Primer combination for detecting helicobacter pylori and application thereof

Abstract

The invention provides a primer combination for detecting helicobacter pylori and application of the primer combination. The primer combination is used for strain identification of the helicobacter pylori and detection of gastric cancer risk sites, antibiotic resistance sites, virulence genotyping and human proton pump inhibitor drug metabolism. On the basis of multiple PCR and targeted high-throughput sequencing technologies, specific targeted amplification primers are designed and mixed, so that the helicobacter pylori identification can be completed, gastric cancer risk sites, antibiotic resistance genes, virulence typing and human proton pump inhibitor drug metabolism genes of the helicobacter pylori can be detected, and finally, the helicobacter pylori can be detected. A quantitative algorithm of approximate concentration of helicobacter pylori is also introduced into the system, so that more sensitive, accurate and comprehensive detection of helicobacter pylori is realized.

Description

Technical Field

[0001] The present invention belongs to the technical field of high-throughput sequencing and gene detection, and specifically relates to a primer combination for detecting Helicobacter pylori and its application, and more particularly to a primer combination for Helicobacter pylori species identification, gastric cancer risk site, antibiotic resistance site, virulence gene typing and human proton pump inhibitor drug metabolism detection and its application. Background Art

[0002] Helicobacter pylori (Hp) is a Gram-negative, microaerophilic bacterium that primarily colonizes the human gastric mucosa and is a major pathogenic factor in chronic gastritis, peptic ulcers, gastric mucosa-associated lymphoid tissue lymphoma, and gastric cancer. H. pylori infection rates are high worldwide. While improved hygiene and widespread antibiotic use have reduced H. pylori infection rates in recent years, drug resistance has become increasingly prominent, posing a challenge to clinical treatment. Currently, H. pylori eradication regimens primarily rely on bismuth-containing quadruple therapy (a proton pump inhibitor, a bismuth agent, and two antibiotics), with individualized treatment tailored to the patient's drug resistance profile and CYP2C19 metabolizer type. Antibiotics commonly used for H. pylori eradication include clarithromycin, levofloxacin, metronidazole, tetracycline, amoxicillin, and furazolidone.

[0003] The pathogenicity of Helicobacter pylori is closely related to its virulence factors, the most important of which are the vacuolar cytotoxin VacA and the cytotoxin-associated protein CagA. Vacuolating cytotoxin (VacA) can cause vacuolar degeneration of epithelial cells, leading to apoptosis. VacA is classified into multiple subtypes based on differences in the gene sequences of its signaling domain (s1 / s2) and intermediate domain (m1 / m2), with virulence decreasing in the order of s1 / m1 > s1 / m2 > s2 / m1 > s2 / m2. CagA, encoded by genes on the HP CagA pathogenicity island, can induce a severe inflammatory response in the host. CagA contains a repeat of the EPIYA amino acid motif at its carboxyl terminus. The EPIYA-C motif is predominantly found in HP strains from Western countries, hence the name "Western type." EPIYA-D is predominantly found in HP strains from East Asian countries, such as China and South Korea, hence the name "East Asian type." The East Asian type of CagA is more virulent and significantly associated with the risk of gastric cancer. Studies have shown that the synergistic effect of CagA and VacA significantly enhances the pathogenicity of Helicobacter pylori, especially infection with CagA East Asian type and VacA s1 / m1 type strains is significantly associated with the risk of gastric cancer.

[0004] The emergence of drug resistance in Helicobacter pylori is an increasingly serious problem. Therefore, accurate detection of drug resistance is crucial for developing personalized treatment plans and improving eradication rates. Currently, the gold standard for drug resistance detection is culture combined with antimicrobial susceptibility testing (DST). This involves culturing H. pylori from a gastric mucosal biopsy sample, followed by in vitro DST to determine its susceptibility to different antibiotics. However, this approach has significant limitations. First, culture requires stringent sample collection, transportation, and incubation conditions. Furthermore, H. pylori grows slowly, typically requiring 7–14 days to obtain results, resulting in a time-consuming process. Second, the success rate of culture is affected by multiple factors, such as recent use of antibiotics or proton pump inhibitors, which can lead to false-negative results. Furthermore, DST is complex and requires high technical expertise in both laboratory equipment and personnel, making it difficult to disseminate in primary care settings. With the continuous development and improvement of molecular detection technologies, molecular-based DST has the advantages of rapidity, high efficiency, high sensitivity and specificity, independence from bacterial activity, and the ability to detect multiple resistance genes and virulence factors. It can serve as an effective alternative to culture-based DST.

[0005] Proton pump inhibitors (PPIs) are one of the core drugs for Helicobacter pylori eradication therapy. They inhibit the H+ / K+-ATPase enzyme in gastric parietal cells, significantly reducing gastric acid secretion and improving antibiotic efficacy. However, the efficacy of PPIs is significantly affected by host CYP2C19 genetic polymorphisms. CYP2C19 is a key enzyme in the metabolism of PPIs, and its genetic polymorphisms can be divided into ultra-rapid metabolizers (UM), extensive metabolizers (EM), intermediate metabolizers (IM), and poor metabolizers (PM). In Helicobacter pylori eradication therapy, selecting the appropriate PPI dose and treatment course based on the patient's CYP2C19 metabolizer type can significantly improve the eradication rate of Helicobacter pylori and reduce adverse reactions. Therefore, personalized treatment plans based on CYP2C19 genotyping have gained increasing attention in recent years.

[0006] Several publications have documented the use of nucleic acid detection methods for Helicobacter pylori detection. For example, CN109797203A discloses a system for detecting Helicobacter pylori using real-time fluorescence quantitative PCR technology. This system can identify Helicobacter pylori, perform CagA virulence typing, and detect resistance to levofloxacin and clarithromycin. However, due to the inherent technical limitations of fluorescence quantitative PCR, only a few sites can be detected simultaneously in a single reaction, resulting in limited detection throughput. This is particularly problematic when detecting a wider range of genetic variants. Furthermore, conventional fluorescence quantitative PCR methods may not be able to meet the needs of rapid testing of large sample sizes.

[0007] CN105368825A discloses a system for detecting Helicobacter pylori using multiplex PCR capillary electrophoresis, which can detect Helicobacter pylori resistance-related tests. However, since this technology determines based on fragment size, it places high demands on the uniformity and specificity of multiplex PCR amplification. CN113604589B discloses a system based on multiplex PCR time-of-flight mass spectrometry detection, which can simultaneously detect Helicobacter pylori resistance sites, virulence gene typing, and proton pump inhibitor metabolic gene typing. Although it can detect multiple targets simultaneously, its detection range is limited by the limitations of pre-designed primers and mass spectrometry detection. It has weak detection capabilities for unknown or new variants and is not sensitive enough for detecting low-abundance pathogens or mixed infections in complex samples.

[0008] Therefore, providing a primer combination and detection method that can complete the identification of Helicobacter pylori and also detect the virulence typing of Helicobacter pylori, gastric cancer risk sites, antibiotic resistance genes and human proton pump inhibitor drug metabolism genes has important application prospects. Summary of the Invention

[0009] In response to the shortcomings of the existing technology, the present invention aims to provide a primer combination for detecting Helicobacter pylori and its application. Based on multiplex PCR and targeted next-generation sequencing (tNGS) technology, the present invention combines specific targeted amplification primers to identify Helicobacter pylori while also detecting virulence typing, gastric cancer risk factors, antibiotic resistance genes, and human proton pump inhibitor drug metabolism genes. Finally, the detection system also incorporates a quantitative algorithm for approximate Helicobacter pylori concentration, enabling more comprehensive detection of Helicobacter pylori.

