Detection method for detecting enteropathogenic bacteria, primer probe group and application

Abstract

The invention discloses a detection method for detecting enteropathogenic bacteria, and relates to the field of molecular biology, specific genes are searched in genomes of salmonella, shigella, vibrio parahaemolyticus, campylobacter jejuni, diarrheagenic escherichia coli, vibrio cholerae and staphylococcus aureus, a beta-actin gene is adopted as a reference gene, and the enteropathogenic bacteria are detected. Primer and probe sequences are designed, a probe group consisting of the primer and probe sequences is used for carrying out real-time fluorescent PCR amplification reaction on nucleic acid in a detected sample, then melting curve analysis is carried out, and whether the pathogenic bacteria exist in the nucleic acid sequence to be detected or not can be judged according to a Tm value. The detection method provided by the invention has the advantages of rapidness, high throughput, accuracy and high sensitivity.

Description

Technical Field

[0001] The present invention relates to the field of molecular biology, and in particular to a detection method, a primer probe set and an application thereof for detecting intestinal pathogenic bacteria. Background Art

[0002] Enteric pathogens mainly enter the digestive tract through contaminated food or water, and eventually colonize in the intestines to cause infectious diarrhea and other diseases. According to statistics, 1.7 billion to 5 billion people suffer from diarrhea every year, and 1.4 million people die from diarrhea. Salmonella, Shigella, Vibrio parahaemolyticus, Campylobacter jejuni, diarrheagenic Escherichia coli, Vibrio cholerae, and Staphylococcus aureus are the main pathogens that cause diarrhea. Therefore, rapid, accurate, and sensitive detection of enteric pathogens has important guiding significance for the clinical diagnosis of diarrhea, timely and effective treatment measures, and prevention of the abuse of antibiotics.

[0003] Existing methods for detecting intestinal pathogens are divided into culture methods, immunological methods, molecular biology methods, and traditional PCR detection methods. The culture method has cumbersome steps and takes several days to get results. It has low sensitivity and is prone to false detection and missed detection. Immunological methods also have problems such as the need to concentrate samples to increase the concentration of bacterial solution and high false positive rates. Molecular biological detection methods are mainly polymerase chain reaction technology, which performs specific detection on a single pathogen. The detection process of a mixture of multiple pathogens is cumbersome, often detecting different pathogens separately and taking a long time; and when multiple primers of multiple pathogens are mixed and detected simultaneously, multiple primers of multiple pathogens are prone to interference, poor specificity, and low sensitivity. Traditional PCR detection methods mainly use a single fluorescent channel to detect a single target, with a small throughput. To detect multiple targets, multiple tests are required, which is time-consuming and has low detection efficiency.

[0004] In response to the problems of time-consuming and low detection efficiency of existing technologies, China's invention patent "A kit for rapid detection of pathogenic bacteria of intestinal infection and its detection method" patent number CN202410759015.X discloses a kit for rapid detection of pathogenic bacteria of intestinal infection and its detection method, and establishes a multiple fluorescent LAMP kit and its detection method for simultaneously detecting four pathogens of Vibrio cholerae, diarrheagenic Escherichia coli, Staphylococcus aureus and Salmonella enterica. This method has the advantages of high efficiency, rapidity, high sensitivity and specificity, but the method has fewer detection items, and a single fluorescent channel only detects a single target.

[0005] Therefore, it is necessary to establish a rapid, high-throughput, accurate and highly sensitive detection method to overcome the limitations of existing molecular biology techniques, so as to detect a variety of intestinal pathogens, quickly obtain diagnostic results, and help clinicians guide medication. It can also be used in population epidemiological surveys, which has very important clinical significance and value. Summary of the invention

[0006] Based on this, the purpose of the present invention is to provide a primer probe set for intestinal pathogenic bacteria detection, which can be applied to intestinal pathogenic bacteria detection and has the advantages of rapidity, high throughput, accuracy and high sensitivity.

[0007] A primer probe set for detecting intestinal pathogenic bacteria, wherein the probe set amplifies specific target sequences of the genomes of Salmonella, Shigella, Vibrio parahaemolyticus, Campylobacter jejuni, diarrheagenic Escherichia coli, Vibrio cholerae, and Staphylococcus aureus, and the probe set uses a β-actin gene as an internal reference gene.

