Primer group and application

Abstract

The embodiment of the invention discloses a primer group and application. Specific primers are designed for a UreA gene, a GyrA gene, a 23SrRNA gene, a PbP1 gene, a 16SrRNA gene, a porD gene, a CagA gene, a vacA gene, a CYP2C19 * 2 gene, a CYP2C19 * 3 gene, a CYP2C19 * 17 gene and mutation sites on the basis of a nucleic acid mass spectrometry technology, the helicobacter pylori detection kit has the advantages of high sensitivity and high accuracy, and helicobacter pylori identification, antibiotic sensitivity / drug resistance detection and virulence gene typing can be quickly and efficiently carried out; the method for detecting the metabolic type of the proton pump inhibitor has a good application prospect.

Description

Technical Field

[0001] The embodiments of the present invention relate to the field of biomedicine technology, and specifically to a primer set and application, and more specifically to a primer set and application for feces-based Helicobacter pylori identification, antibiotic sensitivity / resistance detection, virulence gene typing, and proton pump inhibitor metabolic type detection. Background Art

[0002] Helicobacter pylori (HP) is a Gram-negative microaerophilic pathogen that can infect the human throat, stomach and duodenum. It is spiral-shaped, also known as curved arc or S-shaped. It was first successfully isolated from gastric mucosal biopsy tissue of patients with chronic active gastritis in 1983. It is the only known microorganism that can survive in the human stomach. Helicobacter pylori disease includes gastritis, peptic ulcer, lymphoproliferative gastric lymphoma, etc. caused by Helicobacter pylori infection. Helicobacter pylori infection increases the risk of gastric cancer by 2.7-12 times. If there is no Helicobacter pylori infection, at least 35%-89% of gastric cancer will not occur.

[0003] Traditional culture methods and drug sensitivity tests are the gold standard for Helicobacter pylori identification and drug resistance testing, but Helicobacter pylori grows slowly, has a long culture cycle, and a low culture success rate, making it difficult to promote on a large scale in clinical practice. The common QPCR gene detection method, due to limited throughput, involves very few genes and SNP sites, and there is the possibility of false positives / false negatives, which affects the doctor's judgment. The traditional treatment options for Helicobacter pylori infection - "triple therapy" of two antibiotics + proton pump inhibitors, and "quadruple therapy" of two antibiotics + proton pump inhibitors + bismuth, rely solely on the doctor's experience and the hospital's antibiotic preferences in the selection of antibiotics, and these two treatment methods ignore the detection of virulence typing and proton pump inhibitor metabolic types. Summary of the invention

[0004] To this end, the embodiment of the present invention provides a primer set and application. The present invention carries out the Helicobacter pylori project through individualized treatment, based on fecal DNA gene detection, convenient sampling, avoiding the traditional time-consuming and labor-intensive culture method, and adopting the nucleic acid mass spectrometry method, which has a larger throughput than QPCR, and the detected genes and SNP sites are comprehensive, with high sensitivity and specificity. The Helicobacter pylori detection project of the present invention includes: ① Helicobacter pylori identification; ② Antibiotic sensitivity / resistance detection; ③ Virulence gene typing; ④ Proton pump inhibitor metabolic type; It fully realizes accurate medication guidance, making up for the shortcomings of traditional treatment schemes - "triple therapy" or "quadruple therapy", which only rely on the doctor's experience in the selection of antibiotics, and ignores the shortcomings of virulence typing and proton pump inhibitor metabolic type detection.

