Method for detecting helicobacter pylori virulence typing in fecal samples based on multi-gene combination non-invasive detection

Abstract

The application belongs to the technical field of gene detection, and particularly relates to a method for non-invasive detection of Helicobacter pylori virulence typing in fecal samples based on multiple gene combination, which adopts a primer probe group including an upstream primer with the nucleic acid sequence shown in SEQ ID NO:1, SEQ ID NO:2 and SEQ ID NO:3, a downstream primer with the nucleic acid sequence shown in SEQ ID NO:4, SEQ ID NO:5 and SEQ ID NO:6, and a fluorescent probe with the nucleic acid sequence shown in SEQ ID NO:7, SEQ ID NO:8 and SEQ ID NO:9. The application can be applied to the technical fields of PCR, qPCR, sequencing and the like, has the characteristics of non-invasiveness, rapidness, simplicity, objectivity and high-throughput detection capability, is very suitable for large-scale screening and precise treatment guidance in the clinic, improves the overall level of biological clinical detection, and provides a solid foundation for the wide application of the technology.

Description

Technical Field

[0001] This invention belongs to the field of gene detection technology, specifically relating to a method for non-invasive detection of Helicobacter pylori virulence typing in fecal samples based on multi-gene combination. Background Technology

[0002] Helicobacter pylori (H. pylori) is a spiral-shaped, curved, Gram-negative bacillus. Studies have found that this bacterium is a major pathogenic factor for chronic gastritis and peptic ulcers, and is also associated with the occurrence and development of gastric cancer. According to epidemiological surveys by the World Health Organization, H. pylori is present in the stomachs of more than 50% of the world's population. In 1994, H. pylori was classified as a Group 1 carcinogen by the WHO.

[0003] The core genome of Helicobacter pylori contains approximately 1100 genes, of which about 200 are functionally specific. Its main virulence-related genes include CagA, VacA, UreA / B, babA2, dupA, and omp genes. CagA is closely associated with various gastric diseases, while VacA plays a crucial role in pathological processes by disrupting mitochondrial function and inducing apoptosis. The Ure gene helps bacteria survive in the acidic environment of the stomach, but it has little variation, making it difficult to distinguish virulence differences between strains. The babA2 and dupA genes are associated with certain gastric diseases, but their distribution and expression vary greatly, and their correlation is less clear than that of CagA and VacA. The omp gene family has many members, high variation, and complex genotyping. Therefore, although these genes are important in pathogenic mechanisms, their application in routine genotyping is limited due to their low variation, unclear correlations, and complex detection methods.

[0004] Currently, Helicobacter pylori can be classified into two types: Type I and Type II, based on whether it expresses VacA and CagA proteins. Type I strains are highly virulent, carrying the CagA gene and / or the VacA gene, and expressing at least one of these two proteins, thereby producing at least one toxin, either vacuole toxin (VacA) or cytotoxin-associated protein (CagA). Type II strains, on the other hand, are less virulent, carrying the VacA gene but not the CagA gene, and do not express CagA protein or any active VacA protein.

[0005] The presence of the CagA gene is usually associated with highly virulent strains, but not all highly virulent strains contain the CagA gene. The VacA gene is present in all Helicobacter pylori strains. The expressed VacA protein is a cytotoxin that can induce vacuolation in host cells, inhibit T cell proliferation, and induce apoptosis. The VacA gene is polymorphic, mainly consisting of a signal sequence (S region) and an intermediate sequence (M region). The VacA genotype can be divided into four subtypes: s1m1, s1m2, s2m1, and s2m2. The VacA proteins of the s1m1, s1m2, and s2m1 genotypes have strong cytotoxicity, while the VacA protein of the s2m2 genotype has weaker cytotoxicity and is associated with milder disease manifestations. Based on the above, gene fragments of highly virulent and low-virulence strains are compared to design primers and probes to distinguish between them.