[0010] In order to achieve the purpose of the invention, the present invention adopts the following technical solutions:

[0011] In a first aspect, the present invention provides a primer combination for detecting Helicobacter pylori, wherein the primer combination is used for Helicobacter pylori species identification, gastric cancer risk sites, antibiotic resistance sites, virulence genotyping, and human proton pump inhibitor drug metabolism detection, and the primer combination comprises:

[0012] (1) Primer combination for bacterial species identification, detecting gene ureA;

[0013] (2) Primer combination for detecting gastric cancer risk loci, the detection gene is htrA and the detection locus is S171L;

[0014] (3) Primer combinations for detecting Helicobacter pylori resistance sites, including genes and sites:

[0015] Clarithromycin resistance detection gene 23S rRNA, detection sites include: A2143G and A2142G / A2142C;

[0016] Levofloxacin resistance detection gene gyrA, detection sites include: D91N / D91Y / D91G and N87K / N87I;

[0017] Tetracycline resistance detection gene 16S rRNA, detection sites include: A926G, A965T, G966T and A967C;

[0018] Metronidazole resistance detection gene rdxA, detection sites include: R16H / R16C, G565T and G616A;

[0019] Amoxicillin resistance detection gene pbp1A, detection sites include: V374L, T556S, N562Y, T593A, A1681T, S414R, A1217C and C206T;

[0020] Furazolidone resistance detection genes porD and oorD. The detection sites of porD include: G353A, A356G and C357T / C357G; the detection sites of oorD include: A41G, A122G and C349A / C349G;

[0021] (4) Primer combinations for virulence genotyping, including genes and loci:

[0022] Vacuolating cytotoxin detection gene vacA, detection types include: s1m1, s1m2, s2m1 and s2m2;

[0023] Cytotoxin-associated protein detection gene cagA, detection typing includes: East Asian EPIYA-D and Western EPIYA-C;

[0024] (5) Primer combination for detecting drug metabolism of human proton pump inhibitors, the detection gene is CYP2C19, and the detection sites include: c.-806C>T, c.636G>A and c.681G>A.

[0025] In this study, the inventors, drawing on years of industry experience, identified targets relevant to Helicobacter pylori detection, including Helicobacter pylori identification, virulence typing, gastric cancer risk factors, antibiotic resistance, and human proton pump inhibitor drug metabolism genes. The detection range is shown in Table 1.

[0026] Table 1

[0027]

[0028] The present invention designs primers for specific amplification of selected targets and drug-resistant sites. The optimal amplification conditions of these primers are similar, ensuring that each amplicon can be successfully amplified under a single amplification condition, and the interaction between the primer combinations is small, resulting in less non-specific amplification. These primers are applied to multiplex PCR and targeted high-throughput sequencing technologies, and high-efficiency reactions can be performed in one tube, achieving highly sensitive and accurate detection of Helicobacter pylori within the detection range.

[0029] Preferably, the primer combination for bacterial species identification includes: primers for detecting the ureA gene, the nucleotide sequences of which are shown in SEQ ID NO: 1 and SEQ ID NO: 2.

[0030] Preferably, the primer combination for detecting gastric cancer risk sites includes: primers for detecting the htrA gene, the nucleotide sequences of which are shown in SEQ ID NO:11 and SEQ ID NO:12.

[0031] Preferably, the primer combination for detecting Helicobacter pylori resistance sites includes:

[0032] Primers for detecting 23S rRNA, the nucleotide sequences of which are shown in SEQ ID NO: 13 and SEQ ID NO: 14;

[0033] Primers for detecting gyrA, the nucleotide sequences of which are shown in SEQ ID NO: 15 and SEQ ID NO: 16;

[0034] Primers for detecting 16S rRNA, the nucleotide sequences of which are shown in SEQ ID NO: 17 and SEQ ID NO: 18;

[0035] Primers for detecting the R16H / R16C site of the rdxA gene, the nucleotide sequences of which are shown in SEQ ID NO:29 and SEQ ID NO:30;

[0036] Primers for detecting the G565T and G616A sites of the rdxA gene, the nucleotide sequences of which are shown in SEQ ID NO:31 and SEQ ID NO:32;

[0037] Primers for detecting the V374L site of the pbp1A gene, the nucleotide sequences of which are shown in SEQ ID NO: 19 and SEQ ID NO: 20;

[0038] Primers for detecting the T556S, N562Y, and A1681T sites of the pbp1A gene, the nucleotide sequences of which are shown in SEQ ID NO: 21 and SEQ ID NO: 22;

[0039] Primers for detecting the T593A site of the pbp1A gene, the nucleotide sequences of which are shown in SEQ ID NO: 23 and SEQ ID NO: 24;

[0040] Primers for detecting the S414R and A1217C sites of the pbp1A gene, the nucleotide sequences of which are shown in SEQ ID NO: 25 and SEQ ID NO: 26;

[0041] Primers for detecting the C206T site of the pbp1A gene, the nucleotide sequences of which are shown in SEQ ID NO: 27 and SEQ ID NO: 28;

[0042] Primers for detecting the G353A, A356G, and C357T / C357G sites of the porD gene, the nucleotide sequences of which are shown in SEQ ID NO:33 and SEQ ID NO:34;

[0043] Primers for detecting the A41G site of the oorD gene, the nucleotide sequences of which are shown in SEQ ID NO:35 and SEQ ID NO:36;

[0044] Primers for detecting the A122G site of the oorD gene, the nucleotide sequences of which are shown in SEQ ID NO:37 and SEQ ID NO:38;

[0045] The nucleotide sequences of the primers for detecting the C349A / C349G sites of the oorD gene are shown in SEQ ID NO: 39 and SEQ ID NO: 40.

[0046] Preferably, the primer combination for detecting virulence genotyping includes:

[0047] The nucleotide sequences of primers for detecting vacA genotype s are shown in SEQ ID NO:3 and SEQ ID NO:4, and the nucleotide sequences of primers for detecting vacA genotype m are shown in SEQ ID NO:5 and SEQ ID NO:6;

[0048] Primers for detecting the East Asian EPIYA-D locus of the cagA gene, the nucleotide sequences of which are shown in SEQ ID NO: 7 and SEQ ID NO: 8;

[0049] The nucleotide sequences of primers for detecting the EPIYA-C site of the Western type of the cagA gene are shown in SEQ ID NO: 9 and SEQ ID NO: 10.

[0050] Preferably, the primer combination for detecting human proton pump inhibitor drug metabolism includes:

[0051] Primers for detecting the c.-806C>T site of the CYP2C19 gene, the nucleotide sequences of which are shown in SEQ ID NO:41 and SEQ ID NO:42;

[0052] Primers for detecting the c.636G>A site of the CYP2C19 gene, the nucleotide sequences of which are shown in SEQ ID NO:43 and SEQ ID NO:44;

[0053] The primers for detecting the c.681G>A site of the CYP2C19 gene have nucleotide sequences shown in SEQ ID NO:45 and SEQ ID NO:46.

[0054] Preferably, the primer combination further comprises primers for detecting an internal standard, comprising the nucleotide sequences shown in SEQ ID NO:47 and SEQ ID NO:48.

[0055] The present invention designs primers for specific amplification of selected target sites. These primers have similar optimal amplification conditions, ensuring successful amplification of each amplicon under a single amplification condition. Furthermore, interactions between primer combinations are minimal, enabling all primers to achieve good detection performance in a single system. The primer combination sequences are shown in Table 2 below.

[0056] Table 2

[0057]

[0058]

[0059] Preferably, the 5' end of the primer is connected to a common sequence required for nucleic acid sequencing library construction, the common sequence is complementary to the 3' end of the sequencing adapter of the sequencing platform, and the common sequence is used to connect the sequencing adapter primer during the amplification process.

