Helicobacter pylori virulence gene mutation detection kit and special primer and probe thereof

Abstract

The invention discloses a kit suitable for detecting helicobacter pylori virulence gene mutation in an excrement sample, and belongs to the technical field of microbiological detection. The kit provided by the invention comprises primers / probes / blocking primers / capture probes for specifically detecting mutation sites on the following genes based on PCR (Polymerase Chain Reaction) amplification: a CagA gene, an HtrA gene (Ser171Leuu: 511-513 site information is TTA and TTG) and a CagL gene (As58Tyr: 172-174 site information is TAT, and Lys59Glu: 175-177 site information is GAA). The kit can be used for detecting helicobacter pylori virulence genes and high-virulence mutation sites in samples such as excrement and gastric mucosa of a patient with high sensitivity and high specificity, and can assist clinical implementation of helicobacter pylori radical treatment grading management and treatment.

Description

Technical Field

[0001] The invention belongs to the technical field of microbial detection, and in particular relates to a Helicobacter pylori virulence gene mutation detection kit and special primers and probes thereof. Background Art

[0002] Helicobacter pylori infection can cause a series of diseases, such as gastritis, peptic ulcer, gastric mucosa-associated lymphoid tissue (MALT) lymphoma and gastric cancer. About half of the world's population is currently infected with Helicobacter pylori. In 1994, the International Agency for Research on Cancer (IARC) classified Helicobacter pylori as a Class I carcinogen (a definite carcinogen for human gastric cancer). Eradicating Helicobacter pylori infection is a primary prevention (cause prevention) measure for gastric cancer.

[0003] Helicobacter pylori is a fastidious bacterium. It has strict requirements on the gas environment and nutritional conditions for in vitro isolation and culture, and it grows slowly. Generally, it takes 48 to 72 hours of culture at 37°C under microaerobic conditions to grow into needle-shaped colonies. Helicobacter pylori is a tiny spiral Gram-negative bacterium. The typical culture is S-shaped, C-shaped or seagull-shaped under an optical microscope. The bacteria may become spherical if the culture time is prolonged in vitro, nutrition is deficient or under antibiotic pressure. There are bundles of flagella at the end of the bacteria, which play a key role in bacterial movement; there is no obvious capsule and no spores are produced.

[0004] H. pylori isolates can show a wide diversity in polymorphisms of virulence factor genes, which may determine the increased risk of developing gastrointestinal diseases. Searching for virulence factors in infected H. pylori isolates may identify risk groups for early treatment to control adverse outcomes, especially gastric cancer.

[0005] As a component of the type VI secretion system (T4SS), CagL binds to the basolateral side of the host gastric epithelium with integrin α5β1 to promote CagA translocation leading to carcinogenesis. Helicobacter pylori isolates with CagL amino acid polymorphism Y58 / E59 carry a 4.6-fold increased risk of gastric cancer using higher integrin α5β1. In addition, it has been reported that Helicobacter pylori CagL-Y58 / E59 can activate higher integrin α5β1 under adverse pH conditions, thereby promoting the vicious cycle of hypochloraemic acidosis in gastric carcinogenesis.

[0006] The high temperature requirement A (HtrA) protein is a chaperone and serine protease. H. pylori HtrA consists of a signaling domain, a serine protease domain, and PDZ-1 and PDZ-2 domains. HtrA secreted by H. pylori opens tight junctions and adherens junctions by cleaving the extracellular domains of occludin, mucin-8, and e-cadherin, so that H. pylori can cross the epithelial monolayer to the basolateral membrane to interact with integrin α5β1 and T4SS-injected CagA.

[0007] Studies have shown that Helicobacter pylori isolates carrying HtrA-171Leu are associated with an increased risk of GC, and may work alone or in conjunction with CagL-58Tyr / 59Glu to promote the risk of gastric cancer. The two targets, HtrA-171Leu and CagL-58Tyr / 59Glu, can be used to screen people at risk of eradicating Helicobacter pylori, thereby hindering its carcinogenic process.