[0008] In one embodiment, the probe set comprises:

[0009] (1) Salmonella primer and probe sequences:

[0010] Upstream primer:

[0011] 5'-ACTATACTATTGTTAATTAACACGGTTGAGGATGTTATTCGCAATCG-3';

[0012] Downstream primer: 5′-TCCATCAAATTAGAGTAGGC-3′;

[0013] Probe: 5′-CGTGTACTATACTATTGTTAATTAACACG-3′;

[0014] (2) Shigella primer and probe sequences:

[0015] Upstream primer: 5′-CGTACTAATATTTGCACACGAGTGCGGAGGTCACTAGC-3′;

[0016] Downstream primer: 5′-TCCACTGCCGTCAAGTAA-3′;

[0017] Probe: nCoV19-NP: 5′-CGTGTCGTACTAATATTTGCACACG-3′;

[0018] (3) Primer and probe sequences for Vibrio parahaemolyticus:

[0019] Upstream primer:

[0020] 5'-CCACAGCATCGCAGCACATACACGACTCAACACAAGAAGAGATCGATAA-3';

[0021] Downstream primer: 5′-GATGAGCGGTrGATGTCGTA-3′;

[0022] Probe: 5′-CGTGTCCACAGCATCGCAGCACATACACG-3′;

[0023] (4) Campylobacter jejuni primer and probe sequences:

[0024] Upstream primer:

[0025] 5'-AATAAATTAATATGTAAACTGGCTATTTTATTTTTGAGTGCTTGTG-3';

[0026] Downstream primer: 5′-GCTTTATTGCCATTTGTTTTATTA-3′;

[0027] Probe: 5′-CCAGTAATAAATTAATATGTAAACTGG-3′;

[0028] (5) Primer and probe sequences for diarrheagenic Escherichia coli:

[0029] Upstream primer: 5′-AACTAGCTCGTAGAACAACTGGCAATTTTCAGCCAATATGATTG-3′;

[0030] Downstream primer: 5′-AAACTTCAATCTTGATCAGTTTC-3′;

[0031] Probe: 5′-CCAGTAACTAGCTCGTAGAACAACTGG-3′;

[0032] (6) Primer and probe sequences for Vibrio cholerae:

[0033] Upstream primer:

[0034] 5'-TTAATGCAATATATATATAGTCGTGTTAGGCACGATGATGGAATTG-3';

[0035] Downstream primer: 5′-GACCAGACAATATAGTTTGACCCTCT-3′;

[0036] Probe: 5′-CGACTTTGAATCAATATATATATAGTCG-3′;

[0037] (7) Staphylococcus aureus primer and probe sequences:

[0038] Upstream primer:

[0039] 5'-CTACAATTCTATTAGATCACTCGCAGTGCAATGGGAAATGATATAT-3';

[0040] Downstream primer: 5′-TCTGTAAATTTACCACTAACACCATTCAA-3′;

[0041] Probe: 5′-CGAGTATACACTTCTATTAGATCACTCG-3′;

[0042] (8) Internal reference β-actin gene primer and probe sequences:

[0043] Upstream primer:

[0044] 5'-ACAGCAACAGCAGCACACGACTCGAATGAGCTGCGTGTGCATC-3';

[0045] Downstream primer: 5′-GGCTGGGGTGTTGAAACTC-3′;

[0046] Probe: 5′-CGAGTACAGCAACAGCAGCACACGACTCG-3′.

[0047] In one embodiment, the probe of the probe group is labeled with a fluorescent group at the 5' end and a quenching group at the 3' end, or with a fluorescent group at the 3' end and a quenching group at the 5' end; when not hybridized with the target sequence, the probe itself forms a neck ring structure, so that the fluorescent group and the quenching group are close to each other, and the fluorescence emitted by the fluorescent group is absorbed by the quenching group; when hybridized with the target sequence, the probe neck ring structure dissociates, so that the fluorescent group and the quenching group are separated, and the fluorescence emitted by the fluorescent group cannot be absorbed by the quenching group.