[0005] In order to achieve the above purpose, the embodiment of the present invention provides the following technical solutions:

[0006] According to a first aspect of an embodiment of the present invention, the present invention provides a primer set, comprising a forward primer, a reverse primer and an extension primer:

[0007] For the Helicobacter pylori identification gene UreA: the forward primer has the nucleotide sequence shown in SEQ ID No. 1, the reverse primer has the nucleotide sequence shown in SEQ ID No. 2, and the extension primer has the nucleotide sequence shown in SEQ ID No. 27;

[0008] For levofloxacin gene gyrA 260, 261, 271, 272: the forward primer has the nucleotide sequence shown in SEQ ID No.3, the reverse primer has the nucleotide sequence shown in SEQ ID No.4, and the extension primer has the nucleotide sequence shown in SEQ ID No.28, SEQ ID No.29, SEQ ID No.30, SEQ ID No.31;

[0009] For clarithromycin gene 23SrRNA 2142, 2143, 2144: the forward primer has the nucleotide sequence shown in SEQ ID No.5, the reverse primer has the nucleotide sequence shown in SEQ ID No.6, and the extension primer has the nucleotide sequence shown in SEQ ID No.32, SEQ ID No.33, SEQ ID No.34;

[0010] For amoxicillin gene pbp1 1242, 1454: the forward primer has the nucleotide sequence shown in SEQ ID No.7, the reverse primer has the nucleotide sequence shown in SEQ ID No.8, and the extension primer has the nucleotide sequence shown in SEQ ID No.35 and SEQID No.36;

[0011] For tetracycline gene 16SrRNA 926, 928: the forward primer has the nucleotide sequence shown in SEQ ID No.9, the reverse primer has the nucleotide sequence shown in SEQ ID No.10, and the extension primer has the nucleotide sequence shown in SEQ ID No.37 and SEQID No.38;

[0012] For furazolidone gene porD 356, 357: the forward primer has the nucleotide sequence shown in SEQ ID No.11, the reverse primer has the nucleotide sequence shown in SEQ ID No.12, and the extension primer has the nucleotide sequence shown in SEQ ID No.39 and SEQ ID No.40;

[0013] For the virulence genotyping gene CagA: Due to the variability of gene CagA, two pairs of primers were designed at different positions, wherein one pair of forward primers had a nucleotide sequence as shown in SEQ ID No.13, the reverse primer had a nucleotide sequence as shown in SEQ ID No.14, and the extension primer had a nucleotide sequence as shown in SEQ ID No.41; another pair of forward primers had a nucleotide sequence as shown in SEQ ID No.15, the reverse primer had a nucleotide sequence as shown in SEQ ID No.16, and the extension primer had a nucleotide sequence as shown in SEQ ID No.42;

[0014] For the virulence genotyping gene VacA-S region: the forward primer has the nucleotide sequence shown in SEQ ID No.17, the reverse primer has the nucleotide sequence shown in SEQ ID No.18, and the extension primer has the nucleotide sequence shown in SEQ ID No.43 and SEQ ID No.44;

[0015] For the virulence genotyping gene VacA-m region: the forward primer has the nucleotide sequence shown in SEQ ID No.19, the reverse primer has the nucleotide sequence shown in SEQ ID No.20, and the extension primer has the nucleotide sequence shown in SEQ ID No.45;

[0016] For the proton pump inhibitor metabolizer gene CYP2C19*2: the forward primer has the nucleotide sequence shown in SEQ ID No.21, the reverse primer has the nucleotide sequence shown in SEQ ID No.22, and the extension primer has the nucleotide sequence shown in SEQ ID No.46;

[0017] For the proton pump inhibitor metabolizer gene CYP2C19*3: the forward primer has the nucleotide sequence shown in SEQ ID No.23, the reverse primer has the nucleotide sequence shown in SEQ ID No.24, and the extension primer has the nucleotide sequence shown in SEQ ID No.47;

[0018] For the proton pump inhibitor metabolite type gene CYP2C19*17: the forward primer has the nucleotide sequence shown as SEQ ID No.25, the reverse primer has the nucleotide sequence shown as SEQ ID No.26, and the extension primer has the nucleotide sequence shown as SEQ ID No.48.

[0019] According to a second aspect of an embodiment of the present invention, the present invention provides a kit comprising the primer set as described above.

[0020] According to a third aspect of an embodiment of the present invention, the present invention provides the use of the primer set and the kit as described above in Helicobacter pylori identification, antibiotic sensitivity / resistance detection, virulence gene typing, and proton pump inhibitor metabolic type detection.

