the fluorescence signal Tt value is collected once in each cycle.

[0049] Compare the corresponding identical primer sets and select the RNA primer probe with the smallest Tt value and the highest endpoint fluorescence value; at the same time, select the EmDEA-specific primer pair with the smallest Tt value and the highest endpoint fluorescence value based on the RNA primer probe; the primer set with no amplification signal is used as the negative control, and the EmDEA-specific primer pair is finally screened and determined ( VirR ) and RNA probes ( VirR ).

[0050] Table 2

[0051]

[0052] The results are as follows Figure 1 As shown, the EmDEA specific primer pair designed in this embodiment ( VirR ) and RNA probes ( VirR ), it was found that the Tt value of the VirR-RNA3F4R4 primer-probe combination was low, the fluorescence value was relatively high, and there was no amplification signal in the negative control; therefore, it was finally decided to select the VirR-RNA3F4R4 primer-probe combination for subsequent experiments.

[0053] (2) EmDEA-specific primer pairs ( fosX ) and RNA probes ( fosX )

[0054] In this example, the basic fluorescence isothermal amplification detection kit (Cat. No. RR032) from Suzhou Jingrui Company was used to conduct the following experiment. The EmDEA-specific primer pairs and RNA probes described in Table 1 were added to the reaction system of the EmDEA-specific primer pairs according to the grouping in Table 3. The specific steps were as follows: the DNA samples to be tested were added to the eight tubes. (Listeria monocytogenes fosX The synthetic fragment of drug-resistant gene (plasmid) was used as the amplification template for the experimental group); Forward F / reverse R primers 、 RNA probes , freeze-dried protective agent, centrifuged instantly, let it stand at room temperature for 2 min, and add activation solution along the wall of the eight-tube , instantaneous centrifugation, oscillation mixing for 10 s, instantaneous centrifugation, amplification reaction using a handheld metal bath and a handheld fluorescence detector, 30 cycles at 42°C, reaction time is 30 min, and the fluorescence signal Tt value is collected once in each cycle.

[0055] Compare the corresponding identical primer sets and select the RNA primer probe with the smallest Tt value and the highest endpoint fluorescence value; at the same time, select the EmDEA-specific primer pair with the smallest Tt value and the highest endpoint fluorescence value based on the RNA primer probe; the primer set with no amplification signal is used as the negative control, and the EmDEA-specific primer pair is finally screened and determined ( fosX ) and RNA probes ( fosX ).

[0056] Table 3

[0057]

[0058] The results are as follows Figure 2 As shown, the EmDEA specific primer pair designed in this embodiment ( fosX ) and RNA probes ( fosX ), it was found that the Tt value of the fosX-RNA4F2R3 primer-probe combination was low, the fluorescence value was relatively high, and there was no amplification signal in the negative control; therefore, the fosX-RNA4F2R3 primer-probe combination was finally selected for subsequent experiments.

[0059] Example 2: A method for simultaneously detecting Listeria monocytogenes and its drug-resistant genes fosX Kit

[0060] A method for simultaneous detection of Listeria monocytogenes and its drug-resistant genes fosXThe kit comprises: an EmDEA-specific primer pair and an RNA probe; the EmDEA-specific primer pair comprises VirR-F4, VirR-R4, fosX-F2, and fosX-R3, the nucleotide sequence of VirR-F4 is shown in SEQ ID NO.4, and the nucleotide sequence of VirR-R4 is shown in SEQ ID NO.16; the nucleotide sequence of fosX-F2 is shown in SEQ ID NO.20, and the nucleotide sequence of fosX-R3 is shown in SEQ ID NO.33; the RNA probe comprises VirR-RNA3 and fosX-RNA4, the nucleotide sequence of VirR-RNA3 is shown in SEQ ID NO.9, and the nucleotide sequence of fosX-RNA4 is shown in SEQ ID NO.28;

[0061] The 5' end of the RNA probe is labeled with a reporter group, and the 3' end of the RNA probe is labeled with a quencher group; the reporter group is any one of FAM, HEX, TET, Cyanine dye-, ROX or Texas Red, and the quencher group is any one of TAMRA, BHQ1, BHQ2, BHQ3, Iowa Black or BBQ;

[0062] The reaction system of the EmDEA specific primer pair is as follows: DNA sample to be tested , Forward F / reverse R primers , RNA probes , activating liquid (NTP nucleoside triphosphates and buffer), lyophilized protective agent (the lyophilized protective agent includes a nucleic acid amplification enzyme system and a signal amplification enzyme system; the nucleic acid amplification enzyme system is composed of: recombinase, single-strand binding protein, polymerase and ATP energy regeneration enzyme; the signal amplification enzyme system is composed of: positioning enzyme and cutting enzyme).