[0008] The infection rate of Helicobacter pylori in my country is as high as 40%-60%. Due to Chinese eating habits and living habits, most people are infected with Helicobacter pylori in childhood. The shared dining system makes children more susceptible to Hp infection. Most adult Hp infections are acquired in childhood, mostly in early childhood. After infection, it is generally difficult to clear spontaneously, resulting in lifelong infection. At present, clinical diagnosis and treatment adopts pathological examination of Helicobacter pylori and its drug resistance, and the method of obtaining gastric mucosa through gastroscopy for children and the elderly seriously hinders the general public's awareness of the harm of Helicobacter pylori and the desire to eradicate Helicobacter pylori. Therefore, a non-invasive method is needed to achieve individualized eradication treatment of Hp infection. Detecting Helicobacter pylori through feces is a simple, comfortable, non-invasive and inexpensive method. However, the composition of feces is complex, including gastrointestinal mucosal cells, Hp, various gastrointestinal flora, food residues, protein, fat, pigment, etc., and the genomic DNA in feces will be severely fragmented and degraded, making its content in feces extremely low, making it very difficult to extract high-quality DNA from feces. At the same time, the quality of the extracted DNA is not high and some components in the feces may even inhibit the subsequent PCR reaction.

[0009] Therefore, there is a need in the art for a reagent that can accurately identify the virulence genes of Helicobacter pylori and the mutation sites of different virulence mutants in fecal samples, and can perform preliminary identification of mutant strain types of Helicobacter pylori infection so that targeted treatment measures can be taken to increase the eradication rate of Helicobacter pylori and thus reduce the risk of cancer. Summary of the invention

[0010] In view of one or more problems existing in the prior art, the object of the present invention is to provide a virulence gene and mutation detection reagent for rapidly detecting Helicobacter pylori infection using stool samples.

[0011] The Helicobacter pylori virulence gene mutation detection kit includes reagents for specifically detecting the following gene mutation sites based on the real-time fluorescence PCR amplification method: CagA gene, HtrA gene (Ser171Leu: 511-513 site information is TTA, TTG) and CagL gene (Asp58Tyr: 172-174 site information is TAT, Lys59Glu: 175-177 site information is GAA);

[0012] In some embodiments, the nucleotide sequences of the primers for specifically detecting the gene mutation sites are shown in SEQ ID NOs: 1-8, respectively: CagA gene (SEQ ID NOs: 1-2), HtrA gene (Ser171Leu) (SEQ ID NOs: 3-5), and CagL gene (Asp58Tyr, Lys59Glu) (SEQ ID NOs: 6-8);

[0013] The nucleotide sequences of the detection probes for specifically detecting the genes are shown in SEQ ID NOs: 9-11, respectively: CagA gene (SEQ ID NO: 9), HtrA gene (Ser171Leu) (SEQ ID NO: 10), and CagL gene (Asp58Tyr, Lys59Glu) (SEQ ID NO: 11);

[0014] The nucleotide sequences of the blocking primers for specifically detecting the gene mutation sites are shown in SEQ ID NOs: 12-13, respectively: HtrA gene (Ser171Leu) (SEQ ID NO: 12) and CagL gene (Asp58Tyr, Lys59Glu) (SEQ ID NO: 13);

[0015] The 3' end of the nucleotide sequence of the blocking primer is labeled with one of an amino group, SpacerC3, SpacerC6, and a phosphate group.

[0016] The two ends of the nucleotide sequence of the detection probe carry a fluorescent reporter group and a quencher group respectively.