[0060] Preferably, the common sequence is a sequence of 15-30 bases (for example, 15, 17, 19, 20, 21, 23, 25, 27, 29 or 30, etc.).

[0061] Preferably, the common sequence includes the nucleotide sequence shown in SEQ ID NO:49.

[0062] In the present invention, the 5' end of the primer is connected to the common sequence required for nucleic acid sequencing library construction. The common sequence is complementary to the 3' end of the sequencing adapter of the sequencing platform, and its function is to connect the sequencing adapter primer during the amplification process. To match it, a sequence complementary to the common sequence is set at the 3' end of each sequencing adapter primer, and its function is to connect the target region amplification product. The common sequence is a sequence of 15-30 bases, which should avoid forming secondary structures and avoid high homology with the target genome, ensuring thermodynamic stability and stable binding with other sequences.

[0063] In a specific embodiment of the present invention, the common sequence may be 5'-GACTGCCGCTGGTTGGATG-3' (SEQ ID NO: 49).

[0064] In a second aspect, the present invention provides a method for detecting Helicobacter pylori based on multiplex PCR and targeted high-throughput sequencing technology, the method comprising:

[0065] (1) using the primer combination for detecting Helicobacter pylori described in the first aspect to perform super-multiplex PCR amplification on the test sample, purifying the amplified product, and then amplifying with sequencing adapter primers to construct a super-multiplex PCR library, and the final library structure obtained is sequencing adapter-common sequence-target region-common sequence-sequencing adapter;

[0066] (2) purifying each super-multiplex PCR library, and after purification, mixing the libraries to obtain a library for Helicobacter pylori detection;

[0067] (3) High-throughput sequencing of the Helicobacter pylori detection library was performed using a nucleic acid sequencing platform, and bioinformatics analysis was performed; statistics were collected on the amount of raw data, low-quality data in the raw sequencing results were filtered, valid reads with primer sequences in the sequencing products were screened and retained, and alignment software was used to align the valid read product sequences to a self-built database, and the number of reads covered in each target area was counted according to the BED file; quality control and result determination were performed on the analyzed data;

[0068] The result determination includes: bacterial species identification, gastric cancer risk site and drug resistance site detection result determination, virulence gene typing result determination, and human proton pump inhibitor drug metabolism result determination.

[0069] In the present invention, the detection samples used are feces or gastric mucosal tissue samples, nucleic acid is extracted from them, and the extracted DNA nucleic acid is subjected to super-multiplex PCR amplification of the target region.

[0070] In the present invention, the purification reagent can be any reagent, reagent combination or commercial kit that can purify DNA fragments.

[0071] In the present invention, during the library construction process, the super-multiplex PCR libraries of all samples are purified and then mixed for sequencing. The adapter sequence used for each sample is different. Subsequently, the data off the machine are split according to the adapter sequence to separate the samples.

[0072] In a specific embodiment of the present invention, the purification reagent is a magnetic bead purification reagent.

[0073] Preferably, in step (1), the system for super-multiplex PCR amplification for bacterial species identification further includes a plasmid containing an external reference sequence, wherein the external reference sequence consists of a primer binding region and a non-primer binding region, wherein the primer binding region is the same as the primer binding region of the target region on the Helicobacter pylori genome, and the non-primer binding region is a complementary, reverse or reverse complementary sequence to the non-primer binding region of the target region.

[0074] Preferably, the external reference sequence includes the nucleotide sequence shown in SEQ ID NO: 50.

[0075] In a specific embodiment of the present invention, the non-primer binding region of the external reference sequence is selected as the reverse complementary sequence of the target region, and the nucleotide sequence is shown in SEQ ID NO:50.

[0076] The present invention is directed to a primer PHP-1 for identifying Helicobacter pylori. A corresponding external reference sequence is designed and synthesized in an amplification target region. The external reference sequence consists of a primer binding region and a non-primer binding region, wherein the primer binding region is identical to the primer binding region of the target region on the Helicobacter pylori genome, and the non-primer binding region is a complementary, reverse, or reverse complementary sequence to the non-primer binding region of the target region. The external reference sequence is used to assist in achieving an approximate concentration estimation of Helicobacter pylori.

[0077] The present invention can achieve an approximate concentration estimation of Helicobacter pylori by introducing an external reference sequence into the reaction system and combining it with a detection process algorithm, thereby providing an approximate quantitative result of Helicobacter pylori.

[0078] Preferably, in step (3), the nucleic acid sequencing platform is a second-generation high-throughput sequencing platform, including the Ion Torrent platform, Roche 454 platform, SOLiD sequencing platform, BGI sequencing platform, Zhenmai Bio sequencing platform, Celu Medical sequencing platform and KM MiniSeqDx-CN sequencing platform.

[0079] Preferably, the BGI sequencing platform includes the MGISEQ series or the DNBSEQ series.

[0080] Preferably, in step (3), the software used for the comparison includes Bowtie2, Minimap2, HISAT2 or BWA comparison software;

[0081] Preferably, in step (3), the library is subjected to high-throughput sequencing using the KM MiniSeqDx-CN sequencing platform, and the sequence alignment is performed using the mem alignment algorithm in the BWA software to obtain a BAM format file of the alignment result;

[0082] Preferably, in step (3), the self-built database is a genome matched according to the product sequence of the primer, which is derived from the Nucleotide database of NCBI, and each target region contains at least one genome matched thereto.

[0083] Preferably, the quality control standards include: the minimum sequencing data requirement for each sample is ≥50k, otherwise the original data is judged to be unqualified; the sequencing quality requirement Q30 is ≥75%, otherwise the sequencing quality is judged to be unqualified; the number of internal standard reads detected is ≥50, otherwise the internal standard is judged to be unqualified; the number of external reference sequence reads detected is ≥50, otherwise the external reference sequence is judged to be unqualified; the depth values ​​of the three sites of the human genome CYP2C19 gene detected should all be ≥10, otherwise it is judged to be CYP2C19 typing failure.

[0084] Preferably, the genomes used in the quality control process include: for HP identification primers, the HP genome containing the target gene ureA gene is selected; for virulence typing primers, the HP genome containing the s1 / s2 and m1 / m2 segments of vacA, and the HP genome containing the EPIYA-D and EPIYA-C segments of cagA are respectively selected; for detection primers for gastric cancer risk and antibiotic resistance sites, the HP genome of the corresponding gene segments is selected; for detection primers for proton pump inhibitor drug metabolism genes, the genome of the HG19 version of the human genome CYP2C19 gene segment is selected; for internal standard detection primers, the genome of the human genome ACTB gene segment is selected; for detection primers for external reference sequences, the genome containing the external reference sequence is selected, and the nucleotide sequence is shown in SEQ ID NO: 50.

[0085] Preferably, the criteria for determining the result of the bacterial species identification include: for samples that pass quality control, when the number of Helicobacter pylori reads is ≥50, it is determined to be HP positive, otherwise it is determined to be HP negative; and then the approximate concentration is calculated in combination with the number of reads detected for the external reference sequence.

[0086] Preferably, the calculation formula for calculating the approximate concentration is:

[0087]

[0088] Among them, C R is the concentration of the external reference sequence, V Ris the volume of the external parameter sequence input, V T is the volume of nucleic acid input, N T is the number of reads of Helicobacter pylori detected, N R is the number of reads of the detected external reference sequence, and K is the coefficient.

