CN115029458A - Multiple fluorescent quantitative PCR primer probe group for simultaneously detecting four pathogenic bacteria, method and application - Google Patents

Abstract

The invention relates to a multiplex fluorescence quantitative PCR primer probe group for simultaneously detecting four pathogenic bacteria, a method and application thereof, belonging to the field of molecular biology, wherein the probe primer group comprises a primer pair and a probe aiming at a staphylococcus aureus Nuc gene, a salmonella invA gene, a listeria monocytogenes Hly gene and an escherichia coli O157: H7rfbE gene respectively. The method provided by the invention can be used for simultaneously detecting four pathogenic bacteria, namely staphylococcus aureus, salmonella, listeria monocytogenes and escherichia coli O157: H7 by combining real-time fluorescent quantitative PCR and multiple fluorescent quantitative PCR technologies, so that the detection efficiency is greatly improved, the detection cost is saved, and the requirement of large-scale rapid detection is met.

Description

Multiple fluorescent quantitative PCR primer probe group for simultaneously detecting four pathogenic bacteria, method and application

Technical Field

The invention relates to the field of molecular biology, in particular to a multiple fluorescence quantitative PCR primer probe group for simultaneously detecting four pathogenic bacteria, a method and application thereof.

Background

Staphylococcus aureus (Staphylococcus aureus), Salmonella (Salmonella), Listeria monocytogenes (Listeria monocytogenes), escherichia coli O157: h7 (Enteromorrhhagic E. coli O157: H7) is four common food-borne pathogenic bacteria and is also 4 pathogenic bacteria which are mainly detected in the food safety and sanitation standard in China. Staphylococcus aureus is a common gram-positive bacterium, widely distributed in nature, and can produce enterotoxin as a common food-borne pathogenic bacterium to cause severe diseases such as food poisoning, toxin shock syndrome, osteomyelitis, necrotizing pneumonia, endocarditis and the like. Salmonella gram-negative enterobacteria, whose infections are zoonotic infectious diseases, are mainly caused by eating contaminated food, eggs, poultry and meat products are the main transmission vectors of salmonellosis, and infections are mainly determined by the serotype of Salmonella and the physical condition of eaters, and are most threatened by children, the elderly and immunodeficient individuals. Listeria monocytogenes is a facultative anaerobic gram-positive bacterium, is a pathogen of Listeria, mainly uses food as an infection medium, can still grow and reproduce in an environment at 4 ℃, is one of the most fatal food-borne pathogens, and causes two to three-generation of infected people to die. Escherichia coli O157H 7 belongs to Enterobacteriaceae Escherichia genus gram-negative bacteria, Escherichia coli O157H 7 infection dose is extremely low. The latent period is 3-10 days, and the course of disease is 2-9 days. Severe abdominal pain and watery diarrhea usually occur suddenly, and hemorrhagic diarrhea occurs after several days, with or without fever. Some patients may develop HUS, TTP, etc., and severe cases may lead to death.

In food-borne pathogenic bacteria detection, the traditional separation culture technology is used as a main method technology for identifying and detecting microorganisms for a long time, and the identification purpose is achieved by further performing serum analysis after certain pathogenic bacteria are determined through separation culture, staining microscopic examination, biochemical reaction, hemolytic experiment and the like. With the development of molecular biology, the real-time fluorescent quantitative PCR technology, especially the fluorescent quantitative PCR technology based on TaqMan probe, is gradually applied to the detection of food-borne pathogenic bacteria, compared with the traditional microorganism separation culture identification, the fluorescent quantitative PCR technology has the advantages of high sensitivity, short detection period, strong specificity and the like, and because the combination cross influence and the addition amount of a plurality of pairs of primers and probes are different, currently, single real-time fluorescent quantitation is mostly used for detecting the pathogenic bacteria, and the detection system capable of simultaneously detecting a plurality of target genes is less. The large-scale rapid pathogen detection is required no matter in routine food sampling inspection or in dealing with sudden and serious food safety accidents, so that the establishment of a fluorescent quantitative PCR system for simultaneously detecting staphylococcus aureus, salmonella, listeria monocytogenes and escherichia coli O157: H7 has very important practical significance.