[0017] In some embodiments, the specific detection reagent for the gene mutation site includes:

[0018] (1) Nucleic acid extraction reagent: comprising a solid support containing a specific capture probe, wherein the capture probe is used to capture a specific gene sequence, the nucleotide sequences of the CagA gene, HtrA gene and CagL gene specific capture probes are shown in SEQ ID NOs: 14-16, respectively, and the 3' end of the capture probe nucleotide is an amino group, and there is a spacer between the amino group and the nucleotide, and the spacer is one or a combination of two of (PEG)n and TEG, and the number n in (PEG)n is 3-18;

[0019] (2) Reaction solution: 15mM Tris (pH8.9), 60mM KCl, 4mM MgCl2, 1mM (NH4)2SO4, TritonX1000.01%, PVP 3%, 0.8M betaine, 0.08μM-0.4μM primer, 0.16μM-0.24μM probe, UNG 0.008U / μL, dN(U)TPmix 0.28mM.

[0020] Optionally, the kit also includes:

[0021] (3) Enzyme solution: Hotstart Taq, RNase A.

[0022] In some embodiments, the kit performs gene detection based on a real-time fluorescent PCR amplification method and further includes a reagent for specifically detecting the SUC2 gene, wherein the SUC2 gene serves as an internal control gene and has a nucleotide sequence of SEQ ID NO:17.

[0023] Optionally, the reagent for specifically detecting the SUC2 gene includes a capture probe, a primer and a detection probe for specifically detecting the internal control gene, and their nucleotide sequences are respectively shown in SEQ ID NOs: 18-21, and a fluorescent reporter group and a quencher group are respectively carried at both ends of the nucleotide sequence of the detection probe.

[0024] The 3' end of the capture probe nucleotide is an amino group, and there is a spacer between the amino group and the nucleotide. The spacer is one or a combination of two of (PEG)n and TEG, and n in the (PEG)n is 3-18.

[0025] In some embodiments, the kit further comprises:

[0026] (1) washing solution: containing sodium chloride, polyethylene glycol 8000 and sodium citrate, optionally containing 2-20 mM Tris (pH 7.0), 50-500 mM NaCl, 5-50 mM sodium citrate, 1-10% PEG8000; and / or

[0027] (2) paraffin oil; and / or

[0028] (3) Positive control: artificially synthesized plasmids containing the following genes: CagA gene, HtrA gene (171Leu) and CagL gene (58Tyr, 59Glu); and / or

[0029] (4) Negative control: a system without the following gene nucleic acids: CagA gene, HtrA gene, and CagL gene;

[0030] In some embodiments, the sample comprises stool, gastric mucosa, gastric juice, oral saliva.

[0031] Compared with the existing disclosed technology, the present invention has the following characteristics:

[0032] 1) The present invention uses a coupled capture probe solid support nucleic acid separation and purification technology to quickly, efficiently and with high quality enrich and separate Helicobacter pylori genomic DNA and internal control genes in feces, eliminating the interference of large-scale microbial genomes in the intestinal microecology.

[0033] 2) The genome extracted and separated from feces and the solid support complex are all introduced into the amplification system, achieving no inhibition or interference with the detection system, and realizing the detection of Helicobacter pylori virulence gene mutations with high sensitivity.

[0034] 3) An exogenous internal control system (SUC2 gene) is used to effectively solve the problem of invalid detection caused by low concentration of human genomic DNA from intestinal exfoliated cells as internal control and concentration differences between different individuals.

[0035] 4) Real-time fluorescence PCR technology is used to achieve single-tube detection and detection of virulence genes and mutations of Helicobacter pylori in high-risk gastric cancer patients, which can play a positive role in the graded treatment of Helicobacter pylori. The detection sensitivity of the reagent reaches 400 copies / mL. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] Figure 1 These are the sensitivity and specificity detection results of the HtrA gene (171Leu / TTA).

[0037] Figure 2 These are the sensitivity and specificity detection results of the HtrA gene (171Leu / TTG).

[0038] Figure 3 These are the sensitivity and specificity detection results of the CagL gene (58Tyr / TAT).

[0039] Figure 4 These are the sensitivity and specificity detection results of the CagL gene (59Glu / GAA).

[0040] Figure 5 These are the sensitivity and specificity detection results of the CagA gene.

[0041] Figure 6 The capture probe detection result.