[0006] Helicobacter pylori typing is of great significance in clinical practice. First, it allows for accurate assessment of virulence and individualized treatment. Type I strains are highly virulent, and eradication therapy is recommended for infected individuals, regardless of symptom presence. Type II strains are less virulent, and eradication therapy is chosen based on specific circumstances. Second, it enables early identification of high-risk groups, especially those at high risk for gastric cancer, allowing for more precise intervention and treatment. Third, it helps avoid antibiotic overuse. Typing can prevent unnecessary eradication therapy for low-virulence strains, reducing antibiotic use and the emergence of drug-resistant strains. Fifth, it facilitates the rational allocation of medical resources. Typing helps differentiate between highly virulent strains requiring treatment and low-virulence strains that do not require treatment, thus enabling the rational allocation of medical resources.

[0007] Therefore, there is a strong clinical demand for Helicobacter pylori typing. However, current mainstream testing methods all have significant limitations. First, serological antibody testing, commonly used for Helicobacter pylori infection screening, cannot accurately determine whether a patient is currently infected. Because antibodies persist in the body for a long time, they can still be detected even after infection has been cleared, easily leading to misdiagnosis. Simultaneously, this method cannot be used to assess treatment effectiveness, as antibody levels change slowly and cannot reflect treatment effectiveness in a timely manner, making it difficult to provide real-time feedback. Furthermore, individual differences in antibody responses affect testing accuracy, and it has low sensitivity for early infection detection, easily leading to missed diagnoses. Therefore, although it has some value in initial screening, its application is limited when accurate assessment of infection status and evaluation of treatment effectiveness is required. Gastric mucosal biopsy is the "gold standard" for diagnosing Helicobacter pylori infection. It allows direct observation of the gastric mucosa via gastroscopy and the collection of biopsy tissue from suspicious sites for testing, enabling simultaneous histopathological examination and bacterial culture. However, histopathological examination can be subjective due to staining techniques and observer experience, and may miss early or mild infections. While bacterial culture is highly accurate, it is complex, time-consuming (usually 3-7 days), requires strict laboratory conditions, and is susceptible to false negatives due to antibiotic use. Furthermore, as an invasive method, it can cause patient discomfort and is not easily accepted by patients, making it difficult to use for routine checkups or in healthy individuals. qPCR, a molecular biology technique, amplifies specific gene fragments of Helicobacter pylori to determine infection, enabling early detection and precise identification of infection types. However, current qPCR kits primarily function only for infection assessment and lack the ability to differentiate between highly virulent and low-virulence strains. While the urea breath test is rapid and non-invasive, it cannot perform genotyping. Fecal sample testing eliminates the need for invasive gastroscopy, offering convenient sampling, reflecting the true state of intestinal colonization, stable matrix target abundance, and favorable sample preservation and transportation. Follow-up testing after drug withdrawal is unaffected by short-term interference from proton pump inhibitors and antibiotics. Compared to carbon-13 / 14 breath tests, the waiting period after drug withdrawal is shorter, and results are more stable, allowing for comprehensive virulence gene typing. However, fecal samples contain many impurities, making nucleic acid extraction difficult. High levels of nucleic acid inhibitors and strong PCR inhibition can lead to abnormal amplification curves, delayed Ct values, abnormal internal standards, and false negatives. This has resulted in the current lack of fecal sample-based Helicobacter pylori typing products. In summary, there is currently no non-invasive detection method among mainstream methods that can simultaneously perform symptomatic infection and virulence typing. Summary of the Invention

[0008] The technical problem this invention aims to solve is to address the shortcomings of the existing technology by providing a method for non-invasive detection of Helicobacter pylori virulence typing in fecal samples based on multi-gene combined detection. This method utilizes a DNA template extracted from fecal samples and employs a specifically designed primer-probe set for multi-gene combined detection. It is non-invasive, rapid, simple, and objective, and can effectively reduce the risk of contamination by using a closed-tube reaction. Its high-throughput detection capability makes it highly suitable for large-scale screening and precision treatment guidance in clinical settings. Using qPCR technology, the detection process can be completed within one working day.

[0009] The technical solution adopted in this invention is: a method for non-invasive detection of Helicobacter pylori virulence typing in fecal samples based on multi-gene combined assay, characterized by comprising the following steps:

[0010] Step 1: Collect fecal samples, dilute with preservation solution, grind, extract and purify, and prepare DNA templates;

[0011] Step 2: The DNA template prepared in Step 1 is amplified by qPCR using primer and probe kit reagents, and fluorescence detection is performed in real time to analyze and determine the detection results. The primer and probe kit includes upstream primers for nucleic acid sequences as shown in SEQ ID NO:1, SEQ ID NO:2 and SEQ ID NO:3, downstream primers for nucleic acid sequences as shown in SEQ ID NO:4, SEQ ID NO:5 and SEQ ID NO:6, and fluorescent probes for nucleic acid sequences as shown in SEQ ID NO:7, SEQ ID NO:8 and SEQ ID NO:9.