[0048] In one embodiment, the fluorescent group includes various commonly used fluorescent markers, but is not limited to these fluorescent markers, such as ALEX-350, FAM, VIC, TET, CAL Gold 540, JOE, HEX, CAL FluorOrange560, TAMRA, CAL Fluor Red 590, ROX, CAL Fluor Red 610, TEXAS RED, CAL FluorRed635, Quasar670, CY3, CY5, CY5.5, Quasar 705, etc.

[0049] In one embodiment, the quencher group includes various commonly used quenchers, but is not limited to these quenchers, such as DABCYL, BHQ-1, BHQ-2, ECLIPE, etc.

[0050] The present invention also includes the use of the primer probe set for intestinal pathogen detection in detecting Boehmeria, Shigella, Vibrio parahaemolyticus, Campylobacter jejuni, diarrhea-causing Escherichia coli, Vibrio cholerae, and Staphylococcus aureus.

[0051] The second object of the present invention is to provide a detection method for intestinal pathogenic bacteria detection, comprising the following steps:

[0052] (1) Searching for specific genes in the genomes of Salmonella, Shigella, Vibrio parahaemolyticus, Campylobacter jejuni, diarrheagenic Escherichia coli, Vibrio cholerae, and Staphylococcus aureus, respectively, using the β-actin gene as an internal reference gene, and designing primer and probe sequences;

[0053] (2) Extracting nucleic acid from the test sample, performing melting curve analysis after real-time fluorescence PCR amplification reaction, and judging whether the nucleic acid sequence to be tested contains the above pathogenic bacteria based on the Tm value.

[0054] In one embodiment, the probe is labeled with a fluorescent group at the 5' end and a quencher group at the 3' end, or with a fluorescent group at the 3' end and a quencher group at the 5' end; when not hybridized with the target sequence, the probe itself forms a neck ring structure, so that the fluorescent group and the quencher group are close to each other, and the fluorescence emitted by the fluorescent group is absorbed by the quencher group; when hybridized with the target sequence, the probe neck ring structure dissociates, so that the fluorescent group and the quencher group are separated, and the fluorescence emitted by the fluorescent group cannot be absorbed by the quencher group

[0055] In one embodiment, the PCR amplification reaction system is a 20 μL reaction solution, which includes 4 μL of 5×PCR reaction solution, 25 nM upstream primer, 150 nM downstream primer, 50 nM probe, 5 μL template, and the remaining components are ultrapure water. In the no-template control group, the template in the reaction system is replaced with the same volume of pure water.

[0056] The present invention also includes the application of the above detection method in detecting Boehmeria, Shigella, Vibrio parahaemolyticus, Campylobacter jejuni, diarrheagenic Escherichia coli, Vibrio cholerae and Staphylococcus aureus.

[0057] The beneficial effects of the present invention are:

[0058] (1) The test results are interpreted based on the melting peak temperature. A single channel can detect 2 to 3 targets;

[0059] (2) High detection efficiency. It can detect 7 intestinal pathogens within 2 hours, including Salmonella, Shigella, Vibrio parahaemolyticus, Campylobacter jejuni, diarrheagenic Escherichia coli, Vibrio cholerae, and Staphylococcus aureus. The β-actin gene is used as the internal reference gene.

[0060] For better understanding and implementation, the present invention is described in detail below with reference to the accompanying drawings. BRIEF DESCRIPTION OF THE DRAWINGS

[0061] Figure 1 A melting curve formed in the FAM channel for detecting a Salmonella positive template using the primer probe and reaction system of the present invention;

[0062] Figure 2 A melting curve formed in the FAM channel for detecting Shigella positive template using the primer probe and reaction system of the present invention;

[0063] Figure 3 A melting curve formed in a FAM channel for detecting a positive template of Vibrio parahaemolyticus using the primer probe and reaction system of the present invention;

[0064] Figure 4 A melting curve formed in the VIC channel for detecting a positive template of Campylobacter jejuni using the primer probe and reaction system of the present invention;

[0065] Figure 5 A melting curve formed in the VIC channel for detecting a positive template of diarrheagenic Escherichia coli using the primer probe and reaction system of the present invention;

[0066] Figure 6 For the detection of Vibrio cholerae positive template using the primer probe and reaction system of the present invention, the melting curve formed in the CY5 channel is shown in the figure;

[0067] Figure 7 The melting curve formed in the CY5 channel for detecting the positive template of Staphylococcus aureus using the primer probe and reaction system of the present invention;