Disclosure of Invention

In view of the above problems, the present invention provides a multiplex fluorescence quantitative PCR primer probe set for simultaneously detecting four pathogenic bacteria, and provides a detection method and an application of the primer probe set. The method provided by the invention can be used for simultaneously detecting four pathogenic bacteria, namely staphylococcus aureus, salmonella, listeria monocytogenes and escherichia coli O157: H7 by combining real-time fluorescent quantitative PCR and multiple fluorescent quantitative PCR technologies, so that the detection efficiency is greatly improved, the detection cost is saved, and the requirement of large-scale rapid detection is met.

The invention adopts the specific scheme that:

the multiplex fluorescence quantitative PCR primer probe group for simultaneously detecting the four pathogenic bacteria comprises a primer pair and a probe which respectively aim at a staphylococcus aureus Nuc gene, a salmonella invA gene, a listeria monocytogenes Hly gene and an escherichia coli O157: H7rfbE gene;

wherein, the upstream primer aiming at the staphylococcus aureus Nuc gene is shown as SEQ ID NO: 1, the downstream primer is a primer with a sequence shown as SEQ ID NO: 2, the probe is a probe with a sequence shown in SEQ ID NO: Nuc-P shown in 3; the upstream primer aiming at the salmonella invA gene is shown as SEQ ID NO: 4, the downstream primer is an primer with a sequence shown as SEQ ID NO: 5, probe is shown as SEQ ID NO: 6, invA-P;

the upstream primer aiming at the Hly gene of the listeria monocytogenes is shown as SEQ ID NO: 7, and the downstream primer is a primer with a sequence shown as SEQ ID NO: 8, and the probe is a probe with a sequence shown in SEQ ID NO: 9, Hly-P;

the upstream primer aiming at the Escherichia coli O157H 7rfbE gene is shown as SEQ ID NO: 10, and the downstream primer is an rfbE-F shown in SEQ ID NO: 11, probe is an rfbE-R shown in SEQ ID NO: rfbE-P as shown in 12.

As a further optimization of the scheme, the 5 'end of the probe sequence of the staphylococcus aureus Nuc gene is modified with TexRed, the 5' end of the probe sequence of the salmonella invA gene is modified with Cy5, the 5 'end of the probe sequence of the listeria monocytogenes Hly gene is modified with HEX, and the 5' end of the probe sequence of the escherichia coli O157: H7rfbE gene is modified with FAM; the 3' ends of all the probes are modified with BHQ 1.

The invention also provides a multiplex real-time fluorescent quantitative PCR method for simultaneously detecting four pathogenic bacteria, which comprises the following steps:

step one, extracting genome DNA of a sample to be detected for later use;

step two, adding the genome DNA of a sample to be detected into a multiplex fluorescence quantitative PCR reaction system as a template, wherein the system comprises the primer probe group;

and step three, placing the reaction system in the step two in a fluorescent quantitative PCR instrument for amplification, collecting a fluorescent signal in the PCR amplification process, and judging whether pathogenic bacteria exist or not through fluorescent signal analysis.

The invention also provides a multiple real-time fluorescent quantitative PCR kit for simultaneously detecting four pathogenic bacteria, which comprises PCR reaction liquid, wherein the reaction liquid contains the primer probe group.

As a further optimization of the kit, the reaction liquid bagContaining a reaction solution A and a reaction solution B; the reaction solution A is 2 XPCR Mix, and comprises the following components: taq enzyme 4-8U, dNTP 500nM, Tris-HCl 200mM, KCL 800mM and Mg pH8.3 ²⁺ 10 mM; the reaction solution B comprises the following components: primers Nuc-F, Nuc-R, invA-F, invA-R, Hly-F, Hly-R, rfbE-F, rfbE-R of the four pathogenic bacteria are respectively 500nM and probes Nuc-P, invA-P, Hly-P, rfbE-P are respectively 400 nM.

As further optimization of the kit, the kit also comprises a negative quality control product and a positive quality control product; the negative quality control product is DEPC water, and the positive quality control product is a mixture of plasmids constructed according to target genes of staphylococcus aureus, salmonella, listeria monocytogenes and escherichia coli O157: H7 in a ratio of 1:1:1: 1.

As a further optimization of the kit, the use method comprises the following steps: adding 50-100ng of template into the reaction liquid A10ul and the reaction liquid B1.8ul, supplementing water to 20ul to form a reaction system, and carrying out fluorescent quantitative PCR reaction according to 40 circulation procedures of 5min-10min at 95 ℃, 20s-30s for denaturation at 95 ℃, 30s-35s for annealing/extension at 60 ℃ and 40 cycles.