[0012] The above-mentioned method for non-invasive detection of Helicobacter pylori virulence typing in fecal samples based on multi-gene combination is characterized in that the preservation solution in step one includes tris(hydroxymethyl)aminomethane hydrochloride, EDTA-Na2, NaCl, Tween-20, isopropanol and Proclin 300.

[0013] The above-mentioned method for non-invasive detection of Helicobacter pylori virulence typing in fecal samples based on multi-gene combination is characterized in that the extraction and purification method in step one includes any one of the boiling method, centrifugation column method and magnetic bead method.

[0014] The above-mentioned method for non-invasive detection of Helicobacter pylori virulence typing in fecal samples based on multi-gene combination is characterized in that the primer probe set in step two further includes an upstream primer with a nucleic acid sequence as shown in SEQ ID NO:10, a downstream primer with a nucleic acid sequence as shown in SEQ ID NO:11, and an ATCB internal standard gene probe with a nucleic acid sequence as shown in SEQ ID NO:12.

[0015] The above-mentioned method for non-invasive detection of Helicobacter pylori virulence typing in fecal samples based on multi-gene combination is characterized in that the genes used to detect Helicobacter pylori nucleic acid typing in fecal samples are 16S rRNA, VacA and CagA, SEQ ID NO:1, SEQ ID NO:4 and SEQ ID NO:7 are 16S rRNA sequences, SEQ ID NO:2, SEQ ID NO:5 and SEQ ID NO:8 are VacA sequences, and SEQ ID NO:3, SEQ ID NO:6 and SEQ ID NO:9 are CagA sequences.

[0016] The above-mentioned method for non-invasive detection of Helicobacter pylori virulence typing in fecal samples based on multi-gene combination is characterized in that the 16S rRNA probe 16S-P is labeled with the fluorescent group FAM, the VacA probe VacA-P is labeled with the fluorescent group VIC, the CagA probe CagA-P is labeled with the fluorescent group ROX, the ATCB probe ATCB-P is labeled with the fluorescent group Cy5, and the quenching group is any one of BHQ1, BHQ2, TAMRA, DABCYL, MGB and Eclipse.

[0017] The above-mentioned method for non-invasive detection of Helicobacter pylori virulence typing in fecal samples based on multi-gene combination is characterized in that the concentration of each upstream primer is 0.3 μM, the concentration of each downstream primer is 0.3 μM, and the concentration of each fluorescent probe is 0.2 μM.

[0018] The above-mentioned method for non-invasive detection of Helicobacter pylori virulence typing in fecal samples based on multi-gene combination is characterized in that the reaction program for qPCR amplification in step two is: 95℃, 10min; 95℃, 15s; 60℃, 45s; 45 cycles.

[0019] The above-mentioned method for non-invasive detection of Helicobacter pylori virulence typing in fecal samples based on multi-gene combination is characterized in that the detection results can be determined by amplification curves:

[0020] ① If the 16S rRNA amplification curve shows a value, it indicates that the sample is infected with Helicobacter pylori; based on this, if at least one of the VacA and CagA amplification curves shows a value, it is determined to be an infection of a highly virulent Hp strain; if neither the VacA nor the CagA amplification curves show a value, it is determined to be an infection of a low-virulence Hp strain.

[0021] ②If the 16S rRNA amplification curve does not show any value, it indicates that the sample subject is not infected with Helicobacter pylori.

[0022] Compared with the prior art, the present invention has the following advantages:

[0023] 1. This invention designs a multi-gene joint detection method for Helicobacter pylori virulence typing. It specifically designs multiple sets of highly specific primer-probe sets targeting the 16S rRNA, VacA, and CagA virulence genes of Helicobacter pylori in fecal samples. This overcomes the problems of high levels of nucleic acid inhibitors and strong PCR inhibition in fecal samples, enabling simultaneous detection of the 16S rRNA, VacA, and CagA virulence genes of Helicobacter pylori, and allowing for typing of infected Helicobacter pylori. Furthermore, a primer-probe set for detecting the internal standard ATCB is included. Each primer-probe set does not interfere with the others during amplification and can amplify the specific target, resulting in high detection accuracy.