[0068] Figure 8 A melting curve formed in the CY5 channel for detecting the internal reference β-actin gene template using the primer probe and reaction system of the present invention;

[0069] Fig. 9 The primer probe and reaction system of the present invention are used to detect the melting curve formed in the FAM channel of a template containing Salmonella, Shigella, Vibrio parahaemolyticus, Campylobacter jejuni, diarrheagenic Escherichia coli, Vibrio cholerae, Staphylococcus aureus and an internal reference β-actin gene;

[0070] Fig.10 The primer probe and reaction system of the present invention are used to detect the melting curve formed in the VIC channel of a template containing Salmonella, Shigella, Vibrio parahaemolyticus, Campylobacter jejuni, diarrheagenic Escherichia coli, Vibrio cholerae, Staphylococcus aureus and an internal reference β-actin gene;

[0071] Fig.11 The primer probe and reaction system of the present invention are used to detect the melting curve formed in the CY5 channel of the template containing Salmonella, Shigella, Vibrio parahaemolyticus, Campylobacter jejuni, diarrheagenic Escherichia coli, Vibrio cholerae, Staphylococcus aureus and internal reference β-actin gene. DETAILED DESCRIPTION

[0072] The specific embodiments of the present invention are described in detail below, but it should be understood that the protection scope of the present invention is not limited by the specific embodiments. Unless otherwise expressly indicated, in the entire specification and claims, the term "comprise" or its variations such as "comprises" or "comprising" etc. will be understood to include the stated elements or components, but not to exclude other elements or other components.

[0073] Example 1: Design of primer probe set

[0074] This embodiment provides a PCR primer probe set for detecting multiple intestinal pathogens, and searches for specific genes in the genomes of Salmonella, Shigella, Vibrio parahaemolyticus, Campylobacter jejuni, diarrheagenic Escherichia coli, Vibrio cholerae, and Staphylococcus aureus, respectively. The probe set uses the β-actin gene as an internal reference gene, and the primer and probe sequences are designed. When the probe is not hybridized with the target sequence, the probe itself forms a neck ring structure, so that the fluorescent group and the quenching group are close to each other, and the fluorescence emitted by the fluorescent group is absorbed by the quenching group. When hybridizing with the target sequence, the probe neck ring structure dissociates, so that the fluorescent group and the quenching group are separated, and the fluorescence emitted by the fluorescent group cannot be absorbed by the quenching group.

[0075] The probe can be labeled with a fluorescent group at the 5' end and a quencher group at the 3' end, or the position of the fluorescent group and the position of the quencher group can be interchanged, and the fluorescent group is labeled at the 3' end and the quencher group is labeled at the 5' end. The fluorescent group can use various commonly used fluorescent markers, but are not limited to these fluorescent markers, such as ALEX-350, FAM, VIC, TET, CAL Gold 540, JOE, HEX, CAL Fluor Orange 560, TAMRA, CALFluor Red 590, ROX, CAL Fluor Red 610, TEXAS RED, CAL Fluor Red 635, Quasar670, CY3, CY5, CY5.5, Quasar 705, etc. The quencher group can be applied to various commonly used quenchers, but are not limited to these quenchers, such as DABCYL, BHQ1, BHQ2, BHQ3, ECLIPE, etc.

[0076] In this embodiment, the probe group is specifically:

[0077] Salmonella Primer and Probe Sequences:

[0078] Upstream primer:

[0079] 5'-ACTATACTATTATTAATTAACACGGTTGAGGATGTTATTCGCAATCG-3' (SEQ ID NO: 1);

[0080] Downstream primer: 5'-TCCATCAAATTAGAGTAGGC-3' (SEQ ID NO: 2).

[0081] Probe: 5'-(FAM fluorescein-labeled)-CGTGTACTATACTATTATTAATTAACACG-(BHQ1)-3' (SEQ ID NO: 3).

[0082] Shigella primer and probe sequences:

[0083] Upstream primer: 5′-CGTACTAATCTTTGCACACGAGTGCGGAGGTCACTAGC-3′ (SEQ ID NO: 4);

[0084] Downstream primer: 5'-TCCACTGCCGTCAAGTAA-3' (SEQ ID NO: 5).