Furthermore, collecting fluorescence signals in the PCR amplification process, and carrying out positive judgment through the Ct value of a certain channel gene in a sample to be detected and an S-type amplification curve: a) if the amplification of a certain gene channel of the sample to be detected has an S-shaped amplification curve and Ct is less than or equal to 35, judging that the sample to be detected contains the pathogenic bacteria; b) if a certain gene channel of the sample to be detected does not have an S-shaped amplification curve and Ct is not less than 35, judging that the sample to be detected does not contain the pathogenic bacteria; c) if a certain gene channel of the sample to be detected has an S-shaped amplification curve and Ct is less than or equal to 35 and less than or equal to 40, judging the sample to be uncertain, and re-extracting the nucleic acid for rechecking.

The invention finally provides the application of the primer probe set, the detection method or the kit in any one of the following methods:

(a) detecting at least one pathogenic bacterium of staphylococcus aureus, salmonella, listeria monocytogenes and escherichia coli O157: H7;

(b) preparing a medicament or product for use in the detection of (a).

Has the advantages that: the invention has the advantages that the aim of simultaneously detecting staphylococcus aureus, salmonella, listeria monocytogenes and escherichia coli O157: H7 pathogenic bacteria is realized based on a TaqMan probe multiplex real-time fluorescent quantitative PCR detection technology, the kit has the characteristics of strong specificity, high sensitivity, simplicity, convenience and rapidness, the experimental process is simplified, the detection efficiency can be greatly improved, the detection cost is saved, the requirement of modern large-scale rapid detection of pathogenic bacteria can be met, and the kit has high practical application value.

Drawings

FIG. 1 is a curve of simultaneous positive amplification of four pathogenic bacteria;

FIG. 2 is a graph of a Staphylococcus aureus positive amplification curve;

FIG. 3 is a Salmonella positive amplification curve;

FIG. 4 is a Listeria monocytogenes positive amplification curve;

FIG. 5 shows the amplification curve of Escherichia coli O157: H7;

FIG. 6 is a negative control amplification curve;

FIG. 7 is a multiple fluorescent quantitative PCR standard curve for four pathogenic bacteria;

FIG. 8 is a multiplex fluorescence quantitative PCR sensitivity test for four pathogenic bacteria.

Detailed Description

The invention provides a multiple fluorescence quantitative PCR kit for simultaneously detecting pathogenic bacteria, which comprises a PCR reaction solution A, a reaction solution B, positive quality control and negative control.

The reaction solution A is 2 XPCR Mix and comprises the following components: taq enzyme 4-8U, dNTP 500nM, Tris-HCL 200mM, KCL 800mM, Mg of pH8.3 ²⁺ 10mM。

The reaction liquid B comprises primers and probes aiming at a staphylococcus aureus Nuc gene, a salmonella invA gene, a listeria monocytogenes Hly gene and an escherichia coli O157H 7rfbE gene, the sequences of the primers and the probes are SEQ ID Nos. 1-12 shown in Table 1, wherein SEQ ID Nos. 1, 2, 4, 5, 7, 8, 10 and 11 are primer sequences Nuc-F, Nuc-R, invA-F, invA-R, Hly-F, Hly-R, rfbE-F, rfbE-R respectively, SEQ ID Nos. 3, 6, 9 and 12 are probe sequences Nuc-P, invA-P, Hly-P, rfbE-P respectively, 5 'ends of all the probe sequences are modified with reporter groups of TexRed, Cy5, HEX and FAM respectively, 3' ends are modified with quenching groups of BHQ1 respectively, and simultaneously primers of four pathogenic bacteria Nuc-F, Salmonella in the reaction liquid B, Nuc-R, invA-F, invA-R, Hly-F, Hly-R, rfbE-F, rfbE-R is 500nM each, and probe Nuc-P, invA-P, Hly-P, rfbE-P is 400nM each.

Table 1 probe primer combinations for simultaneous detection of four pathogens according to the present invention.

The negative quality control product is DEPC water, and the positive quality control product is a mixture of plasmids constructed according to target genes of staphylococcus aureus Nuc, salmonella invA, listeria monocytogenes Hly and escherichia coli O157: H7rfbE in a ratio of 1:1:1: 1.