[0024] 2. The method of the present invention can be used for the auxiliary diagnosis of Helicobacter pylori infection, which can help clinicians formulate more precise treatment strategies, significantly improve the eradication efficiency of Helicobacter pylori, and thus effectively curb the rise in the incidence of gastric cancer.

[0025] 3. This invention addresses the differences in virulence among different subtypes of the VacA gene (S1, S2, M1, M2), deeply comparing the sequence characteristics of high- and low-virulence strains, and precisely constructing specific primer sequences for high-virulence VacA strains based on this comparison. For the CagA gene, sequences from highly virulent strains containing this gene were carefully selected for comparison, and highly homologous sequence fragments were screened and used as templates for specific primer design. Simultaneously, corresponding primers were designed for 16S rRNA to ensure specific amplification of Helicobacter pylori without cross-reactivity with other Helicobacter spp. A dedicated ATCB internal standard gene primer is also included for internal standard detection. Each primer set does not interfere with each other during amplification and can achieve amplification of the specific target, resulting in high detection accuracy. The primer-probe set designed in this application is specifically for fecal samples, enabling qPCR amplification detection of Helicobacter pylori in feces, with fast detection speed, high sensitivity, and relatively low cost.

[0026] 4. This invention achieves rapid typing and detection of Helicobacter pylori in fecal samples by diluting them with a preservation solution, grinding them, extracting and purifying them, preparing a DNA template, and then combining it with a primer and probe set and an amplification reaction system. This effectively avoids the invasive procedures associated with traditional invasive sampling.

[0027] 5. The detection method of this invention is non-invasive, rapid, simple, and objective. It utilizes a closed-tube reaction to effectively reduce the risk of contamination and possesses high-throughput detection capabilities, making it highly suitable for large-scale screening and precision medicine guidance in clinical settings. Using qPCR technology, the detection process can be completed within one working day, with highly reliable results. This not only improves the overall level of clinical biological testing but also provides a solid foundation for the widespread application of this technology.

[0028] 6. The primer and probe set, reagent kit, and detection method of this invention can be applied to PCR, qPCR, sequencing, and other technical fields. They are characterized by speed, simplicity, objectivity, and high-throughput detection capabilities, making them highly suitable for large-scale screening and precision medicine guidance in clinical settings. This not only improves the overall level of clinical biological testing but also provides a solid foundation for the widespread application of this technology.

[0029] 7. The detection method of the present invention is a non-invasive detection method, which is very convenient to operate and can obtain the test results within one working day. Moreover, the test results can be judged by detecting the fluorescence change of the reaction system using commonly used fluorescence quantitative PCR detection equipment. It is suitable for large-scale screening and detection and is very suitable for clinical promotion.

[0030] The technical solution of the present invention will be further described in detail below with reference to the accompanying drawings and embodiments. Attached Figure Description

[0031] Figure 1 The results of the 16S rRNA sensitivity test in Experiment Example 1 of this invention;

[0032] Figure 2 The results are the VacA sensitivity test results from Experiment Example 1 of this invention;

[0033] Figure 3 The results are the CagA sensitivity test results from Experiment Example 1 of this invention;

[0034] Figure 4 The results are the ATCB sensitivity test results from Experiment Example 1 of this invention;

[0035] Figure 5 This refers to the specificity test results in Experiment Example 2 of this invention;

[0036] Figure 6 The results of the detection of the highly toxic strain in Experimental Example 3 of this invention;

[0037] Figure 7 This is the detection result of the low-toxicity strain in Experiment Example 3 of this invention. Detailed Implementation

[0038] The following embodiments are provided to better understand the present invention and are not limited to the preferred embodiments described. They do not constitute a limitation on the content and scope of protection of the present invention. Any product that is the same as or similar to the present invention, derived by any person under the guidance of the present invention or by combining the features of the present invention with other prior art, falls within the protection scope of the present invention.