[0021] According to a fourth aspect of an embodiment of the present invention, the present invention provides a method for Helicobacter pylori identification, antibiotic sensitivity / resistance detection, virulence gene typing, and proton pump inhibitor metabolic type detection, characterized in that the method comprises:

[0022] After performing a multiplex PCR amplification reaction on the sample to be tested using the forward primer and reverse primer as described above, a PCR amplification reaction product is obtained;

[0023] The PCR amplification reaction product is subjected to a SAP enzyme digestion reaction, and then a single base extension reaction is performed using the extension primer as described in claim 1, and Helicobacter pylori identification, antibiotic sensitivity / resistance detection, virulence gene typing, and proton pump inhibitor metabolic type detection are performed based on the mass spectrometry detection results.

[0024] Furthermore, the Helicobacter pylori identification and antibiotic sensitivity / resistance detection are performed in the same reaction system.

[0025] Furthermore, the virulence gene typing and proton pump inhibitor metabolic type detection are carried out in the same reaction system.

[0026] Furthermore, the sample to be tested is feces.

[0027] The embodiments of the present invention have the following advantages:

[0028] Easy to operate, high PCR multiplex and conversion success rate: good results can be obtained within 50 PCR multiplexes, and up to 60 PCR multiplexes; >95% of SNP sites can be analyzed and studied using this platform.

[0029] High cost-effectiveness: 10-50 SNP sites can be measured simultaneously without fluorescent labeling, and the consumables cost and sample usage are minimized. High accuracy: By designing primers in both forward and reverse directions, it can effectively detect insertion and deletion INDELs and translocation fusion genes with an accuracy rate of >98%.

[0030] High sample throughput: more than 3,000 samples can be processed per day, and 100,000 genotypes can be typed, with a higher throughput than QPCR.

[0031] High-quality data: Fully automatic data analysis, complete genotyping report, and provide mass spectrometry to obtain sample status and result credibility analysis. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] In order to more clearly illustrate the implementation methods of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for the implementation methods or the description of the prior art. Obviously, the drawings in the following description are only exemplary, and for ordinary technicians in this field, other implementation drawings can be derived from the provided drawings without creative work.

[0033] Figure 1 A simplified diagram of the experimental operation provided by the present invention;

[0034] Figure 2 The positive control Helicobacter pylori identification and antibiotic sensitivity / resistance nucleic acid mass spectrometry provided by the present invention;

[0035] Figure 3 The nucleic acid mass spectra of the positive control virulence genotyping and proton pump inhibitor metabolic type detection provided by the present invention;

[0036] Figure 4 The negative control Helicobacter pylori identification and antibiotic sensitivity / resistance nucleic acid mass spectrometry provided by the present invention;

[0037] Figure 5 The negative control virulence genotyping and proton pump inhibitor metabolic type detection nucleic acid mass spectrometry provided by the present invention;

[0038] Figure 6 Helicobacter pylori identification and antibiotic sensitivity / resistance nucleic acid mass spectrometry of stool sample 1 provided by the present invention;

[0039] Figure 7 Virulence genotyping and proton pump inhibitor metabolic type detection nucleic acid mass spectrometry of the stool sample 1 provided by the present invention;

[0040] Figure 8 Helicobacter pylori identification and antibiotic sensitivity / resistance nucleic acid mass spectrometry of stool sample 2 provided by the present invention;

[0041] Fig. 9 This is the nucleic acid mass spectrogram for virulence genotyping and proton pump inhibitor metabolic type detection of the stool sample 2 provided by the present invention. DETAILED DESCRIPTION

[0042] The following is a description of the implementation of the present invention by specific embodiments. People familiar with the art can easily understand other advantages and effects of the present invention from the contents disclosed in this specification. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0043] If no specific techniques or conditions are specified in the examples, the techniques or conditions described in the literature in the field or the product instructions are used. If no manufacturer is specified for the reagents or instruments used, they are all conventional products that can be purchased through regular channels.