The invention also aims to provide a multiple real-time fluorescence quantitative PCR method for simultaneously detecting four pathogenic bacteria, which comprises the following steps:

(1) extracting the genome DNA of the sample to be detected for later use;

(2) adding the genome DNA of a sample to be detected into a quadruple real-time fluorescent quantitative PCR reaction system by taking the genome DNA of the sample to be detected as a template, wherein the reaction system is as follows: reaction liquid A10ul, reaction liquid B1.8ul, template DNA50-100ng, and water to 20 ul;

(3) placing the reaction system in a fluorescent quantitative PCR instrument, selecting a TexRed channel for detecting a staphylococcus aureus Nuc gene, Cy5 for detecting a salmonella invA gene, a FAM channel for detecting an Escherichia coli O157: H7rfbE gene, a HEX/VIC channel for detecting a Listeria monocytogenes Hly gene, selecting Reference fluorescence (Passive Reference) as none, and setting Sample Volume as 20;

(4) and analyzing results, wherein the negative control has no Ct value and no typical S-type amplification curve, FAM, CY5, TexRed and HEX/VIC channels detected by the positive control have obvious S-type amplification curves, the Ct value is between 20 and 25, and positive judgment is carried out through the Ct value of a certain channel gene in the sample to be detected and the S-type amplification curve. a) If a certain gene channel of the sample to be detected is amplified and has an S-shaped amplification curve and Ct is less than or equal to 35, judging that the sample to be detected is the pathogenic bacterium; b) if a certain gene channel of the sample to be detected does not have an S-shaped amplification curve and Ct is not less than 35, judging that the sample does not contain the pathogenic bacteria; c) if a certain gene channel of the sample to be detected has an S-type amplification curve and Ct is less than or equal to 35 and less than or equal to 40, the sample is judged to be an uncertain sample, and the nucleic acid re-detection needs to be extracted again.

The invention will be elucidated in more detail below by means of specific embodiments and the associated drawings. Unless otherwise stated, the experimental reagent and the experimental instrument involved in the invention are conventional commercial products.

Example 1 a kit for simultaneously detecting staphylococcus aureus, salmonella, listeria monocytogenes, and escherichia coli O157 was prepared.

1. Preparing a reaction solution A: Tris-HCl 200mM, KCL 800mM and Mg at pH8.3 according to Taq enzyme 4-8U, dNTP 500nM ²⁺ Reaction solution A was prepared at a content ratio of 10 mM.

2. Preparing a reaction solution B: 500nM of upstream and downstream primers of four pathogenic bacteria respectively, and 400nM of probes respectively, shaking, mixing uniformly and storing in dark place.

3. Positive quality control product: staphylococcus aureus Nuc, salmonella invA, Listeria monocytogenes Hly, Escherichia coli O157: H7rfbE target gene construct plasmid according to 1:1:1:1 volume ratio mixture.

4. Negative quality control product: DEPC water

Example 2 sample treatment

Genomic DNA of target pathogenic bacterium was determined using GNT from Loyang Gintde Biotech Ltd ^TM The bacterial DNA extraction kit (magnetic bead method) was used for extraction, and the procedures were as described in the specification. The positive quality control plasmids were extracted using a plasmid miniprep kit (DP103) from Tiangen Biochemical technology (Beijing) Ltd, and mixed at a volume ratio of 1:1:1:1 according to the instructions.

Example 3 real-time fluorescent quantitative PCR detection of four pathogenic bacteria

The PCR reaction system is 20ul, wherein the reaction solution A is 10ul, the reaction solution B is 1.8ul, the template to be detected (positive plasmid) is 4ul, and water is added to supplement the reaction solution to 20 ul. The real-time fluorescent quantitative PCR instrument is ABI7500, the reaction program is 95 ℃ for 5min, 95 ℃ for 20s, 60 ℃ for 30s, 40 cycles.

The curve of the simultaneous positive amplification of the four pathogenic bacteria is shown in figure 1, the curves of the amplification of the four pathogenic bacteria and the positive control are shown in figures 2-5, and the negative control is shown in figure 6. As shown in the attached drawing, the positive control and the negative control are controllable, and the four pathogenic bacteria have no non-specific amplification, so that the kit can be used for detecting the four pathogenic bacteria simultaneously.

The results show that: the kit can simultaneously detect four common pathogenic bacteria, namely staphylococcus aureus, salmonella, listeria monocytogenes and escherichia coli O157 within 2 hours, greatly improves the detection efficiency, shortens the detection period, has the characteristics of simple and convenient operation, strong practicability and the like, can meet the requirement of modern large-scale rapid detection of the pathogenic bacteria, and can generate good economic and social benefits.