[0085] Probe: nCoV19-NP: 5'-(FAM fluorescein labeled)-CGTGTCGTACTAATCTTTGCACACG-(BHQ1)-3' (SEQ ID NO: 6).

[0086] V. parahaemolyticus primer and probe sequences:

[0087] Upstream primer:

[0088] 5'-CCACAGCATCTCAGCACATACACGACTCAACACAAGAAGAGATCGATAA-3' (SEQ ID NO: 7);

[0089] Downstream primer: 5'-GATGAGCGGTrGATGTCGTA-3' (SEQ ID NO: 8).

[0090] Probe: 5'-(FAM fluorescein-labeled)-CGTGTCCACAGCATCTCAGCACATACACG-(BHQ1)-3' (SEQ ID NO: 9).

[0091] Campylobacter jejuni primer and probe sequences:

[0092] Upstream primer: 5′-AATAAATTAATATGTTAACTGGCTATTTTATTTTTGAGTGCTTGTG-3′ (SEQ ID NO: 10);

[0093] Downstream primer: 5'-GCTTTATTGCCATTTGTTTTATTA-3' (SEQ ID NO: 11);

[0094] Probe: 5'-(VIC-fluorescein-labeled)-CCAGTAATAAATTAATATGTTAACTGG-(BHQ1)-3' (SEQ ID NO: 12).

[0095] Diarrheagenic Escherichia coli primer and probe sequences:

[0096] Upstream primer: 5′-AACTAGCTCGTAGAACAACTGGCAATTTTCAGCCAATATGATTG-3′ (SEQ ID NO: 13);

[0097] Downstream primer: 5'-AAACTTCAATCTTGATCAGTTTC-3' (SEQ ID NO: 14).

[0098] Probe: 5'-(VIC-fluorescein-labeled)-CCAGTAACTAGCTCGTAGAACAACTGG-(BHQ1)-3' (SEQ ID NO: 15).

[0099] V. cholerae primer and probe sequences:

[0100] Upstream primer: 5′-TTAATGCAATATATATATAGTCGTGTTAGGCACGATGATGGAATTG-3′ (SEQ ID NO: 16);

[0101] Downstream primer: 5'-GACCAGACAATATAGTTTGACCCTCT-3' (SEQ ID NO: 17).

[0102] Probe: 5'-(CY5 fluorescein-labeled)-CGACTTTGAATCAATATATATATATAGTCG-(BHQ3)-3' (SEQ ID NO: 18).

[0103] S. aureus primer and probe sequences:

[0104] Upstream primer: 5′-CTACAATTCTATTAGATCACTCGCAGTGCAATGGGAAATGATATAT-3′ (SEQ ID NO: 19);

[0105] Downstream primer: 5'-TCTGTAAATTTACCACTAACACCATTCAA-3' (SEQ ID NO: 20).

[0106] Probe: 5'-(CY5 fluorescein-labeled)-CGAGTATACACTTCTATTAGATCACTCG-(BHQ3)-3' (SEQ ID NO: 21).

[0107] Internal reference β-actin gene primer and probe sequences:

[0108] Upstream primer: 5′-ACAGCAACAGCAGCACACGACTCGAATGAGCTGCGTGTGCATC-3′ (SEQ ID NO: 22);

[0109] Downstream primer: 5'-GGCTGGGGTGTTGAAACTC-3' (SEQ ID NO: 23).

[0110] Probe: 5'-(CY5 fluorescein-labeled)-CGAGTACAGCAACAGCAGCACACGACTCG-(BHQ3)-3' (SEQ ID NO: 24).

[0111] Example 2: Construction of detection reaction system

[0112] In this embodiment, a detection system capable of detecting or distinguishing Salmonella, Shigella, Vibrio parahaemolyticus, Campylobacter jejuni, diarrheagenic Escherichia coli, Vibrio cholerae, and Staphylococcus aureus is constructed.

[0113] The specific detection steps are:

[0114] (1) Extraction of nucleic acid from test samples:

[0115] Use a commercial fecal microbial nucleic acid extraction kit to extract nucleic acids (RNA / DNA) from the test samples. The specific operations can be carried out according to the instructions provided by the kit manufacturer.