[0063] Example 3: A method for simultaneously detecting Listeria monocytogenes and its drug-resistant genes fosX The kit is used to detect Listeria monocytogenes and its drug resistance genes fosX Applications

[0064] (1) Specificity detection

[0065] In this example, TaKaRa MiniBEST Bacteria Genomic DNA Extraction Kit Ver.3.0 (Tkara, 9763) was used to extract Gram-negative bacteria (G - ): Escherichia coli ( Escherichia coli ), Pseudomonas aeruginosa ( Pseudomonas aeruginosa ) and Salmonella Enteritidis ( Salmonella Enteritidis ); Gram-positive bacteria (G + ): Staphylococcus aureus ( Staphylococcus aureus ), Bacillus subtilis ( Bacillus subtilis ) and Streptococcus pneumoniae ( Streptococcus pneumoniae ) to extract DNA, and to obtain DNA and Listeria monocytogenes VirR The gene fragment plasmids were used as templates, TE was used as a negative control, and RNase-free water was used as a blank control. The kit prepared in Example 2 was used for detection.

[0066] The results are as follows Figure 3-4 As shown, three Gram-negative bacteria (G - ) and three Gram-positive bacteria (G + ) was tested for specificity, and the results showed that none of the six bacteria tested were detected, only Listeria monocytogenes and its drug-resistant genes were detected. fosX Detected; indicating that the kit provided by the present invention is effective for Listeria monocytogenes and its drug-resistant genes fosX The detection has a high specificity.

[0067] (2) Repeatability test

[0068] This example is to evaluate the stability and reliability of the test kit detection system prepared in Example 2, using Listeria monocytogenes VirR Genes and fosX The synthetic fragment (plasmid) of the drug-resistant gene was used as the experimental amplification template for technical repeat testing.

[0069] The results are as follows Figure 5-6 As shown, the detection system (VirR-RNA3F4R4 and fosX-RNA4F2R3) of the kit provided by the present invention has good repeatability, good stability and reliability.

[0070] (3) Sensitivity detection

[0071] In this example, Listeria monocytogenes VirR Genes and fosX Drug resistance gene synthetic fragment (plasmid copy number 1×10 6 ) was used as a template and the plasmid was serially diluted using RNase-free water. The copy number was 1×10 5 , 1×10 4 , 1×10 3 , 500 plasmid as template for sensitivity detection;

[0072] Add the DNA samples to be tested separately (Copy number is 1×10 5 , 1×10 4 , 1×10 3 500 of Listeria monocytogenes VirR Genes and fosX drug-resistant gene synthesis fragment plasmid), Forward F / reverse R primers (VirR-RNA3F4R4 and fosX-RNA4F2R3 primer sets), RNA probes , freeze-dried protective agent, centrifuged instantly, let it stand at room temperature for 2 min, and add activation solution along the wall of the eight-tube , instantaneous centrifugation, oscillation mixing for 10 s, instantaneous centrifugation, amplification reaction using a handheld metal bath and a handheld fluorescence detector, 30 cycles at 42°C, reaction time is 30 min, and the fluorescence signal Tt value is collected once in each cycle.

[0073] The results are as follows Figure 7-8 As shown, the kit provided by the present invention can detect a DNA sample with a minimum copy number of 500, indicating that the kit provided by the present invention has high sensitivity and the gradient diluted plasmid has a good effect on the machine.

[0074] (4) Testing of actual samples

[0075] S1: 24 Listeria monocytogenes DNA samples randomly selected from the laboratory were used as test samples. High-efficiency lysis buffer was added and reacted at 95°C for 10 minutes to obtain Listeria monocytogenes DNA.

[0076] S2: The Listeria monocytogenes DNA obtained in S1 was added to the EmDEA-specific primer pair reaction system in the kit prepared in Example 2. An amplification reaction was performed using a handheld metal bath and a handheld fluorescence detector. The plasmid was used as a standard and TE was used as a negative control. The detection results were determined based on the fluorescence signal Tt value.