Example 4 specificity and sensitivity of multiplex real-time fluorescent quantitative PCR of four pathogenic bacteria

(1) And (3) specific test of probes and primers:

after the 25 known strains are rapidly amplified, extracting genome DNA, and performing multiplex qPCR amplification detection on the 25 known strains by using the PCR system and the reaction conditions determined in the example 3, wherein the detection result completely conforms to the expected design: the four target pathogenic bacteria have amplification curves, and other non-target bacteria have no amplification condition. The experimental result of specificity detection shows that the probe and the primer designed by the experiment have high species specificity.

Table 2: and (3) multiple real-time fluorescent quantitative PCR specificity test results.

(2) Establishing a multiplex real-time fluorescent quantitative PCR standard curve and testing the sensitivity:

the DNA template of each target bacterium was extracted and the concentration and purity (A260/A280) were measured as in example 2 above, and the ratio of Staphylococcus aureus: 136.47ng/ul and 1.832, Salmonella: 205.38ng/ul and 1.896, Listeria monocytogenes: 75.61ng/ul and 1.923, Escherichia coli O157: H7: 175.82ng/ul and 1.786. Each template DNA was started at 50ng/ul using ddH ₂ O was diluted to 0.005ng/ul in 10-fold gradients, multiplex qPCR amplifications were performed according to optimized reaction system and procedure, standard curves were established and their sensitivity was determined, 3 replicates per dilution gradient. The standard curves of the four pathogenic bacteria are shown in fig. 7, and the linear equation of the standard curve of staphylococcus aureus is as follows: 5.0091x +6.6386, R ² 0.9988; the linear equation of the standard curve of the salmonella is as follows: 5.3573x +1.4095, R ² 0.9996; the linear equation of the standard curve of the listeria monocytogenes is as follows: 4.0238x +4.8358, R ² 0.9992; the standard curve linear equation of Escherichia coli O157H 7 is as follows: 5.4304x +1.6392, R ² 0.9996. The results show that R ² Are all larger than 0.99, and the logarithm (X axis) of the template DNA concentration and the Ct value (Y axis) have good linear relation. The sensitivity of the multiplex fluorescence quantitative PCR is shown in figure 8, the Ct value is less than or equal to 35 as a positive judgment result, and the minimum detection concentration of the four pathogenic bacteria is 0.005 ng/ul.

EXAMPLE 5 detection of artificially contaminated sample

Due to the diversity of food and the complexity of food matrix, various substances inhibiting PCR reaction may exist in the food, so in order to test the wide applicability of the method, the multiple fluorescence quantitative PCR system established by the method is used for testing the four pathogenic bacteria in artificially contaminated food. According to the national standard (GB 29921 and 2021 pathogenic bacteria limit in food safety national standard prepackaged food), GB 31607 and 2021 pathogenic bacteria limit in food safety national standard bulk instant food), the corresponding food is purchased, and the types of prepackaged and bulk food are basically covered. Adding 100ul of bacterial suspension into each 25g/25ml sample, artificially polluting the food sample by using four pathogenic bacteria, increasing bacteria in a TSB culture medium for 6-8 hours, extracting bacterial gene DNA according to the sample processing method in the embodiment 2, and performing multiplex fluorescence quantitative PCR detection on the reaction system and the reaction program in the embodiment 3, wherein the results are shown in the table 3.

Table 3: and (5) detecting the artificially contaminated sample.

It should be noted that the above-mentioned embodiments illustrate rather than limit the scope of the invention, which is defined by the appended claims. It will be apparent to those skilled in the art that certain insubstantial modifications and adaptations of the present invention can be made without departing from the spirit and scope of the invention.

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Claims (9)

1. The multiple fluorescent quantitative PCR primer probe group for simultaneously detecting four pathogenic bacteria is characterized in that: the probe primer group comprises a primer pair and a probe which respectively aim at a staphylococcus aureus Nuc gene, a salmonella invA gene, a listeria monocytogenes Hly gene and an escherichia coli O157: H7rfbE gene;

wherein, the upstream primer aiming at the staphylococcus aureus Nuc gene is shown as SEQ ID NO: 1, the downstream primer is a primer with a sequence shown as SEQ ID NO: 2, the probe is a probe with a sequence shown in SEQ ID NO: Nuc-P as shown in 3; the upstream primer aiming at the salmonella invA gene is shown as SEQ ID NO: 4, the downstream primer is an primer with a sequence shown as SEQ ID NO: 5, probe is shown as SEQ ID NO: 6, invA-P;

the upstream primer aiming at the Hly gene of the listeria monocytogenes is shown as SEQ ID NO: 7, and the downstream primer is a primer with a sequence shown in SEQ ID NO: 8, and the probe is an Hly-R shown in SEQ ID NO: 9, Hly-P;

the upstream primer aiming at the Escherichia coli O157H 7rfbE gene is shown as SEQ ID NO: 10, and the downstream primer is an rfbE-F shown in SEQ ID NO: 11, probe is an rfbE-R shown in SEQ ID NO: rfbE-P as shown in 12.