[0039] The following terms or definitions are provided merely to aid in understanding the invention. These definitions should not be construed as having a scope less than that understood by those skilled in the art.

[0040] Unless otherwise defined below, all technical and scientific terms used in the specific embodiments of this invention are intended to have the same meaning as commonly understood by those skilled in the art. While it is believed that the following terms will be well understood by those skilled in the art, the following definitions are set forth to better explain the invention.

[0041] As used in this invention, the terms “comprising,” “including,” “having,” “containing,” or “involving” are inclusive or open-ended and do not exclude other unlisted elements or method steps.

[0042] The term “consisting of” is considered a preferred embodiment of the term “comprising”. If a group is defined below as comprising at least a certain number of embodiments, this should also be understood to disclose a group that preferably consists only of these embodiments.

[0043] When referring to a singular noun, the indefinite or definite article used, such as "a" or "a kind of," "the," includes the plural form of the noun.

[0044] The terms "approximately" and "generally" in this invention refer to a range of accuracy that, as would be understood by those skilled in the art, still guarantees the technical effects of the discussed features. This term typically indicates a deviation from the indicated value of ±10%, preferably ±5%.

[0045] The term "nucleic acid" or "nucleic acid sequence" as used in this invention refers to any molecule, preferably a polymeric molecule, containing units of ribonucleic acid, deoxyribonucleic acid, or similar molecules. The nucleic acid may be single-stranded or double-stranded. A single-stranded nucleic acid may be the nucleic acid of one strand of denatured double-stranded DNA. Alternatively, a single-stranded nucleic acid may be a single-stranded nucleic acid derived from any double-stranded DNA.

[0046] The present application will be further described in detail below with reference to the embodiments.

[0047] Example 1

[0048] This example illustrates the design and synthesis of upstream primers, downstream primers, and fluorescent probes.

[0049] This invention provides a primer and probe set for non-invasive detection of Helicobacter pylori virulence typing in fecal samples based on multi-gene combination. The set includes primers and probes for detecting 16S rRNA, VacA, CagA, and the internal control ATCB. The main research content is as follows: based on the sequence characteristics of the 16S rRNA, CagA, and VacA genes in the HP genome, PCR primers are designed to amplify the target fragments for typing detection. In addition, the ATCB gene is designed as an IC internal control to monitor the entire experimental process. Multiple sequences of the 16S rRNA, CagA, VacA, and ATCB genes are searched and downloaded from the Genebank database. MEGA alignment is used to identify the sequence with the highest homology for each target. Primers are designed using Primer Premier 5.0 based on the sequences with the highest homology for each target, ensuring that the Tm values ​​of the typing amplification primers are similar; thus, each typing primer can be amplified under the same conditions. The designed primers were synthesized by Sangon Biotech (Shanghai) Co., Ltd. After synthesis, the primer sequences were verified by our personnel, and then dissolved and diluted to the required concentration. This invention screened numerous experiments to identify genotypic amplification primers capable of efficient and stable amplification of nucleic acids in fecal samples. Changes in the length and position of the primers in this invention would reduce the sensitivity and reproducibility of this kit. Through individual screening of primers for each genotype and optimization of primer combinations for each genotype, the optimized final primer and probe sequences used in this invention were determined as follows:

[0050] The primer and probe set for detecting 16S rRNA includes:

[0051] 16S-F, sequence as shown in SEQ ID NO:1; 16S-R, sequence as shown in SEQ ID NO:4; 16S-P, sequence as shown in SEQ ID NO:7;

[0052] The primer and probe set for detecting VacA includes:

[0053] VacA-F, sequence as shown in SEQ ID NO:2; VacA-R, sequence as shown in SEQ ID NO:5; VacA-P, sequence as shown in SEQ ID NO:8;

[0054] The primer and probe set for detecting CagA includes:

[0055] CagA-F, sequence as shown in SEQ ID NO:3; CagA-R, sequence as shown in SEQ ID NO:6; CagA-P, sequence as shown in SEQ ID NO:9;

[0056] The primer and probe set for detecting ATCB includes:

[0057] ATCB-F, sequence as shown in SEQ ID NO:10; ATCB-R, sequence as shown in SEQ ID NO:11; ATCB-P, sequence as shown in SEQ ID NO:12;

[0058] The specific sequence of this application is shown in Table 1 below.