[0044] Example 1

[0045] Nucleic acid mass spectrometry technology is a detection technology based on time-of-flight mass spectrometry. It can identify and distinguish different nucleic acid sequences by measuring the molecular weight of nucleic acid molecules. It has the advantages of high throughput, more genes and SNP sites detected, and more comprehensiveness.

[0046] The present invention uses nucleic acid mass spectrometry technology to specifically detect the urease gene UreA infected with Helicobacter pylori to determine whether Helicobacter pylori is infected; accurately detect the Helicobacter pylori GyrA gene, 23SrRNA gene, PbP1 gene, 16SrRNA gene and porD gene loci to determine the resistance of antibiotics levofloxacin, clarithromycin, amoxicillin, tetracycline, and furazolidone; accurately distinguish the differences in pathogenic strains of Helicobacter pylori, type I and type II; detect CYP2C19 gene polymorphism to determine enzyme activity and then guide the precise use of proton pump inhibitors (PPI). Proton pump inhibitors (PPI) are currently the most effective inhibitors of gastric acid secretion and are widely used to treat gastroesophageal reflux, peptic ulcer disease and Helicobacter pylori infection. Omeprazole, lansoprazole and pantoprazole are first-generation PPI drugs, and rabeprazole, esomeprazole and dexlansoprazole are second-generation PPI drugs. The first generation of PPIs are mainly metabolized by CYP2C19 enzymes. CYP2C19 gene polymorphism has an important impact on their individual differences in pharmacokinetics, stability of efficacy and drug interactions. The second generation of PPIs do not use CYP2C19 as the main metabolizing enzyme, so they are less affected by CYP2C19 gene polymorphism. This product uses proton pump inhibitors scientifically and rationally by detecting CYP2C19 gene polymorphism to accelerate the successful eradication of Helicobacter pylori.

[0047] This example studies the correlation between gene loci and diseases.

[0048] HP identification: Detect the urease gene UreA of Helicobacter pylori to determine whether the patient is infected with Helicobacter pylori.

[0049] Antibiotic sensitivity / resistance: In this study, 90 samples were tested by PCR+Sanger sequencing. The mutation sites of Helicobacter pylori resistance to levofloxacin were 260 (A>T), 261 (C>A), 271 (G>A), and 272 (A>G) of the GyrA gene; the mutation sites of Helicobacter pylori resistance to clarithromycin appeared in the 23SrRNA gene, and the mutation sites included 2142 (A>G), 2143 (A>G), and 2144 (A>G); the mutation sites of Helicobacter pylori resistance to amoxicillin were 1242 (C>G) and 1454 (A>G) in the PBP1 gene; the mutation sites of Helicobacter pylori resistance to tetracycline appeared in the 16SrRNA gene, 926 (A>G) and 928 (A>C); the mutation sites of Helicobacter pylori resistance to furazolidone appeared in the porD gene, 356 (A>G) and 357 (C>G). The results of the antibiotic sensitivity / resistance studies are shown in Table 1 below.

[0050] Table 1

[0051]

[0052] Virulence gene typing: HP virulence factors include motility, adhesion, urease activity, phosphopolysaccharide endotoxin-like activity, proteolytic enzymes, phospholipase A, etc. These common virulence factors are shared by HP, while the other two virulence factors only exist in 50%~60% of HP. They are 90kDa vacuolar toxin (VacA) and 120kDa cytotoxin-associated protein (CagA). According to the genotype and phenotype of these two virulence factors, HP is divided into two major categories: Type I HP contains both vacA gene and CagA gene and expresses active VacA and CagA proteins, and Type II HP contains only vacA gene, does not contain cagA gene, and does not express active VacA and CagA proteins.