2. The primer probe set of claim 1, wherein: the 5 'end of the probe sequence of the staphylococcus aureus Nuc gene is modified with TexRed, the 5' end of the probe sequence of the salmonella invA gene is modified with Cy5, the 5 'end of the probe sequence of the listeria monocytogenes Hly gene is modified with HEX, and the 5' end of the probe sequence of the escherichia coli O157H 7rfbE gene is modified with FAM; the 3' ends of all the probes are modified with BHQ 1.

3. A multiple real-time fluorescent quantitative PCR method for simultaneously detecting four pathogenic bacteria is characterized in that: the method comprises the following steps:

step one, extracting genome DNA of a sample to be detected for later use;

step two, taking the genome DNA of a sample to be detected as a template and adding the template into a multiplex fluorescence quantitative PCR reaction system, wherein the system comprises the primer probe group in claim 2;

and step three, placing the reaction system in the step two in a fluorescent quantitative PCR instrument for amplification, collecting a fluorescent signal in the PCR amplification process, and judging whether pathogenic bacteria exist or not through fluorescent signal analysis.

4. A multiple real-time fluorescent quantitative PCR kit for simultaneously detecting four pathogenic bacteria is characterized in that: the kit comprises a PCR reaction solution containing the primer probe set according to claim 2.

5. The kit of claim 4, wherein: the reactionThe liquid comprises a reaction liquid A and a reaction liquid B; the reaction solution A is 2 XPCR Mix, and comprises the following components: taq enzyme 4-8U, dNTP 500nM, Tris-HCl 200mM, KCL 800mM and Mg pH8.3 ²⁺ 10 mM; the reaction solution B comprises the following components: 500nM each of primers Nuc-F, Nuc-R, invA-F, invA-R, Hly-F, Hly-R, rfbE-F, rfbE-R and 400nM each of probes Nuc-P, invA-P, Hly-P, rfbE-P of four pathogenic bacteria.

6. The kit of claim 5, wherein: the kit also comprises a negative quality control product and a positive quality control product; the negative quality control product is DEPC water, and the positive quality control product is a mixture of plasmids constructed according to target genes of staphylococcus aureus, salmonella, listeria monocytogenes and escherichia coli O157: H7 in a ratio of 1:1:1: 1.

7. The kit of claim 6, wherein: the method for detecting pathogenic bacteria by adopting the kit comprises the following steps: adding 50-100ng of template into reaction liquid A10ul and reaction liquid B1.8ul, supplementing water to 20ul to form a reaction system, and carrying out fluorescent quantitative PCR reaction according to 40 circulation procedures of 5min-10min at 95 ℃, 20s-30s for denaturation at 95 ℃, 30s-35s for annealing/extension at 60 ℃ and 40 cycles; and (3) collecting a fluorescence signal in the PCR amplification process, and carrying out positive judgment through the Ct value of a certain channel gene in the sample to be detected and the S-shaped amplification curve.

8. The kit of claim 7, wherein: the judgment standard is as follows: a) if the amplification of a certain gene channel of the sample to be detected has an S-shaped amplification curve and Ct is less than or equal to 35, judging that the sample to be detected contains pathogenic bacteria; b) if a certain gene channel of the sample to be detected does not have an S-shaped amplification curve and Ct is not less than 35, judging that the sample to be detected does not contain the pathogenic bacteria; c) if a certain gene channel of the sample to be detected has an S-shaped amplification curve and Ct is less than or equal to 35 and less than or equal to 40, judging the sample to be uncertain, and re-extracting the nucleic acid for rechecking.

9. Use of a primer probe set according to claim 2, a detection method according to claim 3 or a kit according to any one of claims 4 to 8 in any one of:

(a) detecting at least one pathogenic bacterium of staphylococcus aureus, salmonella, listeria monocytogenes and escherichia coli O157: H7;

(b) preparing a medicament or product for use in the detection of (a).

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