[0089] Preferably, the criteria for determining the results of the gastric cancer risk site and drug-resistant site detection include: for samples determined to be HP positive, the bioinformatics software samtools is used to perform site depth and base statistics on the aligned BAM result file to obtain the number of reads of the reference base and the mutant base of the target site. When the site to be tested covers the number of original reads, that is, the depth value is ≥10, the site detection is determined to be successful, otherwise it is determined to be insufficient in depth; for the site where the detection is successful, the number of reads of the reference base and the mutant base is obtained. When the number of reads of the mutant base accounts for the proportion, that is, the mutation ratio is ≥10%, it is determined that a mutation is detected, otherwise it is determined that no mutation is detected; for the site where a mutation is detected, it is determined to be at high risk of gastric cancer or resistant to the corresponding antibiotic according to its significance, otherwise it is determined to be at low risk of gastric cancer or sensitive to the corresponding antibiotic.

[0090] Preferably, the quality control judgment criteria for the detection of virulence gene typing include: for samples judged to be HP positive, determining the typing based on the comparison of the detected vacA and cagA gene sequences with the reference sequence; comparing the s segment product sequence of vacA with the reference sequence to determine the s1 / s2 type, and comparing the m segment product sequence of vacA with the reference sequence to determine the m1 / m2 type, and finally combining them to obtain the vacA typing results s1m1 / s1m2 / s2m1 / s2m2; comparing the product sequence of the cagA segment with the reference sequence to determine the type of East Asian type EPIYA-D or Western type EPIYA-C.

[0091] Preferably, the quality control judgment criteria for detecting human proton pump inhibitor drug metabolism include: for samples that pass the quality control, using the bioinformatics software samtools to perform site depth and base statistics on the aligned BAM result file, obtaining the number of reads of the reference base and the mutant base of the target site, determining whether the site is detected according to the set threshold, and calculating the proportion of the mutant base, and determining the genotype result according to the mutation frequency; when the depth proportion of the mutant base is <0.25, it is determined to be wild type; when the depth proportion of the mutant base is in the interval [0.25,0.75], it is determined to be heterozygous; otherwise, when the depth proportion of the mutant base is >0.75, it is determined to be homozygous mutant.

[0092] In the present invention, the corresponding relationships between the detection sites of human proton pump inhibitor drug metabolism and the haplotype combination results and metabolic type results are shown in Table 3 below.

[0093] Table 3

[0094] c.-806C>T c.636G>A c.681G>A Haplotype combination Metabolic results T / T G / G G / G *17 / *17 Ultra-rapid metabolizer (UM) C / T G / G G / G *1 / *17 Extensive metabolizer (EM) C / C G / G G / G *1 / *1 Normal Metabolizer (NM) C / C G / G A / G *1 / *2 Intermediate metabolizer (IM) C / C A / G G / G *1 / *3 Intermediate metabolizer (IM) C / T G / G A / G *2 / *17 Intermediate metabolizer (IM) C / T A / G G / G *3 / *17 Intermediate metabolizer (IM) C / C G / G A / A *2 / *2 Poor metabolizer (PM) C / C A / G A / G *2 / *3 Poor metabolizer (PM) C / C A / G A / A *2 / *3 Poor metabolizer (PM) C / C A / A A / G *2 / *3 Poor metabolizer (PM) C / C A / A A / A *2 / *3 Poor metabolizer (PM) C / C A / A G / G *3 / *3 Poor metabolizer (PM)

[0095] In a third aspect, the present invention provides a kit comprising the primer combination for detecting Helicobacter pylori described in the first aspect.

[0096] Preferably, the kit further comprises any one or a combination of at least two of a plasmid containing an external reference sequence, a sequencing adapter primer or a PCR amplification enzyme.

[0097] Preferably, the external reference sequence includes the nucleotide sequence shown in SEQ ID NO: 50.

[0098] Preferably, a sequence complementary to the common sequence is provided at the 3' end of the sequencing adapter primer for connecting to the amplified product of the target region.

[0099] In a fourth aspect, the present invention provides use of the primer combination for detecting Helicobacter pylori described in the first aspect or the kit described in the third aspect in preparing a product for detecting Helicobacter pylori.

[0100] The numerical range described in the present invention includes not only the point values ​​listed above, but also any point values ​​between the above numerical ranges that are not listed. Due to space limitations and for the sake of simplicity, the present invention no longer exhaustively lists the specific point values ​​included in the range.

[0101] Compared with the prior art, the present invention has the following beneficial effects:

[0102] (1) Using stool or gastric mucosal tissue samples, a single test can complete the identification of Helicobacter pylori, virulence typing, gastric cancer risk points, antibiotic resistance, and human proton pump inhibitor drug metabolism type. Compared with single test items, it can greatly improve the detection efficiency.

[0103] (2) Based on multiplex PCR amplification and enrichment technology, combined with high-throughput sequencing methods, the detection of mutation sites can not only display the specific mutation ratio as a reference, but also ensure high detection sensitivity.

[0104] (3) The detection system introduces a quantitative method, which can perform approximate quantitative detection of the concentration of Helicobacter pylori and better indicate the severity of infection.

[0105] (4) The solution of the present invention uses high-throughput sequencing technology. The entire detection process takes about 12 hours. The detection throughput is large and hundreds of samples can be detected at one time. It is particularly suitable for use on a large scale, especially for people undergoing routine physical examinations. BRIEF DESCRIPTION OF THE DRAWINGS

[0106] Figure 1 This is a schematic diagram of external reference sequence design.

[0107] Figure 2 This is the library peak map. DETAILED DESCRIPTION

[0108] The technical solution of the present invention is further described below by way of specific embodiments. It should be understood by those skilled in the art that the embodiments are merely to help understand the present invention and should not be regarded as specific limitations of the present invention.

[0109] If no specific techniques or conditions are specified in the examples, the experiments were carried out according to the techniques or conditions described in the literature in the field or according to the product instructions. If no manufacturer is specified for the reagents or instruments used, they are all conventional products that can be purchased through regular channels.

[0110] Example 1

[0111] Clinical sample testing process

[0112] The primer set was used to detect the collected clinical samples as follows:

[0113] 1. Sample collection: Fecal samples and gastric mucosal tissue samples were retrospectively collected in the gastroenterology department and endoscopy department of the hospital. A total of 50 fecal samples were collected, including 40 Helicobacter pylori-positive samples and 10 Helicobacter pylori-negative samples. Twenty gastric mucosal samples were collected, including 18 Helicobacter pylori-positive samples and 2 Helicobacter pylori-negative samples. Helicobacter pylori was isolated and cultured from all 18 positive samples and survived.

[0114] 2. Sample pretreatment: For fecal samples, take a soybean-sized fecal sample into a 1.5mL centrifuge tube, add 1mL of normal saline, shake and mix, and take 500μL of the lower solid-liquid mixture for extraction; for gastric mucosal tissue samples, transfer the tissue sample to a new 1.5mL centrifuge tube, add 0.5mL of normal saline and mix for extraction.

[0115] 3. Nucleic acid extraction: Use nucleic acid extraction or purification reagents (Guangzhou Jinqirui Biotechnology Co., Ltd., registration number Yuesui Xiebei 20220884) according to the instructions for extraction.

[0116] 4. Preparation of Primer Mix: According to the ratios in Table 4 below, mix all primers to make 120 μL of a mixture for later use, with 1.5 μL of primer mix required for each reaction. To prepare 120 μL of primer mix, dilute the primers to 100 μM, then pipette 2.4 μL of PHP-1F and PHP-1R, then 2 μL of PHP-2F and PHP-2R, and so on, until finally pipetting 3.2 μL of PHP-24F and PHP-24R, respectively. Mix thoroughly. Any remaining primer mix should be stored at 4°C and used within one week. For long-term storage, keep at -20°C.