[0059] Table 1 Sequence List

[0060]

[0061] The primer and probe set designed in this invention is specifically designed for the genotyping detection of Helicobacter pylori in fecal samples, and can simultaneously detect 16S rRNA, VacA, CagA, and ATCB in fecal samples. This primer and probe set can be widely applied in the field of fluorescent qPCR amplification for the detection of Helicobacter pylori.

[0062] Example 2

[0063] This embodiment illustrates a kit for non-invasive detection of Helicobacter pylori virulence typing in fecal samples based on multi-gene combination, including the primer and probe set described in Example 1, with each upstream primer having a concentration of 0.3 μM, each downstream primer having a concentration of 0.3 μM, and each fluorescent probe having a concentration of 0.2 μM.

[0064] Centrifuge the synthesized qPCR primers / probes from Example 1 at 6000 rpm for 5 min; open the cap, add nuclease-free water, dilute the primers and probes to 100 μM, and vortex thoroughly to mix before use.

[0065] qPCR amplification reagents include:

[0066] 0.06 μL of the upstream primer (100 μM) shown in SEQ ID NO.1;

[0067] 0.06 μL of the upstream primer (100 μM) shown in SEQ ID NO.2;

[0068] 0.06 μL of the upstream primer (100 μM) shown in SEQ ID NO.3;

[0069] 0.06 μL of the downstream primer (100 μM) shown in SEQ ID NO.4;

[0070] 0.06 μL of the downstream primer (100 μM) shown in SEQ ID NO.5;

[0071] 0.06 μL of the downstream primer (100 μM) shown in SEQ ID NO.6;

[0072] 0.04 μL of the fluorescent probe (100 μM) shown in SEQ ID NO.7;

[0073] 0.04 μL of the fluorescent probe (100 μM) shown in SEQ ID NO.8;

[0074] 0.04 μL of the fluorescent probe (100 μM) shown in SEQ ID NO.9;

[0075] 0.06 μL of the upstream primer (100 μM) shown in SEQ ID NO.10;

[0076] 0.06 μL of the downstream primer (100 μM) shown in SEQ ID NO.11;

[0077] 0.04 μL of the fluorescent probe (100 μM) shown in SEQ ID NO.12;

[0078] PCR reaction buffer 10 μL;

[0079] 4.36 μL of nuclease-free water.

[0080] Example 3

[0081] This embodiment provides a method for non-invasive detection of Helicobacter pylori virulence typing in fecal samples based on multi-gene combined detection, including the following steps:

[0082] Step 1: Collect fecal samples, dilute with preservation solution, grind, extract and purify, and prepare DNA templates. The specific steps for the dilution method are as follows: weigh 0.2g~1g of feces and add it to the preservation solution at a ratio of 1g:5mL; the specific steps for grinding are as follows: grind with a grinder / homogenizer for 20~40s or vortex mixer for 10min to ensure the sample is evenly dispersed in the preservation solution; extraction and purification can be performed using any one of the following methods: boiling method, centrifugation column method, and magnetic bead method; the preservation solution is prepared as follows: add 1.3228g of tris(hydroxymethyl)aminomethane hydrochloride, 2.5224g of EDTA-Na2, 5g of NaCl, 1.5mL of L-ween-20, 1.3mL of isopropanol, and 1.5mL of Proclin 300 to purified water, mix thoroughly, and bring the volume to 1L.

[0083] Step 2: The DNA template prepared in Step 1 was amplified by qPCR using the qPCR amplification reagent of Example 2. The qPCR amplification reaction program was as follows: 95℃, 10 min; 95℃, 15 s; 60℃, 45 s; 45 cycles. Fluorescence was collected in real time during the reaction, and the detection results were analyzed and determined.

[0084] The test results can be determined by the amplification curve:

[0085] ① If the 16S rRNA amplification curve shows a value, it indicates that the sample is infected with Helicobacter pylori; based on this, if at least one of the VacA and CagA amplification curves shows a value, it is determined to be an infection of a highly virulent Hp strain; if neither the VacA nor the CagA amplification curves show a value, it is determined to be an infection of a low-virulence Hp strain.