[0042] Figure 7 The results of genome sensitivity testing.

[0043] Figure 8 This is the result of cross-reaction detection.

[0044] Fig. 9 To interfere with the test results. DETAILED DESCRIPTION

[0045] The present invention will be further described below in conjunction with the accompanying drawings.

[0046] Case study 1: Design of primers and probes for virulence gene mutation sites

[0047] First, multiple complete sequences of three genes, including CagA gene (NCBI: AB017921.1-017923.1, AB015404.1-015415.1), CagL gene sequence (NCBI: MK014461.1-MK014478.1) and HtrA gene sequence (NCBI: MH926335.1-MH926354.1), were downloaded from the Genebank database, and DNAMAN software was used for comparison and analysis. Upstream and downstream primers were designed for detecting the CagA gene; primers were designed for the TTA / TTG genotype at the 511-513 site of the HtrA gene, the TAT genotype at the 172-174 site of the CagL gene, and the GAA genotype at the 175-177 site. Mutation points were artificially introduced at the +2bp site upstream of the 3 ends of the primers for the HtrA gene and CagL gene to enhance specific detection. At the same time, blocking primers were designed for the wild-type TCA at the 511-513 site of the HtrA gene, and blocking primers were designed for the wild-type AAA at the 175-177 site of the CagL gene. The downstream primers and detection probes for amplification are universal for the mutant wild type. Finally, the designed primer probes are used to detect the genes and their sites in the fecal samples and gastric mucosal samples (breath C13 positive). If there is a signal, relevant sequencing is performed, and the sequence is analyzed again to determine whether the designed primer probe covers the detection site and perform relevant correction design, and multiple combination tests, sensitivity and specificity tests are performed to finally obtain the primers and probes of the present invention.

[0048] The specific designed primer and probe information is as follows:

[0049]

[0050]

[0051]

[0052] Note: The 3' end of the nucleotide sequence of the blocking primer is labeled with a phosphate group.

[0053] After the primers / probes / blocking primers of each detection gene are individually combined and screened to obtain the best (sensitivity / specificity) primer pair, double combination and triple combination are performed to obtain the best multiple combination primer, probe and blocking primer combination. The best combination information is as follows:

[0054]

[0055]

[0056] Case study 2: Primer / probe / blocking primer optimization

[0057] Set the concentrations of primers / probes / blocking primers as follows:

[0058]

[0059]

[0060] Through orthogonal combination testing, according to the sensitivity and specificity test results of each detection site, the optimal primer / probe / blocking primer concentration is obtained as follows:

[0061]

[0062]

[0063] The sensitivity and specificity results of each detected gene or site are shown in Figure 1-5 As shown in the figure, the sensitivity of the mutation detection sites of CagA gene, CagL gene and HtrA gene can reach 10 copies / μL, and there is no non-specificity between the mutation detection sites and the wild type.

[0064] Case Study 3: Capture Probe Design and Screening Optimization

[0065] Three capture probes were designed for each gene targeting the conserved regions of the CagA gene, HtrA gene, CagL gene and the exogenous internal control gene SUC2, and confirmed fecal positive samples were used to screen the capture probes.

[0066] The designed capture probes are as follows:

[0067]

[0068]

[0069] Note: The 3' end of the capture probe nucleotide is an amino group, and there is a spacer between the amino group and the nucleotide. The spacer is (PEG)n, where n is 9.

[0070] After the designed capture probes are delivered to the synthesis company for synthesis, the HtrA gene, CagL gene and SUC2 gene capture probes are connected to the solid support (nanomagnetic bead particles) (by binding with N-hydroxysuccinimide and 1-ethyl-3-(3-dimethylaminopropylcarbodiimide)) according to the following connection method, the 3-amino-modified capture probe is coupled to the surface carboxyl-modified magnetic beads, and then the three coupled magnetic beads are mixed in equal proportions for the capture of specific genes in stool samples. The capture probe concentration is 0.5-50μΜ, 2.5-1g / L nanomagnetic bead particles.

