Helicobacter pylori detection compositions and kits
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- GETEIN BIOTECH
- Filing Date
- 2024-12-10
- Publication Date
- 2026-06-12
AI Technical Summary
Existing Helicobacter pylori detection methods are insufficient in accuracy and speed, and cannot fully meet the needs of clinical diagnosis. In particular, invasive and non-invasive detection methods each have their own shortcomings and cannot be simultaneously effective.
Using the Helicobacter pylori-specific 16S rDNA gene and the human β-Globin gene as internal reference genes, the primer and probe set is designed with fluorescent reporter and quencher groups to achieve rapid and accurate detection through a microfluidic chip. It includes nucleic acid release agents, RPA basic reagents and activators, and provides positive and negative controls to monitor the sample processing.
It achieves highly sensitive Helicobacter pylori detection, simplifies the operation process, reduces the professional requirements, is suitable for rapid diagnosis of various sample types, has high repeatability and accuracy, and is suitable for POCT environments.
Smart Images
Figure CN122189208A_ABST
Abstract
Description
Technical Field
[0001] This invention relates to the field of medical testing technology, and in particular to a Helicobacter pylori detection composition and kit. Background Technology
[0002] Helicobacter pylori (H. pylori) is a Gram-negative, microaerobic bacterium that parasitizes the stomach and duodenum. Infection is very common, with a global infection rate exceeding 50% in the general population. Factors influencing H. pylori infection rates include economic status, living conditions, education level, occupation, and drinking habits; generally, infection rates are higher in developing countries than in developed countries. Studies have found that the detection rate of H. pylori in gastric mucosal biopsy specimens from patients with chronic gastritis can reach 80%–90%, while it is even higher in patients with peptic ulcers, reaching over 95%, or even approaching 100%. In gastric cancer, due to the metaplasia of local epithelial cells, the reported detection rate of H. pylori varies.
[0003] Diagnostic methods for Helicobacter pylori infection are divided into two main categories based on whether the sampling is invasive: invasive and non-invasive methods. The former refers to methods that rely on endoscopic sampling, including histological examination, bacterial culture, rapid urease test (RUT), and Helicobacter pylori nucleic acid detection; the latter includes serological (antibody) testing, fecal Helicobacter pylori antigen detection or gene testing, and urea breath test (UBT), etc. Different diagnostic methods have their own advantages and limitations.
[0004] For the diagnosis of current Helicobacter pylori infection, the most recommended non-invasive Helicobacter pylori testing method in clinical practice is the UBT method, which is non-invasive, rapid, and accurate. However, it also has certain limitations; it can only reflect past or present Helicobacter pylori infection status and cannot be used as an immediate assessment after treatment. Monoclonal antibody fecal antigen testing can be used as an alternative. A positive serological antibody test indicates past infection and can be considered a current infection in untreated individuals. This method has a certain lag, requiring the patient to produce antibodies before detection. For invasive testing methods, if the subject has indications for endoscopy but no contraindications for biopsy, and gastroscopy requires a biopsy, the RUT test is recommended. For gastritis biopsy specimens, a positive histological staining is sufficient to diagnose Helicobacter pylori infection; for negative specimens, immunohistochemical staining can be performed.
[0005] In summary, existing Helicobacter pylori detection methods have certain limitations and cannot fully meet the needs of clinical diagnosis. Currently, there is a need in clinical practice to find more accurate, rapid, and effective detection methods that can accommodate both invasive and non-invasive sample types to better serve clinical diagnosis and treatment. Summary of the Invention
[0006] In view of the shortcomings of the prior art, the purpose of this invention is to provide a Helicobacter pylori detection composition and kit to solve the problem of low detection efficiency in the prior art.
[0007] To achieve the above objectives, the present invention provides the following technical solution:
[0008] A Helicobacter pylori detection composition comprising:
[0009] Helicobacter pylori primer and probe set and internal reference gene β-Globin primer and probe set;
[0010] The Helicobacter pylori primer and probe set includes a 16S rDNA forward primer, a 16S rDNA reverse primer, and a 16S rDNA probe; the sequence of the 16S rDNA forward primer is shown in SEQ ID NO.1, the sequence of the 16S rDNA reverse primer is shown in SEQ ID NO.6, and the sequence of the 16S rDNA probe is shown in SEQ ID NO.7.
[0011] The internal reference gene β-Globin primer and probe set includes a β-Globin forward primer, a β-Globin reverse primer, and a β-Globin probe; the sequence of the β-Globin forward primer is shown in SEQ ID NO.9, the sequence of the β-Globin reverse primer is shown in SEQ ID NO.13, and the sequence of the β-Globin probe is shown in SEQ ID NO.14.
[0012] Furthermore, the 16S rDNA probe is modified with a fluorescent reporter group at a distance of 32 nt from the 5' end and a fluorescent quencher group at a distance of 14 nt from the 3' end; the bases between the fluorescent reporter group and the fluorescent quencher group are replaced with THF as exonuclease cleavage sites; and the 3' end of the probe is labeled with the modification group.