[0117] Table 4

[0118]

[0119] 5. Preparation of external reference sequence: Based on the identification primer PHP-1 for Helicobacter pylori, the corresponding external reference sequence was designed in the amplification target region ( Figure 1 ), the reverse complementary sequence of the target region was selected as the non-primer binding region of the external reference sequence, and the nucleotide sequence was shown in SEQ ID NO: 50. The synthesized external reference sequence was used to construct a plasmid, and after the plasmid was fixed, it was diluted to 1.0×10 4 copies / mL for future use.

[0120] 6. Library Preparation

[0121] (1) PCR 1: Perform super-multiplex PCR amplification on the nucleic acid template. A 25 μL library amplification reaction system was prepared by combining 1.5 μL of multiplex primer mix, 11.5 μL of PCR reaction buffer, 2 μL of plasmid containing an external reference sequence, and 10 μL of nucleic acid to obtain the target region fragment. The PCR reaction 1 amplification program is shown in Table 5.

[0122] Table 5

[0123]

[0124] (2) After purification of the PCR 1 reaction product, a 25 μL library amplification reaction system was prepared by combining 1 μL of adapter primers, 12.5 μL of PCR reaction buffer, and 11.5 μL of purified enriched product. The resulting library structure was "sequencing adapter-common sequence-target region-common sequence-sequencing adapter." The PCR reaction 2 amplification procedure is shown in Table 6.

[0125] Table 6

[0126]

[0127] (3) Each super-multiplex PCR library is purified by magnetic beads, and after purification, the libraries are mixed to obtain a library for Helicobacter pylori detection.

[0128] 7. Sequencing: The sequencer used for sequencing was KMMiniSeqDx-CN produced by Guangzhou Jinqirui Biotechnology Co., Ltd., and the universal sequencing reaction kit (Guangzhou Jinqirui Biotechnology Co., Ltd., product number: KS107-CXR, specification MR100) was used. The library was sequenced according to the instructions.

[0129] Figure 2 This is the peak map of the library, from Figure 2 It can be seen that there is a peak around 200-370bp, and the peak area in this region accounts for a relatively high proportion, indicating that the library construction is successful and the library quality is good.

[0130] Example 2

[0131] The sequencing data obtained by testing the samples to be tested using the primer sets in Table 2 can be analyzed as follows.

[0132] 1. Bioinformatics analysis: For the fastQ files obtained by sequencing, first use fastp software to count the raw data volume and filter out low-quality data in the raw sequencing results. Then, filter and retain valid reads containing primer sequences in the sequencing products. Then, use the mem alignment algorithm of BWA alignment software to align the valid read product sequences to the self-built database, and count the number of reads covered in each target region based on the BED file. Among them, the above-mentioned self-built database is a genome matched according to the product sequence of the primer, which is derived from the Nucleotide database of NCBI. Each target region contains at least one matching genome, and multiple genomes are added as much as possible so that all possible product sequences of the primer can be perfectly aligned to the database. Specifically, for the identification primers of HP, the HP genome containing the target gene ureA gene is selected, for the typing of virulence, the HP genome containing the s1 / s2 and m1 / m2 segments of vacA, and the HP genome containing the EPIYA-D and EPIYA-C segments of cagA are selected respectively, for the detection of gastric cancer risk and antibiotic resistance sites, the HP genome of the corresponding gene segment is selected, for the detection of proton pump inhibitor drug metabolism genes, the genome of the CYP2C19 gene segment of the HG19 version of the human genome is selected, for the detection of internal standards, the genome of the ACTB gene segment of the human genome is selected, and for the detection of external reference sequences, the genome containing the external reference sequence is selected. The nucleotide sequence is shown in SEQ ID NO: 50. The BED file format is a text file format widely used in bioinformatics. It is specifically used to describe features and regions on the genome. It is generated based on the primer theory alignment to the genomic region position information of the self-built database.

[0133] 2. Sample quality control: The data obtained from the analysis were quality controlled. The minimum sequencing data requirement for each sample was ≥50k, otherwise the raw data was judged to be unqualified; the sequencing quality requirement Q30 was ≥75%, otherwise the sequencing quality was judged to be unqualified; the number of internal standard reads detected was ≥50, otherwise the internal standard was judged to be unqualified; the number of external reference sequence reads detected was ≥50, otherwise the external reference sequence was judged to be unqualified; the depth values ​​of the three sites of the human genome CYP2C19 gene detected should all be ≥10, otherwise the CYP2C19 typing was judged to be unsuccessful.

[0134] 3. Identification and quantification of HP: For samples that have passed quality control, if the number of Helicobacter pylori reads is ≥50, it is considered HP positive, otherwise it is considered HP negative. At the same time, the approximate concentration is calculated based on the number of reads detected in the external reference sequence:

[0135]

[0136] Among them, C R is the concentration of the external reference sequence, V R is the volume of the external parameter sequence input, V T is the volume of nucleic acid input, N T is the number of reads of Helicobacter pylori detected, N R is the number of reads of the detected external reference sequence, and K is the coefficient.

[0137] According to the detection process of Example 1, C R =1.0×10 4 copies / mL, V R =2μL, V T =10μL, and the coefficient K is set to 20.

[0138] 4. HP virulence typing: For HP-positive samples, the typing is determined based on the alignment of the detected vacA and cagA gene sequences with the reference sequence. Specifically, the vacA s segment product sequence is compared with the reference sequence to determine the s1 / s2 type, and the vacA m segment product sequence is compared with the reference sequence to determine the m1 / m2 type. These are combined to obtain the vacA typing results of s1m1 / s1m2 / s2m1 / s2m2. Similarly, the cagA segment product sequence is compared with the reference sequence to determine the East Asian type (EPIYA-D) / Western type (EPIYA-C).

[0139] 5. HP-positive gastric cancer risk and drug-resistant loci detection: For samples tested positive for HP, the bioinformatics software samtools was used to analyze the site depth and base counts of the aligned BAM result files. When the tested site covered the original number of reads (i.e., the depth value was ≥10), the site detection was considered successful; otherwise, it was considered insufficient depth. For successfully detected sites, the number of reads of the reference base and the mutant base was obtained. When the proportion of reads of the mutant base (i.e., the mutation ratio) was ≥10%, a mutation was detected; otherwise, no mutation was detected. For sites with detected mutations, the significance of the detected mutations was determined as high risk for gastric cancer or resistance to the corresponding antibiotic. Otherwise, the risk was determined as low risk for gastric cancer or sensitivity to the corresponding antibiotic.

[0140] 6. Detection of Proton Pump Inhibitor Metabolism Genes: For samples that passed quality control, the bioinformatics software samtools was used to analyze the site depth and base counts of the aligned BAM result files. The number of reads for the reference base and the mutant base at the target site was obtained. When the depth ratio of the mutant base was <0.25, the gene was considered wild-type. When the depth ratio of the mutant base was in the range [0.25, 0.75], the gene was considered heterozygous. Otherwise, when the depth ratio of the mutant base was >0.75, the gene was considered homozygous mutant. The correspondence between the genotype results of the detected sites, the haplotype combination results, and the metabolic type results is shown in Table 3.

[0141] 7. In this example, the 70 samples from Example 1 were analyzed according to the above process, and the statistical results are shown in the table below. The results show that the primer set of the present invention can detect both stool and gastric mucosal samples with a high detection success rate, and the HP identification results are highly consistent with clinical results. Table 7 shows the statistical results of the 70 samples tested.

[0142] Table 7

[0143]

[0144] Example 3

[0145] Quantitative Analysis of Helicobacter pylori. Based on Example 2, this example calculated the approximate concentration of Helicobacter pylori for stool and gastric mucosal samples using the concentration estimation formula in Example 2 for samples that passed quality control and were determined to be HP-positive. A total of 58 positive samples were collected, and some of the data are shown in Table 8 below.