[0116] (2) Configuration of PCR amplification reaction system:

[0117] The nucleic acid extracted in step (1) was used as a template, and pure water was used as a no-template control (NTC). A PCR amplification reaction system was constructed using the primers and probes in Example 1, wherein the PCR amplification reaction system was a 20 μL reaction solution, wherein the 20 μL reaction solution included 4 μL of 5×PCR reaction solution, 25 nM upstream primer, 150 nM downstream primer, 50 nM probe, 5 μL template, and the remaining components were ultrapure water. For the no-template control group, the template in the reaction system was replaced with the same volume of pure water.

[0118] Among them, the 5×PCR reaction solution was purchased from Nanjing Novozymes Biotech Co., Ltd., and the upstream primer, downstream primer, and probe were any one of the 8 sets of upstream primers, downstream primers, and probes in Example 1.

[0119] (3) PCR amplification and melting curve analysis:

[0120] After sealing the PCR tube cap, gently mix the sample, centrifuge briefly, and transfer to the tray of the SLAN real-time fluorescence quantitative PCR instrument of Shanghai Hongshi Medical Technology Co., Ltd.

[0121] The program used was: reverse transcription at 55°C for 15 minutes; pre-denaturation at 95°C for 30 seconds; denaturation at 95°C for 10 seconds, annealing and extension at 60°C for 30 seconds, for a total of 45 cycles without collecting fluorescence; denaturation at 95°C for 2 minutes, annealing and extension at 50°C for 1 minute; melting curve analysis at 50-80°C, with a heating rate of 0.04°C per second, collecting fluorescence, and obtaining a melting curve.

[0122] Data Analysis:

[0123] The SLAN real-time fluorescence quantitative PCR analysis software of Shanghai Hongshi Medical Technology Co., Ltd. was used to interpret the results by the Tm value of the melting curve. The primer probe and reaction system of the present invention were used to detect the Salmonella positive template. The melting curve formed in the FAM channel was as follows: Figure 1 The primer probe and reaction system of the present invention are used to detect Shigella positive template, and the melting curve formed in the FAM channel is as shown in FIG. Figure 2 The primer probe and reaction system of the present invention are used to detect the positive template of Vibrio parahaemolyticus, and the melting curve formed in the FAM channel is as shown in FIG. Figure 3 The primer probe and reaction system of the present invention are used to detect the positive template of Campylobacter jejuni, and the melting curve formed in the VIC channel is as shown in FIG. Figure 4 The primer probe and reaction system of the present invention are used to detect the positive template of diarrhea-causing Escherichia coli, and the melting curve formed in the VIC channel is as shown in FIG. Figure 5 The primer probe and reaction system of the present invention are used to detect the positive template of Vibrio cholerae, and the melting curve formed in the CY5 channel is as shown in FIG. Figure 6 The primer probe and reaction system of the present invention are used to detect the positive template of Staphylococcus aureus, and the melting curve formed in the CY5 channel is as shown in FIG. Figure 7 The primer probe and reaction system of the present invention are used to detect the internal reference β-actin gene template, and the melting curve formed in the CY5 channel is as shown in FIG. Figure 8 The primer probe and reaction system of the present invention are used to detect the template containing Salmonella, Shigella, Vibrio parahaemolyticus, Campylobacter jejuni, diarrheagenic Escherichia coli, Vibrio cholerae, Staphylococcus aureus and the internal reference β-actin gene. The melting curves formed in the FAM, VIC and CY5 channels are shown as follows. Fig. 9 , 10 As shown in Figures 1 and 11, the corresponding melting curves can be analyzed to determine the fluorescence channel and Tm value corresponding to the target. The specific results are shown in Table 1 below.