[0071] Specific capture and detection operations:

[0072] (1) Take 5 g of fecal sample and add it to 20 mL / tube of sample preservation solution (self-produced), shake and mix for 1-2 min until homogenized, centrifuge the sample preservation tube at 4500 rpm for 3 min, transfer 200 μL of supernatant to a 2 mL centrifuge tube, add 200 μL of lysis solution (Tris-HCl 300 mM, SDS 2%), lyse for 10 min, add 100 μL of nucleic acid extraction reagent (0.3 M sodium chloride, 3 M sodium citrate, 0.8 mg / mL coupled capture probe magnetic beads (4 kinds of mixed)), then add 10 μL of 1 M sodium hydroxide, shake and mix, incubate at 95 degrees for 5 min, and at room temperature for 10 min.

[0073] (2) Place the centrifuge tube on a magnetic bead separation device and let it stand for 2-5 minutes. After the magnetic beads are adsorbed on the tube wall, place the centrifuge tube on the magnetic bead separation device, discard the liquid, and retain the magnetic beads. Add 0.8 mL of washing solution (5 mM Tris (pH 7.0), 200 mM NaCl, 20 mM sodium citrate, 5% PEG8000) and 0.2 mL of paraffin oil, shake evenly, let it stand for 2-5 minutes, discard the liquid, and retain the magnetic beads.

[0074] (3) The centrifuge tube is removed from the magnetic bead separation device, leaving behind the magnetic bead-nucleic acid complex for later use.

[0075] (4) Add 30 μL of amplification detection solution to each centrifuge tube containing magnetic beads-nucleic acid complexes: 15 mM Tris (pH 8.9), 60 mM KCl, 4 mM MgCl2, 1 mM (NH4)2SO4, TritonX100 0.01%, PVP 3%, 0.8 M betaine, 0.16 μM-0.4 μM primer, 0.16 μM probe, UNG 0.008 U / μL, dN(U)TPmix 0.28 mM, enzyme solution (HotstartTaq, RNase A), and oscillate to resuspend the magnetic beads;

[0076] (5) Transfer 25 μL of amplification detection solution to a PCR reaction tube and place it in a real-time fluorescence PCR instrument (program: 50 degrees for 2 minutes, 95 degrees for 3 minutes; 45 cycles (95 degrees for 10 seconds, 58 degrees for 45 seconds)) for amplification detection. Select the FAM, HEX and CY5 channels for the fluorescence channel.

[0077] The optimal capture probe can be obtained through the detection results as follows:

[0078]

[0079] The capture probe screening test results are shown in Figure 6 As can be seen from the figure, the capture efficiency of the CagA gene capture probe is T1>T2>T3, the capture efficiency of the HtrA gene capture probe is T1>T2>T3, the capture efficiency of the CagL gene capture probe is T1>T2>T3, and the capture efficiency of the SUC2 gene capture probe is T1>T2>T3. Therefore, the best capture probes for the four genes are selected as shown in the table above.

[0080] Case study 4: Optimization of amplification system

[0081] Since the present invention is used to detect the genotype of Helicobacter pylori in fecal samples, a small amount of inhibitors will exist after nucleic acid separation and purification, and the enhancement of PCR amplification efficiency and anti-inhibition ability are mainly related to the ratio of each component in the reaction system. The concentration of each component in the PCR reaction system is orthogonally designed and tested, and the specific concentration settings are as follows:

[0082] 10mM Tris: pH8.0, pH8.3, pH8.6, pH8.9;

[0083] 15mM Tris: pH8.0, pH8.3, pH8.6, pH8.9;

[0084] 20mM Tris: pH8.0, pH8.3, pH8.6, pH8.9;

[0085] 25mM Tris: pH8.0, pH8.3, pH8.6, pH8.9;

[0086] KCl: 40mM, 50mM, 60mM, 70mM;

[0087] MgCl2: 2mM, 3mM, 4mM, 5mM;