[0053] Proton pump inhibitor metabolic type: Proton pump inhibitors (PPIs) are currently the most powerful class of drugs for acid suppression and are widely used in the treatment of digestive system diseases. The bioavailability and metabolism of PPIs are mainly affected by the drug metabolizing enzyme CYP2C19. The main enzymes that play a role in drug metabolism are CYP2C19*2, CYP2C19*3, and CYP2C19*17. There are four different metabolic types: ultra-fast metabolizer (*1 / *17, *17 / *17), which may cause a significant increase in enzyme activity due to abnormal replication or amplification of the functional region gene; fast metabolizer (*1 / *1), the functional region gene is normally expressed, expressing normal active enzymes, which is the metabolic phenotype of the normal population; intermediate metabolizer (*1 / *2, *1 / *3, *17 / *2, *17 / *3), may carry a functional defective allele or a non-functional allele, and the activity of drug metabolizing enzymes is slightly lower than normal; slow metabolizer (*2 / *2, *2 / *3, *3 / *3), may carry two invalid alleles, resulting in a significant decrease in the activity of drug metabolizing enzymes. Genotyping of CYP2C19 can guide doctors to use PPI drugs accurately.

[0054] Example 2

[0055] In this embodiment, specific primers are designed for UreA gene, GyrA gene, 23SrRNA gene, PbP1 gene, 16SrRNA gene, porD gene, CagA gene, vacA gene, CYP2C19*2 gene, CYP2C19*3 gene, CYP2C19*17 gene and mutation sites. The specific primers include forward primers and reverse primers for multiplex PCR reactions, and extension primers for single base extension reactions.

[0056] The multiplex PCR primer set information is shown in Table 2 below.

[0057] Table 2

[0058]

[0059] The single base extension primer set information is shown in Table 3 below.

[0060] Table 3

[0061]

[0062] Among them, sequence 36 is a thio-modified primer, and * indicates that the oxygen atom in the phosphodiester bond between the corresponding two bases is replaced by a sulfur atom.

[0063] Example 3

[0064] This embodiment provides a method for Helicobacter pylori identification, antibiotic sensitivity / resistance detection, virulence gene typing, and proton pump inhibitor metabolic type detection, comprising the following steps:

[0065] 1. Nucleic Acid Purification

[0066] (1) Sample type: feces;

[0067] (2) Sampling and preservation: Use Kangwei Century CWY041 lysis-type feces preservation tube and follow its instructions.

[0068] (3) DNA extraction and purification: Use the Kangwei Century CWY095 lysis extraction kit according to its instructions.

[0069] 2. Multiplex PCR Amplification

[0070] Helicobacter pylori identification and antibiotic sensitivity / resistance detection were performed in the same PCR reaction system; virulence gene typing and proton pump inhibitor metabolic type detection were performed in another PCR reaction system. The multiplex PCR reaction reagents were taken out from -20°C and melted at room temperature. According to the actual number of samples, the PCR reaction system was configured according to Table 4, and thermal cycling was performed according to Table 5. At the same time, multiplex PCR reactions were performed using DNA of ACTT 11637 strain as a positive control and water as a negative control.

[0071] Table 4 Multiplex PCR amplification system

[0072]

[0073] Table 5 Multiplex PCR reaction conditions

[0074]

[0075] 3. SAP enzyme digestion reaction

[0076] The SAP reaction reagent was taken out from -20°C and melted at room temperature. After the multiplex PCR reaction was completed, the SAP reaction system was configured. The identification of Helicobacter pylori, drug resistance evaluation, virulence gene typing, and proton pump inhibitor metabolic type detection all used the system in Table 6 and the reaction conditions in Table 7, that is, the PCR reaction products of Helicobacter pylori identification and antibiotic sensitivity / resistance detection were mixed with the PCR reaction products of virulence gene typing and proton pump inhibitor metabolic type detection and then subjected to SAP enzyme digestion reaction.

[0077] Table 6 SAP reaction system

[0078]

[0079] Table 7 SAP reaction conditions

[0080]

[0081] 4. Single base extension reaction

[0082] The single base extension reaction reagent was taken out from -20°C and melted at room temperature. After the SAP reaction was completed, the PCR single base extension reaction system, Helicobacter pylori identification, drug resistance evaluation, virulence gene typing, and proton pump inhibitor metabolic type detection all used the following system in Table 8 and reaction conditions in Table 9.