[0086] ②If the 16S rRNA amplification curve does not show any value, it indicates that the sample subject is not infected with Helicobacter pylori.

[0087] Experimental Example 1

[0088] This experimental example is used to verify the sensitivity of the method of the present invention.

[0089] To evaluate the sensitivity of the detection method, synthetic plasmids targeting different Helicobacter pylori targets (16S rRNA, VacA, CagA, ATCB) were serially diluted and mixed with fecal matrix from individuals confirmed to be Helicobacter pylori negative, preparing a series of sensitivity assessment samples. The final samples contained Helicobacter pylori concentrations of 1,000 cfu / mL, 100 cfu / mL, 10 cfu / mL, and 1 cfu / mL, respectively, with sterile double-distilled water used as a negative control. These samples were tested according to the method in Example 3, and the results are as follows:

[0090] Detection results of 16S rRNA, VacA, CagA, and ATCB (as shown below) Figure 1 , Figure 2 , Figure 3 , Figure 4 As shown in the figure): when the concentration of 16S rRNA / VacA / CagA / ATCB target nucleotides in the sample is as low as 1 cfu / ml, a clear detection result can still be obtained, showing extremely high detection sensitivity.

[0091] Experiment Example 2

[0092] This experimental example is used to verify the specificity of the method of the present invention.

[0093] Nucleic acids were extracted from Campylobacter jejuni, Klebsiella pneumoniae, Staphylococcus aureus, Bacillus atrophicus, Enterococcus faecalis, Streptococcus pneumoniae, Salmonella paratyphi B, Escherichia coli, Proteus, Candida albicans, Helicobacter felineus, Bifidobacterium breve, and Lactobacillus plantarum, respectively. Plasmids targeting different Helicobacter pylori targets were used as positive controls, and deionized water (ddH2O) was used as a negative control. The samples used for specificity assessment and the negative control samples were tested according to the method in Example 3.

[0094] Test results as follows Figure 5 As shown, from Figure 5 It can be observed that the Helicobacter pylori detection system used does not have cross-reaction with other common pathogens, demonstrating good specificity.

[0095] Experimental Example 3

[0096] This experimental example is used to verify the accuracy of the method of the present invention.

[0097] Nucleic acid extraction and testing were performed on 77 clinical samples according to the method in Example 3. The Jiangsu Kangwei Century Helicobacter pylori Nucleic Acid Detection Kit (PCR-fluorescent probe method), a commercially available reagent, was used as the control reagent. The detection method was strictly performed according to the kit instructions. The detection results were compared with the actual clinical sample results, and the accuracy was calculated. The detection results are shown in Table 2.

[0098] Table 2 Accuracy Verification Test Results

[0099]

[0100] As can be seen from the table, the detection results of the present invention are accurate and reliable.

[0101] Experiment Example 4

[0102] This experimental example is used to verify the ability of the primers and probes designed in this invention to distinguish between highly toxic and low-toxic strains.

[0103] This invention involves four targets of Helicobacter pylori genes. Highly virulent strains (ATCC26695, ATCC 43504) and lowly virulent strains (ATCC 51932) reported in the literature were selected as test samples. The primers and probes designed in this invention were used to verify the targets according to the method in Example 3, and first-generation sequencing was performed simultaneously.

[0104] Figure 6 This indicates that the highly virulent strains reported in the literature, when tested using the method of this invention, contain the CagA and vacA genes, consistent with the literature reports. First-generation sequencing results show that ATCC 26695 and ATCC 43504 belong to Hp type I strains. This demonstrates that the test results of this method are consistent with the content reported in the literature.

[0105] Figure 7 This indicates that the low-virulence strain reported in the literature, when tested using the method of this invention, shows that the low-virulence strain (ATCC 51932) does not contain the CagA and vacA genes, consistent with the literature reports. First-generation sequencing results show that ATCC 51932 belongs to Hp type II strains. This demonstrates that the test results of this method are consistent with the content reported in the literature.

[0106] The above description is merely a specific embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any person skilled in the art can easily conceive of various equivalent modifications or substitutions within the technical scope disclosed in the present invention, and these modifications or substitutions should all be covered within the scope of protection of the present invention. Therefore, the scope of protection of the present invention should be determined by the scope of the claims.