[0088] (NH4)2SO4: 0.5mM, 1mM, 1.5mM, 2mM;

[0089] TritonX100: 0.005%, 0.01%, 0.015%, 0.02%;

[0090] PVP: 1%, 2%, 3%, 4%;

[0091] Betaine: 0.6M, 0.8M, 1.0M, 1.2M;

[0092] dN(U)TPmix: 0.2mM, 0.24mM, 0.28mM, 0.32mM;

[0093] UNG: 0.004U / μL, 0.008U / μL, 0.012U / μL, 0.016U / μL;

[0094] After a large number of orthogonal optimization tests, the optimal concentration of the reaction solution system was obtained:

[0095] 15mM Tris (pH8.9), 60mM KCl, 4mM MgCl2, 1mM (NH4)2SO4, TritonX1000.01%, PVP3%, 0.8M betaine, UNG 0.008U / μL, dN(U)TPmix 0.28mM, UNG 0.008U / μL.

[0096] The sensitivity test results of the confirmation system are shown in Figure 7 As shown in the figure, the sensitivity of using the optimized reaction system to detect Helicobacter pylori of different genotypes simulated by fecal matrix can reach 400 copies / mL.

[0097] Implementation Case 5: Cross-reactivity Validation

[0098] The optimized and verified reaction system was used to select Campylobacter jejuni, Bacillus subtilis, Escherichia coli, Enterobacter cloacae, Proteus vulgaris, Candida albicans, Enterococcus faecalis, Klebsiella, Helicobacter helminthii, Pseudomonas aeruginosa, Clostridium, Staphylococcus aureus, Streptococcus pneumoniae, Salmonella, Acinetobacter baumannii, Fusobacterium nucleatum subsp. nucleatum, Bacteroides fragilis, Bifidobacterium, Lactococcus lactis, Shigella, and Vibrio parahaemolyticus in the gastrointestinal system for cross-reaction detection, and the results showed that there were no specific problems. The experimental results are shown in Figure 8 .

[0099] Implementation Case 6: Interference Test Verification

[0100] Simulated samples were used for interference experiment verification. The interference substances were: clarithromycin (2.9 mg / L), amoxicillin (4.42 mg / L), levofloxacin (1.6 mg / L), metronidazole benzoate (16.6 mg / L), tetracycline hydrochloride (4 mg / L), furazolidone (3.3 mg / L), and hemoglobin (160 g / L). Compared with the control group, the △ct value was ≤2, and these interference substances had no effect on the test results. The experimental results are shown in Fig. 9 .

[0101] Implementation Case 8: Clinical Sample Verification

[0102] At the same time, 50 cases of C 13 Gastric mucosa and fecal samples of patients with positive or negative breath (UBT) test, and confirm that the patients have not received antibiotics, proton pump inhibitors, or bismuth-related treatment within one month. Gastric mucosa and fecal samples are simultaneously tested by the detection method of the present invention and Sanger sequencing is used to determine the mutation type.

[0103] The specific capture and detection steps refer to Example 3, and the amplification reagent adopts the determined formula after optimization in Example 4.

[0104]

[0105]

[0106]

[0107] From the comparison results of gastric mucosal samples and fecal samples, the two are consistent, and the sequencing type is also consistent with the detected type, indicating that the present invention can be used for non-invasive fecal detection of virulence mutations of Helicobacter pylori infection in the stomach.