[0146] Table 8

[0147]

[0148] The results show that the primer set and external reference sequence of the present invention, combined with the concentration estimation formula, can successfully estimate the approximate concentration of Helicobacter pylori-positive samples. This approximate concentration can reveal the degree of Helicobacter pylori infection to a certain extent and has certain guiding significance for monitoring the course of the disease.

[0149] Example 4

[0150] Verification of detection site accuracy. Based on Example 1, this example performed whole-genome sequencing on 18 strains isolated and cultured from HP-positive gastric mucosal tissue samples. Through bioinformatics analysis, the sequencing sequences were assembled to obtain the virulence typing, gastric cancer risk sites, and drug resistance site results of these 18 Helicobacter pylori strains. The results of the detection using this primer set system in Example 2 were analyzed for consistency with the whole-genome sequencing results. The results are shown in Table 9 below, which compares the positive rates of the detection sites.

[0151] Table 9

[0152]

[0153]

[0154] From the above results, it can be seen that the detection results of the primer set of the present invention for HP virulence typing, gastric cancer risk sites and drug resistance sites of clinical samples are consistent with the results of whole genome sequencing, indicating that the primer set system of the present invention can successfully and correctly detect the target sites.

[0155] Example 5

[0156] Frequency Analysis of Proton Pump Inhibitor Metabolism Sites. Based on Example 2, this example analyzed the frequency of genotypes detected at three sites on the CYP2C19 gene for 70 quality-control-qualified samples. These frequencies were compared with the frequencies of the Chinese Beijing population (CHB) and the Chinese Southern population (CHS) in the 1000 Genomes database, as shown in the following table. The 1000 Genomes data is derived from the Ensembl database (http: / / grch37.ensembl.org / index.html). Table 10 compares the frequencies of CYP2C19 detection sites with those in the 1000 Genomes database.

[0157] Table 10

[0158]

[0159] For the genotype results of the three loci in the CYP2C19 gene, based on the correspondence table between the locus genotype results and the haplotype combination results and the metabolite results in Example 2, the haplotype combination results of 70 samples were obtained. Frequency statistics were performed on these haplotype combinations and compared with the frequencies of East Asian populations (EAS) in the PharmGKB database (Pharmacogenetics and Pharmacogenomics KnowledgeBase), as shown in the following table. The PharmGKB database website is (https: / / www.pharmgkb.org / ). Table 11 shows the comparison of CYP2C19 haplotype combinations with the PharmGKB database frequencies.

[0160] Table 11

[0161] CYP2C19 haplotype combinations PharmGKB-EAS Detection frequency of the present invention (70 samples) *17 / *17 0.0％ 0.0％ *1 / *17 2.4％ 1.4％ *1 / *1 41.1％ 32.9％ *1 / *2 33.8％ 44.3％ *1 / *3 8.6％ 5.7％ *2 / *17 1.2％ 2.9％ *3 / *17 0.3％ 1.4％ *2 / *2 8.0％ 8.6％ *2 / *3 4.1％ 2.9％ *3 / *3 0.5％ 0.0％

[0162] From the above results, it can be seen that the population frequency of the CYP2C19 gene site detected by the primer set of the system of the present invention is close to the frequency of the Chinese population in the 1000 Genomes database, and the haplotype combination results detected show a similar trend with the frequency of the East Asian population in the PharmGKB database, which indirectly demonstrates the accuracy of the detection site and the detection results of the proton pump inhibitor metabolic type of the system of the present invention.

[0163] Example 6

[0164] Specificity test. To verify the specificity of the primer set of the present invention, other bacteria with similar ecological niches to Helicobacter pylori or that may appear in gastric mucosa or fecal samples were selected for testing. In this example, nucleic acids from cultures of Escherichia coli, Enterococcus faecalis, Staphylococcus aureus, Campylobacter jejuni, Proteus, Clostridium difficile, and Salmonella typhi were mixed with human genomic DNA as templates, and the final concentration of each strain was 1.0×10 5 copies / mL, and the primer set system of the present invention was used for detection, and no false positive results were found, indicating that the primer set system of the present invention has good specificity.

[0165] Example 7

[0166] Detection limit test. This example uses Helicobacter pylori culture samples to test the detection limit performance of the system of the present invention. The following experimental design is performed:

[0167] 1. Gradient dilution: Helicobacter pylori culture nucleic acid was diluted with human cell suspension (1.0×10 6 cells / mL) nucleic acid was serially diluted as artificial simulated samples, and the final concentrations of Helicobacter pylori were 100 copies / mL, 200 copies / mL, 400 copies / mL, 800 copies / mL and 1600 copies / mL, for a total of 5 gradients.

[0168] 2. Set up repetitions: For the five simulated samples with concentration gradients, perform 20 repetitions respectively, and then test the above samples according to the scheme in Example 1, and obtain a total of 100 library experimental results.

[0169] 3. According to the analysis scheme in Example 2, the test results are statistically summarized; when calculating the detection rate, the following three rules are used for judgment: 1) If the number of reads in the identification target area is ≥50, it is judged as a positive detection, otherwise it is judged as negative, that is, missed detection. 2) If the number of reads in the virulence typing target area is ≥10, it is judged as virulence detected, otherwise it is judged as no virulence detected. Only when all virulence genes of a sample are judged as virulence detected, the sample is judged as virulence detected, otherwise it is judged as missed detection. 3) Only when the gastric cancer risk site and all drug resistance sites of a sample meet the site depth value ≥10, the site detection of the sample is judged to be successful, otherwise it is judged as missed detection.

[0170] 4. According to the above process, the statistical results are shown in Table 12 below. Table 12 shows the detection rate statistics at different dilution concentrations.

[0171] Table 12

[0172] Statistical classification 100 copies / mL 200 copies / mL 400 copies / mL 800 copies / mL 1600 copies / mL Number of repetitions 20 20 20 20 20 HP identification detection rate 60％(12 / 20) 100％(20 / 20) 100％(20 / 20) 100％(20 / 20) 100％(20 / 20) HP toxicity detection rate 5％(1 / 20) 40％(8 / 20) 100％(20 / 20) 100％(20 / 20) 100％(20 / 20) HP site detection rate 0％(0 / 20) 70％(14 / 20) 95％(19 / 20) 100％(20 / 20) 100％(20 / 20)

[0173] From the results of nucleic acid dilution and 20 repetitions, it can be seen that the primer set system of the present invention has an identification detection rate of 100% (20 / 20) for Helicobacter pylori at a concentration of 200 copies / mL. At a concentration of 400 copies / mL, the detection rate for the virulence, gastric cancer risk sites and drug resistance sites of Helicobacter pylori can reach more than 95%. Therefore, it is believed that the primer set system of the present invention has a high detection sensitivity.

[0174] Example 8

[0175] System adjustment performance study. This embodiment designs multiple pairs of candidate primers for each target, and conducts a large amount of research, and finally obtains the best amplification conditions close to each other, with little mutual interference between primers and no obvious non-specific amplification, ensuring that each amplicon can successfully amplify the primer combination under the same amplification conditions. During the experiment, the system was adjusted according to the ratio of primer dimers detected in the system and the detection rate of each target site. Specifically, first, the primer combination with a single pair of dimers having a mean value higher than 8% in the system was replaced so that the overall dimer ratio of the system was not higher than 25%, and secondly, samples identified as strongly positive (number of reads detected ≥ 1000) for Helicobacter pylori were replaced, but primers with virulence, gastric cancer risk sites and drug-resistant sites that were missed or with lower detection performance were replaced. Finally, the primer coefficient of the internal reference was adjusted so that different sample types could be detected successfully.