Claims

1. A method for non-invasive detection and virulence typing of Helicobacter pylori in fecal samples based on multi-gene combination, characterized in that, Includes the following steps: Step 1: Collect fecal samples, dilute with preservation solution, grind, extract and purify, and prepare DNA templates; Step 2: The DNA template prepared in Step 1 is amplified by qPCR using primer and probe kit reagents, and fluorescence detection is performed in real time to analyze and determine the detection results. The primer and probe kit includes upstream primers for nucleic acid sequences as shown in SEQ ID NO:1, SEQ ID NO:2 and SEQ ID NO:3, downstream primers for nucleic acid sequences as shown in SEQ ID NO:4, SEQ ID NO:5 and SEQ ID NO:6, and fluorescent probes for nucleic acid sequences as shown in SEQ ID NO:7, SEQ ID NO:8 and SEQ ID NO:

9.

2. The method for non-invasive detection and virulence typing of Helicobacter pylori in fecal samples based on multi-gene combined assay as described in claim 1, characterized in that, The preservation solution described in step one includes tris(hydroxymethyl)aminomethane hydrochloride, EDTA-Na2, NaCl, Tween-20, isopropanol, and Proclin 300.

3. The method for non-invasive detection and virulence typing of Helicobacter pylori in fecal samples based on multi-gene combined detection according to claim 1, characterized in that, The extraction and purification methods described in step one include any one of the following: boiling method, centrifugal column method, and magnetic bead method.

4. The method for non-invasive detection and virulence typing of Helicobacter pylori in fecal samples based on multi-gene combined assay according to claim 1, characterized in that, The primer and probe set described in step two also includes an upstream primer of the nucleic acid sequence shown in SEQ ID NO:10, a downstream primer of the nucleic acid sequence shown in SEQ ID NO:11, and an ATCB internal control gene probe of the nucleic acid sequence shown in SEQ ID NO:

12.

5. The method for non-invasive detection and virulence typing of Helicobacter pylori in fecal samples based on multi-gene combined detection according to claim 1, characterized in that, The genes used to detect Helicobacter pylori nucleic acid typing in fecal samples are 16S rRNA, VacA, and CagA. SEQ ID NO:1, SEQ ID NO:4, and SEQ ID NO:7 are 16S rRNA sequences, SEQ ID NO:2, SEQ ID NO:5, and SEQ ID NO:8 are VacA sequences, and SEQ ID NO:3, SEQ ID NO:6, and SEQ ID NO:9 are CagA sequences.

6. The method for non-invasive detection and virulence typing of Helicobacter pylori in fecal samples based on multi-gene combined assay as described in claim 5, characterized in that, The 16S rRNA probe 16S-P is labeled with the fluorescent group FAM, the VacA probe VacA-P is labeled with the fluorescent group VIC, the CagA probe CagA-P is labeled with the fluorescent group ROX, the ATCB probe ATCB-P is labeled with the fluorescent group Cy5, and the quenching group is any one of BHQ1, BHQ2, TAMRA, DABCYL, MGB and Eclipse.

7. The method for non-invasive detection and virulence typing of Helicobacter pylori in fecal samples based on multi-gene combined assay according to claim 1, characterized in that, The concentrations of all upstream primers and downstream primers were 0.3 μM, and the concentrations of all fluorescent probes were 0.2 μM.

8. The method for non-invasive detection and virulence typing of Helicobacter pylori in fecal samples based on multi-gene combined assay according to claim 1, characterized in that, The reaction program for qPCR amplification in step two is as follows: 95℃, 10 min; 95℃, 15 s; 60℃, 45 s; 45 cycles.

9. The method for non-invasive detection and virulence typing of Helicobacter pylori in fecal samples based on multi-gene combined assay according to claim 1, characterized in that, The detection results can be determined using amplification curves: ① If the 16S rRNA amplification curve shows a value, it indicates that the sample is infected with Helicobacter pylori; based on this, if at least one of the VacA and CagA amplification curves shows a value, it is determined to be an infection of a highly virulent Hp strain; if neither the VacA nor the CagA amplification curves show a value, it is determined to be an infection of a low-virulence Hp strain. ②If the 16S rRNA amplification curve does not show any value, it indicates that the sample subject is not infected with Helicobacter pylori.

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