Claims

1. A Helicobacter pylori virulence gene mutation detection kit, characterized in that: The kit includes reagents for specifically detecting the following genes and gene mutation sites: CagA gene, HtrA gene: 511-513 site information is TTA, TTG and CagL gene: 172-174 site information is TAT, 175-177 site information is GAA; The nucleotide sequences of the primers for specifically detecting the gene and gene mutation site are shown in SEQ ID NOs: 1-8 respectively: The primers for the CagA gene are shown in SEQ ID NO: 1-2; The primers for TTA and TTG at sites 511-513 of the HtrA gene are shown in SEQ ID NOs: 3-5; The primers for TAT at sites 172-174 and GAA at sites 175-177 of the CagL gene are shown in SEQ ID NOs: 6-8; The nucleotide sequences of the detection probes for specifically detecting the gene are shown in SEQ ID NOs: 9-11 respectively: The detection probe of CagA gene is shown in SEQ ID NO:9; The detection probe for TTA and TTG at sites 511-513 of the HtrA gene is shown in SEQ ID NO: 10; The detection probes for TAT at sites 172-174 and GAA at sites 175-177 of the CagL gene are shown in SEQ ID NO: 11; The nucleotide sequences of the blocking primers for specifically detecting the gene mutation sites are shown in SEQ ID NOs: 12-13 respectively: The blocking primer of HtrA gene (Ser171Leu) is shown in SEQ ID NO:12; The blocking primer of CagL gene (Asp58Tyr, Lys59Glu) is as follows: The blocking primer of SEQ ID NO: 13 is as follows; The 3' end of the nucleotide sequence of the blocking primer is labeled with one of an amino group, SpacerC3, SpacerC6, and a phosphate group; The two ends of the nucleotide sequence of the detection probe carry a fluorescent reporter group and a quencher group respectively.

2. The Helicobacter pylori virulence gene mutation detection kit according to claim 1, characterized in that: The kit also includes: (1) Nucleic acid extraction reagent: comprising a solid support containing a specific capture probe, wherein the capture probe is used to capture a specific gene sequence, the nucleotide sequences of the CagA gene, HtrA gene and CagL gene specific capture probes are shown in SEQ ID NOs: 14-16, respectively, and the 3' end of the capture probe nucleotide is an amino group, and there is a spacer between the amino group and the nucleotide, wherein the spacer is one or a combination of two of (PEG)n and TEG, and n in (PEG)n is 3-18; (2) Reaction solution: 15mM Tris pH8.9, 60mM KCl, 4mM MgCl2, 1mM (NH4)2SO4, TritonX1000.01%, PVP 3%, 0.8M betaine, 0.08μM-0.4μM primer, 0.16μM-0.24μM probe, UNG 0.008U / μL, dN(U)TPmix 0.28mM.

3. The Helicobacter pylori virulence gene mutation detection kit according to claim 1, characterized in that: The kit also includes: Enzyme solution: Hotstart Taq, RNase A.

4. The Helicobacter pylori virulence gene mutation detection kit according to claim 1, characterized in that: The kit detects the gene based on the real-time fluorescent PCR amplification method, and the kit also includes a reagent for specifically detecting the SUC2 gene, the SUC2 gene is used as an internal control gene, and its nucleotide sequence is SEQ ID NO: 17; The reagent for specifically detecting the SUC2 gene includes a capture probe, a primer and a detection probe for specifically detecting the internal control gene, and the nucleotide sequences thereof are respectively shown in SEQ ID NOs: 18-21, and the two ends of the nucleotide sequence of the detection probe carry a fluorescent reporter group and a quencher group respectively; The 3' end of the capture probe nucleotide is an amino group, and there is a spacer between the amino group and the nucleotide. The spacer is one or a combination of two of (PEG)n and TEG, and n in the (PEG)n is 3-18.

5. The Helicobacter pylori virulence gene mutation detection kit according to any one of claims 1 to 4, characterized in that: The kit also includes: (1) washing solution: comprising sodium chloride, polyethylene glycol 8000 and sodium citrate; and / or (2) paraffin oil; and / or (3) Positive control: artificially synthesized plasmids containing the following genes: CagA gene, HtrA gene (171Leu) and CagL gene (58Tyr, 59Glu); and / or (4) Negative control: a system that does not contain the following gene nucleic acids: CagA gene, HtrA gene and CagL gene.

6. The Helicobacter pylori virulence gene mutation detection kit according to any one of claims 1 to 4, characterized in that: The samples used include stool, gastric mucosa, gastric juice, and oral saliva.

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