[0176] The detection results of the primer system (intermediate system) in Table 13 below are used as an example for comparison. In addition to using the primers in the table below to replace the corresponding target primers in Table 2 (final system) of the present invention, the detection primers for other target sites are the same as those in Table 2.

[0177] Table 13

[0178]

[0179] Three HP-positive stool samples and three HP-positive gastric mucosal tissue samples were selected, and the simulated samples described in Example 7 with final concentrations of 400 copies / mL and 800 copies / mL were selected. Each was repeated three times, for a total of 12 samples. The intermediate system and the final system of the present invention were used for testing. The comparative results are shown in Table 14 below. Table 14 shows the performance comparison of the detection results of the intermediate system and the final system.

[0180] Table 14

[0181]

[0182] The results show that in the intermediate system, the average proportion of overall dimers is 44%, and the average maximum proportion of single dimer combinations is 16%, which does not meet the detection requirements. In addition, the number of internal standard sequences detected is low, and there are samples that fail site and virulence detection. The detection results of the final system of the present invention show that the average proportion of overall dimers is 15%, and the average maximum proportion of single dimer combinations is 5%. All types of samples are successfully detected, and there are no failed sites, indicating that the final version of the primer system of the present invention has better detection performance.

[0183] The applicant declares that the above is only a specific embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Those skilled in the art should understand that any changes or substitutions that can be easily thought of by those skilled in the art within the technical scope disclosed by the present invention fall within the scope of protection and disclosure of the present invention.

Claims

1. A primer combination for detecting Helicobacter pylori, characterized in that: The primer combination is used for Helicobacter pylori species identification, gastric cancer risk sites, antibiotic resistance sites, virulence gene typing and human proton pump inhibitor drug metabolism detection, and the primer combination includes: (1) Primer combination for bacterial species identification, detecting gene ureA; (2) Primer combination for detecting gastric cancer risk loci, the detection gene is htrA and the detection locus is S171L; (3) Primer combinations for detecting Helicobacter pylori resistance sites, including genes and sites: Clarithromycin resistance detection gene 23S rRNA, detection sites include: A2143G and A2142G / A2142C; Levofloxacin resistance detection gene gyrA, detection sites include: D91N / D91Y / D91G and N87K / N87I; Tetracycline resistance detection gene 16S rRNA, detection sites include: A926G, A965T, G966T and A967C; Metronidazole resistance detection gene rdxA, detection sites include: R16H / R16C, G565T and G616A; Amoxicillin resistance detection gene pbp1A, detection sites include: V374L, T556S, N562Y, T593A, A1681T, S414R, A1217C and C206T; Furazolidone resistance detection genes porD and oorD. The detection sites of porD include: G353A, A356G and C357T / C357G; the detection sites of oorD include: A41G, A122G and C349A / C349G; (4) Primer combinations for detecting virulence genotyping, including: Vacuolating cytotoxin detection gene vacA, detection types include: s1m1, s1m2, s2m1 and s2m2; Cytotoxin-associated protein detection gene cagA, detection typing includes: East Asian EPIYA-D and Western EPIYA-C; (5) Primer combination for detecting drug metabolism of human proton pump inhibitors, the detection gene is CYP2C19, and the detection sites include: c.-806C>T, c.636G>A and c.681G>A.

2. The primer combination for detecting Helicobacter pylori according to claim 1, characterized in that: The primer combination for bacterial species identification includes: primers for detecting the ureA gene, the nucleotide sequences of which are shown in SEQ ID NO: 1 and SEQ ID NO:

2.

3. The primer combination for detecting Helicobacter pylori according to claim 1 or 2, characterized in that: The primer combination for detecting gastric cancer risk sites includes: primers for detecting the htrA gene, the nucleotide sequences of which are shown in SEQ ID NO: 11 and SEQ ID NO:

12.

4. The primer combination for detecting Helicobacter pylori according to any one of claims 1 to 3, characterized in that: The primer combination for detecting Helicobacter pylori resistance sites includes: Primers for detecting 23S rRNA, the nucleotide sequences of which are shown in SEQ ID NO: 13 and SEQ ID NO: 14; Primers for detecting gyrA, the nucleotide sequences of which are shown in SEQ ID NO: 15 and SEQ ID NO: 16; Primers for detecting 16S rRNA, the nucleotide sequences of which are shown in SEQ ID NO: 17 and SEQ ID NO: 18; Primers for detecting the R16H / R16C site of the rdxA gene, the nucleotide sequences of which are shown in SEQ ID NO:29 and SEQ ID NO:30; Primers for detecting the G565T and G616A sites of the rdxA gene, the nucleotide sequences of which are shown in SEQ ID NO:31 and SEQ ID NO:32; Primers for detecting the V374L site of the pbp1A gene, the nucleotide sequences of which are shown in SEQ ID NO: 19 and SEQ ID NO: 20; Primers for detecting the T556S, N562Y, and A1681T sites of the pbp1A gene, the nucleotide sequences of which are shown in SEQ ID NO: 21 and SEQ ID NO: 22; Primers for detecting the T593A site of the pbp1A gene, the nucleotide sequences of which are shown in SEQ ID NO: 23 and SEQ ID NO: 24; Primers for detecting the S414R and A1217C sites of the pbp1A gene, the nucleotide sequences of which are shown in SEQ ID NO:25 and SEQ ID NO:26; Primers for detecting the C206T site of the pbp1A gene, the nucleotide sequences of which are shown in SEQ ID NO: 27 and SEQ ID NO: 28; Primers for detecting the G353A, A356G, and C357T / C357G sites of the porD gene, the nucleotide sequences of which are shown in SEQ ID NO:33 and SEQ ID NO:34; Primers for detecting the A41G site of the oorD gene, the nucleotide sequences of which are shown in SEQ ID NO:35 and SEQ ID NO:36; Primers for detecting the A122G site of the oorD gene, the nucleotide sequences of which are shown in SEQ ID NO:37 and SEQ ID NO:38; The nucleotide sequences of the primers for detecting the C349A / C349G sites of the oorD gene are shown in SEQ ID NO: 39 and SEQ ID NO:

40.

5. The primer combination for detecting Helicobacter pylori according to any one of claims 1 to 4, characterized in that: The primer combination for detecting virulence genotyping includes: The nucleotide sequences of primers for detecting vacA genotype s are shown in SEQ ID NO:3 and SEQ ID NO:4, and the nucleotide sequences of primers for detecting vacA genotype m are shown in SEQ ID NO:5 and SEQ ID NO:6; Primers for detecting the East Asian EPIYA-D locus of the cagA gene, the nucleotide sequences of which are shown in SEQ ID NO: 7 and SEQ ID NO: 8; The nucleotide sequences of primers for detecting the Western-type EPIYA-C site of the cagA gene are shown in SEQ ID NO: 9 and SEQ ID NO:

10.

6. The primer combination for detecting Helicobacter pylori according to any one of claims 1 to 5, characterized in that: The primer combination for detecting human proton pump inhibitor drug metabolism includes: Primers for detecting the c.-806C>T site of the CYP2C19 gene, the nucleotide sequences of which are shown in SEQ ID NO:41 and SEQ ID NO:42; Primers for detecting the c.636G>A site of the CYP2C19 gene, the nucleotide sequences of which are shown in SEQ ID NO:43 and SEQ ID NO:44; The primers for detecting the c.681G>A site of the CYP2C19 gene have nucleotide sequences shown in SEQ ID NO:45 and SEQ ID NO:

46.