[0083] Table 8 Single base extension reaction system

[0084]

[0085] Table 9 Single base extension reaction conditions

[0086]

[0087] 5. Mass spectrometry detection

[0088] (1) Resin purification

[0089] 1. Resin purification solution preparation: To facilitate purification, the resin can be prepared into an aqueous solution first, adding 400 mg of resin to 1 mL of nuclease-free water;

[0090] 2. Add resin purification solution: oscillate and mix the resin purification solution, add 18μL to each reaction; the resin is easy to settle, oscillate and mix once every 5 seconds;

[0091] 3. Purification: Place the sample in a tumbling mixer and mix at 20 rpm for 30 min;

[0092] 4. Centrifugation: After completion, centrifuge at 2000 rpm for 1 min and the supernatant is ready for testing.

[0093] (2) Sample

[0094] In the middle of the matching target plate well, add 0.5 μL of matrix, wait for at least 20 minutes, and after it is fully dried, add 0.7 μL of supernatant, wait for at least 20 minutes, fully dry the crystals, and then measure on the machine.

[0095] 6. Results Analysis

[0096] The present invention detected a total of 11 genes and 19 loci, and the results are shown in Tables 10 and 11 below.

[0097] Table 10

[0098]

[0099] Table 11

[0100]

[0101] Example 4

[0102] According to the method of Example 3, 53 stool samples were tested, and the test results were compared with the sequencing results of the "gold standard". The results are shown in Table 12 below. The results show that the accuracy (conformity) of the sequencing results and the test results of the present invention = (52 / 53) × 100% = 98.11%, which can prove the reliability and accuracy of the results of the present invention. The positive control is the DNA of the ACTT 11637 strain, which is used to monitor whether the multiple PCR and single base extension are normal, and the negative control is a blank control to monitor whether there is contamination and exclude false positives.

[0103] Table 12 Comparison of nucleic acid mass spectrometry detection results and sequencing results of 53 stool samples

[0104]

[0105]

[0106]

[0107]

[0108]

[0109]

[0110]

[0111]

[0112]

[0113]

[0114] Repeatability verification

[0115] Four samples were randomly selected for testing. On the first day, each sample was tested three times in one batch, on the second day, another batch was tested, and on the third day, another batch was tested to verify the intra-batch and inter-batch repeatability. The results showed that the consistency of the results of the three repeated tests of the four samples reached 100%. The intra-batch repeatability results and the inter-batch repeatability verification results are shown in Tables 13 and 14 below.

[0116] Table 13 Intra-batch repeatability results

[0117]

[0118] Table 14 Inter-batch repeatability results

[0119]

[0120] Although the present invention has been described in detail above by general description and specific embodiments, it is obvious to those skilled in the art that some modifications or improvements can be made to the present invention. Therefore, these modifications or improvements made without departing from the spirit of the present invention all belong to the scope of protection claimed by the present invention.