[0124]

[0125]

[0126] Table 1 Fluorescence channels and Tm values ​​corresponding to the targets

[0127] Example 3: Detection limit test of the detection method

[0128] In order to verify the detection limit of the detection system in the above embodiment, different concentrations of Salmonella, Shigella, Vibrio parahaemolyticus, Campylobacter jejuni, diarrheagenic Escherichia coli, Vibrio cholerae, and specific genes of Staphylococcus aureus and internal reference β-actin gene plasmid were used as detection samples, and the detection was carried out according to the reaction system and reaction procedure in the above embodiment. Among them, the concentrations of each target in the detection sample were 1.00E+04, 1.00E+03, 200, and 100 copies / mL, respectively. The samples of 1.00E+04 and 1.00E+03 copies / mL were repeated 3 times, and the samples of 200 and 100 copies / mL were repeated 20 times. The detection rate was calculated, and the lowest concentration with a detection rate ≥95% was the detection limit. The results are shown in the following table:

[0129]

[0130]

[0131] The detection system in the above embodiment can detect the specific genes of Salmonella, Shigella, Vibrio parahaemolyticus, Campylobacter jejuni, diarrheagenic Escherichia coli, Vibrio cholerae, and Staphylococcus aureus and the internal reference β-actin gene plasmid samples in the range of 200 to 1.00E+04 copies / mL, indicating that the detection system in the above embodiment has a good detection effect for Salmonella, Shigella, Vibrio parahaemolyticus, Campylobacter jejuni, diarrheagenic Escherichia coli, Vibrio cholerae, Staphylococcus aureus, and the internal reference β-actin gene, and the detection limit is 200 copies / mL.

[0132] The above-mentioned embodiments only express several implementation methods of the present invention, and the description is relatively specific and detailed, but it cannot be understood as limiting the scope of the invention patent. It should be pointed out that for ordinary technicians in this field, several modifications and improvements can be made without departing from the concept of the present invention, and the present invention is also intended to include these modifications and modifications.

Claims

1. A primer probe set for detecting intestinal pathogenic bacteria, characterized in that: The probe group is used to amplify specific target sequences of the genomes of Salmonella, Shigella, Vibrio parahaemolyticus, Campylobacter jejuni, diarrheagenic Escherichia coli, Vibrio cholerae, and Staphylococcus aureus, and the probe group uses the β-actin gene as an internal reference gene.

2. The primer probe set for intestinal pathogen detection according to claim 1, characterized in that: The probe set includes: (1) Salmonella primer and probe sequences: Upstream primer: 5'-ACTATACTATTGTTAATTAACACGGTTGAGGATGTTATTCGCAATCG-3'; Downstream primer: 5′-TCCATCAAATTAGAGTAGGC-3′; Probe: 5′-CGTGTACTATACTATTGTTAATTAACACG-3′; (2) Shigella primer and probe sequences: Upstream primer: 5′-CGTACTAATATTTGCACACGAGTGCGGAGGTCACTAGC-3′; Downstream primer: 5′-TCCACTGCCGTCAAGTAA-3′; Probe: nCoV19-NP: 5′-CGTGTCGTACTAATATTTGCACACG-3′; (3) Primer and probe sequences for Vibrio parahaemolyticus: Upstream primer: 5'-CCACAGCATCGCAGCACATACACGACTCAACACAAGAAGAGATCGATAA-3'; Downstream primer: 5′-GATGAGCGGTrGATGTCGTA-3′; Probe: 5′-CGTGTCCACAGCATCGCAGCACATACACG-3′; (4) Campylobacter jejuni primer and probe sequences: Upstream primer: 5'-AATAAATTAATATGTAAACTGGCTATTTTATTTTTGAGTGCTTGTG-3'; Downstream primer: 5′-GCTTTATTGCCATTTGTTTTATTA-3′; Probe: 5′-CCAGTAATAAATTAATATGTAAACTGG-3′; (5) Primer and probe sequences for diarrheagenic Escherichia coli: Upstream primer: 5′-AACTAGCTCGTAGAACAACTGGCAATTTTCAGCCAATATGATTG-3′; downstream primer: 5′-AAACTTCAATCTTGATCAGTTTC-3′; Probe: 5′-CCAGTAACTAGCTCGTAGAACAACTGG-3′; (6) Primer and probe sequences for Vibrio cholerae: Upstream primer: 5'-TTAATGCAATATATATATAGTCGTGTTAGGCACGATGATGGAATTG-3'; Downstream primer: 5′-GACCAGACAATATAGTTTGACCCTCT-3′; Probe: 5′-CGACTTTGAATCAATATATATATAGTCG-3′; (7) Staphylococcus aureus primer and probe sequences: Upstream primer: 5'-CTACAATTCTATTAGATCACTCGCAGTGCAATGGGAAATGATATAT-3'; Downstream primer: 5′-TCTGTAAATTTACCACTAACACCATTCAA-3′; Probe: 5′-CGAGTATACACTTCTATTAGATCACTCG-3′; (8) Internal reference β-actin gene primer and probe sequences: Upstream primer: 5'-ACAGCAACAGCAGCACACGACTCGAATGAGCTGCGTGTGCATC-3'; Downstream primer: 5′-GGCTGGGGTGTTGAAACTC-3′; Probe: 5′-CGAGTACAGCAACAGCAGCACACGACTCG-3′.