[0077] The kit prepared in Example 2 was used to test the effect of on-site detection of actual samples. 24 Listeria monocytogenes DNA samples were randomly selected from the laboratory for detection. The results are as follows: Figure 9-10 As shown, all 24 randomly selected samples were accurately detected, and Listeria monocytogenes and its drug-resistant genes were successfully detected. fosX , no false negative or false positive results occurred. It can be seen that the kit provided by the present invention can be used for clinical detection of Listeria monocytogenes and its drug resistance genes fosX, It can achieve fast, accurate and highly sensitive detection, and is suitable for food safety and clinical infection prevention and control.

[0078] Any matters not described in detail in this specification are well known to those skilled in the art. Although the present invention has been disclosed above with reference to preferred embodiments, these are not intended to limit the present invention. Anyone skilled in the art may make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention shall be determined by the claims.

Claims

1. A method for simultaneous detection of Listeria monocytogenes and its drug-resistant genes fosX The kit is characterized in that The kit includes: an EmDEA-specific primer pair and an RNA probe; The EmDEA-specific primer pair includes F4, R4, fosX-F2, and fosX-R3, wherein the nucleotide sequence of F4 is shown in SEQ ID NO.4, and the nucleotide sequence of R4 is shown in SEQ ID NO.16; the nucleotide sequence of fosX-F2 is shown in SEQ ID NO.20, and the nucleotide sequence of fosX-R3 is shown in SEQ ID NO.33; The RNA probe includes RNA3 and fosX-RNA4, the RNA3 nucleotide sequence is shown in SEQ ID NO.9, and the fosX-RNA4 nucleotide sequence is shown in SEQ ID NO.

28.

2. The kit according to claim 1, wherein The 5' end of the RNA probe is labeled with a reporter group, and the 3' end of the RNA probe is labeled with a quencher group; the reporter group is any one of FAM, HEX, TET, Cyanine dye, ROX or Texas Red, and the quencher group is any one of TAMRA, BHQ1, BHQ2, BHQ3, Iowa Black or BBQ.

3. The kit according to claim 1, wherein The reaction system of the EmDEA specific primer pair is as follows: 7 μL of the DNA sample to be tested, 1 μL each of 10 μM forward F and reverse R primers, 1 μL of 1 μM RNA probe, 10 μL of activation solution, and lyophilized protective agent; The freeze-dried protective agent comprises a nucleic acid amplification enzyme system and a signal amplification enzyme system; the nucleic acid amplification enzyme system comprises: recombinase, single-chain binding protein, polymerase and ATP energy regeneration enzyme; the signal amplification enzyme system comprises: positioning enzyme and cutting enzyme.

4. The kit according to claim 3, wherein The activation solution is composed of: NTP nucleoside triphosphate and buffer.

5. A method for using the kit according to any one of claims 3 to 4 for non-diagnostic purposes, characterized in that: The method of use comprises the following steps: S1: Collect the sample to be tested, add lysis buffer, and react at 95°C for 10 min to obtain the DNA of the sample to be tested; S2: Add the sample DNA obtained in S1 to the reaction system of the EmDEA specific primer pair, perform the amplification reaction using a handheld metal bath and a handheld fluorescence detector, and determine the test result based on the fluorescence signal Tt value.

6. The method of use according to claim 5, wherein: The amplification reaction conditions are: 30 cycles at 42° C., and a reaction time of 10-30 min.

7. The method of use according to claim 5, wherein: The lysis solution contains pH=8 Tris, NaCl, EDTA, SDS, PVP-40, and a broad-spectrum protease.

8. The method of use according to claim 5, characterized in that: The judgment criteria for the fluorescence signal Tt value in S2 are: If no fluorescent signal Tt value appears during the amplification cycle, the test result is negative, indicating that the sample is not infected with Listeria monocytogenes or does not contain Listeria monocytogenes resistance genes. fosX ; If the fluorescent signal Tt value appears during the amplification cycle, the test result is positive, indicating that the sample is infected with Listeria monocytogenes or contains Listeria monocytogenes resistance genes. fosX .

9. The kit according to any one of claims 1 to 4 for detecting Listeria monocytogenes and its drug resistance genes fosX The application does not involve the diagnosis and treatment of diseases.

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