Claims

1. A primer set, characterized in that: Includes forward primer, reverse primer and extension primer: For the Helicobacter pylori identification gene UreA: the forward primer has the nucleotide sequence shown in SEQ ID No. 1, the reverse primer has the nucleotide sequence shown in SEQ ID No. 2, and the extension primer has the nucleotide sequence shown in SEQ ID No. 27; For levofloxacin gene gyrA 260, 261, 271, 272: the forward primer has the nucleotide sequence shown in SEQ ID No.3, the reverse primer has the nucleotide sequence shown in SEQ ID No.4, and the extension primer has the nucleotide sequence shown in SEQ ID No.28, SEQ ID No.29, SEQ ID No.30, SEQ ID No.31; For clarithromycin gene 23SrRNA 2142, 2143, 2144: the forward primer has the nucleotide sequence shown in SEQ ID No.5, the reverse primer has the nucleotide sequence shown in SEQ ID No.6, and the extension primer has the nucleotide sequence shown in SEQ ID No.32, SEQ ID No.33, SEQ ID No.34; For amoxicillin gene pbp1 1242, 1454: the forward primer has the nucleotide sequence shown in SEQ ID No.7, the reverse primer has the nucleotide sequence shown in SEQ ID No.8, and the extension primer has the nucleotide sequence shown in SEQ ID No.35 and SEQ ID No.36; For tetracycline gene 16SrRNA 926, 928: the forward primer has the nucleotide sequence shown in SEQ ID No.9, the reverse primer has the nucleotide sequence shown in SEQ ID No.10, and the extension primer has the nucleotide sequence shown in SEQ ID No.37 and SEQ ID No.38; For furazolidone gene porD 356, 357: the forward primer has the nucleotide sequence shown in SEQ ID No.11, the reverse primer has the nucleotide sequence shown in SEQ ID No.12, and the extension primer has the nucleotide sequence shown in SEQ ID No.39 and SEQ ID No.40; For the virulence genotyping gene CagA: the forward primer has the nucleotide sequence shown in SEQ ID No.13, the reverse primer has the nucleotide sequence shown in SEQ ID No.14, and the extension primer has the nucleotide sequence shown in SEQ ID No.41; the forward primer has the nucleotide sequence shown in SEQ ID No.15, the reverse primer has the nucleotide sequence shown in SEQ ID No.16, and the extension primer has the nucleotide sequence shown in SEQ ID No.42; For the virulence genotyping gene VacA-S region: the forward primer has the nucleotide sequence shown in SEQ ID No.17, the reverse primer has the nucleotide sequence shown in SEQ ID No.18, and the extension primer has the nucleotide sequence shown in SEQ ID No.43 and SEQ ID No.44; For the virulence genotyping gene VacA-m region: the forward primer has the nucleotide sequence shown in SEQ ID No.19, the reverse primer has the nucleotide sequence shown in SEQ ID No.20, and the extension primer has the nucleotide sequence shown in SEQ ID No.45; For the proton pump inhibitor metabolizer gene CYP2C19*2: the forward primer has the nucleotide sequence shown in SEQ ID No.21, the reverse primer has the nucleotide sequence shown in SEQ ID No.22, and the extension primer has the nucleotide sequence shown in SEQ ID No.46; For the proton pump inhibitor metabolizer gene CYP2C19*3: the forward primer has the nucleotide sequence shown in SEQ ID No.23, the reverse primer has the nucleotide sequence shown in SEQ ID No.24, and the extension primer has the nucleotide sequence shown in SEQ ID No.47; For the proton pump inhibitor metabolite type gene CYP2C19*17: the forward primer has the nucleotide sequence shown as SEQ ID No.25, the reverse primer has the nucleotide sequence shown as SEQ ID No.26, and the extension primer has the nucleotide sequence shown as SEQ ID No.

48.

2. A kit, characterized in that: Comprising the primer set as claimed in claim 1.

3. Use of the primer set of claim 1 and the kit of claim 2 in identification of Helicobacter pylori, detection of antibiotic sensitivity / resistance, virulence gene typing, and detection of proton pump inhibitor metabolic types.

4. A method for Helicobacter pylori identification, antibiotic sensitivity / resistance detection, virulence gene typing, and proton pump inhibitor metabolic type detection, characterized in that: The method comprises: After performing a multiple PCR amplification reaction on the sample to be tested using the forward primer and the reverse primer as described in claim 1, a PCR amplification reaction product is obtained; The PCR amplification reaction product is subjected to a SAP enzyme digestion reaction, and then a single base extension reaction is performed using the extension primer as described in claim 1, and Helicobacter pylori identification, antibiotic sensitivity / resistance detection, virulence gene typing, and proton pump inhibitor metabolic type detection are performed based on the mass spectrometry detection results.

5. The method according to claim 4, characterized in that The Helicobacter pylori identification and antibiotic sensitivity / resistance detection are carried out in the same reaction system.

6. The method according to claim 4, characterized in that The virulence gene typing and proton pump inhibitor metabolic type detection are carried out in the same reaction system.

7. The method according to claim 4, characterized in that The sample to be tested is feces.

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