3. The primer probe set for detecting intestinal pathogenic bacteria according to claim 2, characterized in that: The probes of the probe group are labeled with a fluorescent group at the 5' end and a quenching group at the 3' end, or with a fluorescent group at the 3' end and a quenching group at the 5' end; when not hybridized with the target sequence, the probe itself forms a neck ring structure, so that the fluorescent group and the quenching group are close to each other, and the fluorescence emitted by the fluorescent group is absorbed by the quenching group; when hybridized with the target sequence, the probe neck ring structure dissociates, so that the fluorescent group and the quenching group are separated, and the fluorescence emitted by the fluorescent group cannot be absorbed by the quenching group.

4. The primer probe set for detecting intestinal pathogenic bacteria according to claim 3, characterized in that: The fluorescent group includes various commonly used fluorescent markers, but is not limited to these fluorescent markers, such as ALEX-350, FAM, VIC, TET, CAL Gold 540, JOE, HEX, CAL Fluor Orange 560, TAMRA, CAL Fluor Red 590, ROX, CALFluor Red 610, TEXAS RED, CAL Fluor Red 635, Quasar670, CY3, CY5, CY5.5, Quasar 705, etc.

5. The primer probe set for detecting intestinal pathogenic bacteria according to claim 4, characterized in that: The quenching group includes various commonly used quenchers, but is not limited to these quenchers, such as DABCYL, BHQ-1, BHQ-2, ECLIPE, etc.

6. Use of the primer probe set for intestinal pathogen detection according to any one of claims 1 to 5 in detecting Boehmeria, Shigella, Vibrio parahaemolyticus, Campylobacter jejuni, diarrheagenic Escherichia coli, Vibrio cholerae, and Staphylococcus aureus.

7. A detection method for intestinal pathogenic bacteria detection, characterized in that: The steps include: (1) Searching for specific genes in the genomes of Salmonella, Shigella, Vibrio parahaemolyticus, Campylobacter jejuni, diarrheagenic Escherichia coli, Vibrio cholerae, and Staphylococcus aureus, respectively, using the β-actin gene as an internal reference gene, and designing primer and probe sequences; (2) Extracting nucleic acid from the test sample, performing melting curve analysis after real-time fluorescence PCR amplification reaction, and judging whether the nucleic acid sequence to be tested contains the above pathogens based on the Tm value.

8. The detection method for intestinal pathogenic bacteria detection according to claim 7, characterized in that: The probe is labeled with a fluorescent group at the 5' end and a quencher group at the 3' end, or with a fluorescent group at the 3' end and a quencher group at the 5' end; when not hybridized with the target sequence, the probe itself forms a neck ring structure, so that the fluorescent group and the quencher group are close to each other, and the fluorescence emitted by the fluorescent group is absorbed by the quencher group; when hybridized with the target sequence, the probe neck ring structure dissociates, so that the fluorescent group and the quencher group are separated, and the fluorescence emitted by the fluorescent group cannot be absorbed by the quencher group.

9. The detection method for intestinal pathogenic bacteria detection according to claim 7, characterized in that: The PCR amplification reaction system is a 20 μL reaction solution, which includes 4 μL of 5×PCR reaction solution, 25 nM upstream primer, 150 nM downstream primer, 50 nM probe, 5 μL template, and the remaining components are ultrapure water. In the no-template control group, the template in the reaction system is replaced with the same volume of pure water.

10. Use of the detection method for intestinal pathogenic bacteria detection according to any one of claims 7 to 9 in detecting Boehmeria, Shigella, Vibrio parahaemolyticus, Campylobacter jejuni, diarrheagenic Escherichia coli, Vibrio cholerae, and Staphylococcus aureus.

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