7. The primer combination for detecting Helicobacter pylori according to any one of claims 1 to 6, characterized in that: The primer combination also includes primers for detecting internal standards, including the nucleotide sequences shown in SEQ ID NO:47 and SEQ ID NO:48; Preferably, the 5' end of the primer is connected to a common sequence required for nucleic acid sequencing library construction, the common sequence is complementary to the 3' end of the sequencing adapter of the sequencing platform, and the common sequence is used to connect the sequencing adapter primer during the amplification process; Preferably, the common sequence is a sequence of 15-30 bases; Preferably, the common sequence includes the nucleotide sequence shown in SEQ ID NO:

49.

8. A method for detecting Helicobacter pylori based on multiplex PCR and targeted high-throughput sequencing technology, characterized in that: The method comprises: (1) using the primer combination for detecting Helicobacter pylori described in any one of claims 1 to 7 to perform super-multiplex PCR amplification on the test sample, purifying the amplified product, and then amplifying with sequencing adapter primers to construct a super-multiplex PCR library, wherein the final library structure obtained is sequencing adapter-common sequence-target region-common sequence-sequencing adapter; (2) purifying each super-multiplex PCR library, and after purification, mixing the libraries to obtain a library for Helicobacter pylori detection; (3) High-throughput sequencing of the Helicobacter pylori detection library using a nucleic acid sequencing platform; statistics on the amount of raw data, filtering low-quality data in the raw sequencing results, screening and retaining valid reads containing primer sequences in the sequencing products, using alignment software to align the valid read product sequences to a self-built database, and counting the number of reads covering each target area based on the BED file; quality control and result determination of the analyzed data; The result determination includes: bacterial species identification, gastric cancer risk site and drug resistance site detection result determination, virulence gene typing result determination, and human proton pump inhibitor drug metabolism result determination; Preferably, in step (1), the super-multiplex PCR amplification system for bacterial species identification further includes a plasmid containing an external reference sequence, wherein the external reference sequence consists of a primer binding region and a non-primer binding region, wherein the primer binding region is the same as the primer binding region of the target region on the Helicobacter pylori genome, and the non-primer binding region is a complementary, reverse, or reverse complementary sequence to the non-primer binding region of the target region; Preferably, the external reference sequence includes the nucleotide sequence shown in SEQ ID NO: 50; Preferably, in step (3), the nucleic acid sequencing platform is a second-generation high-throughput sequencing platform, including the Ion Torrent platform, the Roche 454 platform, the SOLiD sequencing platform, the BGI sequencing platform, the Zhenmai Bio sequencing platform, the Celu Medical sequencing platform, and the KM MiniSeqDx-CN sequencing platform; Preferably, the BGI sequencing platform includes the MGISEQ series or the DNBSEQ series; Preferably, in step (3), the software used for the comparison includes Bowtie2, Minimap2, HISAT2 or BWA comparison software; Preferably, in step (3), the library is subjected to high-throughput sequencing using the KM MiniSeqDx-CN sequencing platform, and the sequence alignment is performed using the mem alignment algorithm in the BWA software to obtain a BAM format file of the alignment result; Preferably, in step (3), the self-built database is a genome matched according to the product sequence of the primer, which is derived from the Nucleotide database of NCBI, and each target region contains at least one genome matched thereto; Preferably, the quality control standards include: a minimum sequencing data requirement of ≥50k for each sample, otherwise the raw data is judged to be unqualified; a sequencing quality requirement Q30 ≥75%, otherwise the sequencing quality is judged to be unqualified; the number of internal standard reads detected is ≥50, otherwise the internal standard is judged to be unqualified; the number of external reference sequence reads detected is ≥50, otherwise the external reference sequence is judged to be unqualified; the depth values of the three sites of the human genome CYP2C19 gene detected should all be ≥10, otherwise the CYP2C19 typing is judged to be unsuccessful; Preferably, the genomes used in the quality control process include: for HP identification primers, an HP genome containing the target gene ureA gene is selected; for virulence typing primers, HP genomes containing the s1 / s2 and m1 / m2 segments of vacA, and the EPIYA-D and EPIYA-C segments of cagA are selected respectively; for detection primers for gastric cancer risk and antibiotic resistance sites, HP genomes of corresponding gene segments are selected; for detection primers for proton pump inhibitor drug metabolism genes, a genome of the CYP2C19 gene segment of the HG19 version of the human genome is selected; for internal standard detection primers, a genome of the ACTB gene segment of the human genome is selected; for detection primers for external reference sequences, a genome containing an external reference sequence is selected, and the nucleotide sequence is shown in SEQ ID NO: 50; Preferably, the criteria for determining the result of the bacterial species identification include: for a sample that has passed quality control, when the number of Helicobacter pylori reads detected is ≥50, it is determined to be HP positive, otherwise it is determined to be HP negative; and then the approximate concentration is calculated in combination with the number of reads detected in the external reference sequence; Preferably, the calculation formula for calculating the approximate concentration is: Among them, C R is the concentration of the external reference sequence, V R is the volume of the external parameter sequence input, V T is the volume of nucleic acid input, N T is the number of Helicobacter pylori read sequences detected, N R is the number of read sequences of the detected external reference sequence, and K is the coefficient; Preferably, the criteria for determining the results of the gastric cancer risk site and drug-resistant site detection include: for samples determined to be HP positive, using the bioinformatics software samtools to perform site depth and base statistics on the aligned BAM result file, obtaining the number of reads of the reference base and the mutant base of the target site, and when the site to be tested covers the original number of reads, that is, the depth value is ≥10, it is determined that the site detection is successful, otherwise it is determined that the depth is insufficient; for the site that is successfully detected, the number of reads of the reference base and the mutant base is obtained, and when the proportion of the number of reads of the mutant base, that is, the mutation ratio, is ≥10%, it is determined that a mutation is detected, otherwise it is determined that no mutation is detected; for the site where a mutation is detected, it is determined to be a high risk of gastric cancer or resistant to the corresponding antibiotic according to its significance, otherwise it is determined to be a low risk of gastric cancer or sensitive to the corresponding antibiotic; Preferably, the quality control judgment criteria for the detection of virulence genotyping include: for samples judged to be HP positive, determining the typing based on the comparison of the detected vacA and cagA gene sequences with the reference sequence; comparing the vacA s segment product sequence with the reference sequence to determine the s1 / s2 type, and comparing the vacA m segment product sequence with the reference sequence to determine the m1 / m2 type, and finally combining them to obtain the vacA typing result s1m1 / s1m2 / s2m1 / s2m2; comparing the cagA segment product sequence with the reference sequence to determine the type of East Asian EPIYA-D or Western EPIYA-C; Preferably, the quality control judgment criteria for detecting human proton pump inhibitor drug metabolism include: for samples that pass quality control, using the bioinformatics software samtools to perform site depth and base statistics on the aligned BAM result file, obtaining the number of reads of the reference base and the mutant base of the target site, determining whether the site is detected according to the set threshold, and calculating the proportion of the mutant base, and determining the genotype result according to the mutation frequency; when the depth proportion of the mutant base is <0.25, it is determined to be wild type; when the depth proportion of the mutant base is in the interval [0.25,0.75], it is determined to be heterozygous; otherwise, when the depth proportion of the mutant base is >0.75, it is determined to be homozygous mutant.

9. A kit, characterized in that The kit comprises the primer combination for detecting Helicobacter pylori according to any one of claims 1 to 7; Preferably, the kit further comprises any one or a combination of at least two of a plasmid containing an external reference sequence, a sequencing adapter primer, or a PCR amplification enzyme; Preferably, the external reference sequence includes the nucleotide sequence shown in SEQ ID NO: 50; Preferably, a sequence complementary to the common sequence is provided at the 3' end of the sequencing adapter primer for connecting to the amplified product of the target region.

10. Use of the primer combination for detecting Helicobacter pylori according to any one of claims 1 to 7 or the kit according to claim 9 in preparing a product for detecting Helicobacter pylori.

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