[0013] Furthermore, the fluorescent reporter group is selected from ALEX-350, FAM, VIC, TET, CAL Fluor Gold540, JOE, HEX, CAL Flour Orange 560, TAMRA, Cal Fluor Red 590, ROX, CAL Fluor 20Red 610, TEXASRED, CAL Flour Red 635, Quasar 670, Cy3, Cy5, Cy5.5, or Quasar 705; the fluorescent quencher group is selected from TAMRA, BHQ1, BHQ2, or MGB; and the modifying group is selected from amino groups, phosphate groups, biotin, biotin-TEG, or C3-spacer.
[0014] Furthermore, it also includes positive and negative control groups; the positive and negative control groups include a positive control and a negative control; the positive control includes the pUC57 vector plasmid of the 16S rDNA gene and the pUC57 vector plasmid of the human β-Globin internal reference gene; the negative control includes the pUC57 empty vector plasmid of any gene.
[0015] A Helicobacter pylori detection kit includes:
[0016] The detection composition described above;
[0017] In addition, nucleic acid release agents, RPA basic reagents and activators.
[0018] Furthermore, the nucleic acid releasing agent comprises: guanidine salt 0.05-2M; Tris 25-75mM; DMSO 1-2wt%; polyethylene glycol 0.5-1wt%; trehalose 0.1-0.5wt%; Tween 0.1-0.5wt%; and proteinase K 0.06-0.12mg / mL.
[0019] Furthermore, the RPA base reagent contains an enzyme mixture; the enzyme mixture includes exonuclease III, DNA polymerase, single-stranded DNA binding protein, recombinase, and phosphokinase.
[0020] Furthermore, the activator comprises: 10-20 mM magnesium acetate, 0.5-1.5 wt% trehalose, 2-4 wt% sucrose, 0.5-1.5 wt% glycine, 0.05-1 wt% sorbitol, and 1-3 wt% polyethylene glycol.
[0021] Furthermore, the final concentrations of the forward primer F and the reverse primer R in the detection composition are both 400 nM, and the final concentration of the probe P is 120 nM.
[0022] Compared with the prior art, the beneficial effects of the present invention are as follows:
[0023] This invention utilizes the highly conserved 16S rDNA gene from Helicobacter pylori, which can detect Helicobacter pylori with high specificity; the selected gene has a high expression level, giving the product high sensitivity; at the same time, the human β-Globin gene is also selected as an internal reference gene, combined with positive and negative controls, which can effectively monitor the sample nucleic acid pretreatment process and the performance of the detection reagent. Attached Figure Description
[0024] Figure 1 This is a screening and amplification effect diagram of each primer and probe set for Helicobacter pylori 16S rDNA in this invention;
[0025] Figure 2This is a diagram showing the screening and amplification results of various primer and probe sets for the human β-Globin gene in this invention. Detailed Implementation
[0026] The preferred embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only a part of the embodiments of the present invention, and not all of them. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are within the scope of protection of the present invention. Experimental methods not specified with specific conditions in the embodiments are generally performed under conventional conditions or as recommended by the manufacturer.
[0027] A Helicobacter pylori detection composition comprising:
[0028] Helicobacter pylori primer and probe set and internal reference gene β-Globin primer and probe set;
[0029] The Helicobacter pylori primer and probe set includes a 16S rDNA forward primer, a 16S rDNA reverse primer, and a 16S rDNA probe; the sequence of the 16S rDNA forward primer is shown in SEQ ID NO.1, the sequence of the 16S rDNA reverse primer is shown in SEQ ID NO.6, and the sequence of the 16S rDNA probe is shown in SEQ ID NO.7.
[0030] The internal reference gene β-Globin primer and probe set includes a β-Globin forward primer, a β-Globin reverse primer, and a β-Globin probe; the sequence of the β-Globin forward primer is shown in SEQ ID NO.9, the sequence of the β-Globin reverse primer is shown in SEQ ID NO.13, and the sequence of the β-Globin probe is shown in SEQ ID NO.14.
[0031] The specific sequence is as follows:
[0032] SEQ ID No.1: 5'-CCGTGCCAGCAGCCGCGGTAATACGGAGGGTG-3'
[0033] SEQ ID No.6: 5'-CAAATGCAGTTCTATGGTTAAGCCATAGGATT-3'
[0034] SEQ ID No.7: 5'-AATCACTGGGCGTAAAGAGCGCGTAGGCGGGATAG TCAGTCAGGTGTGAA-3'
[0035] SEQ ID No.9: 5'-CATCTATTGCTTACATTTGCTTCTGACACAA-3'
[0036] SEQ ID No.13: 5'-CCAATAGGCAGAGAGAGTCAGTGCCTATCAGAAA-3'
[0037] SEQ ID No.14: 5'-TCTGCCGTTACTGCCCTGTGGGGCAAGGTGAACGTGGATGAAGTTGGTGGTG-3'
[0038] The detection composition of the present invention also includes positive and negative control groups; the positive and negative control groups include a positive control and a negative control; the positive control includes the pUC57 vector plasmid of the 16S rDNA gene and the pUC57 vector plasmid of the human β-Globin internal reference gene; the negative control includes the pUC57 empty vector plasmid of any gene.
[0039] This invention utilizes the highly conserved 16S rDNA gene from Helicobacter pylori, enabling highly specific detection of the bacterium. The selected gene exhibits high expression levels, resulting in high product sensitivity. Furthermore, the human β-Globin gene is used as an internal reference gene. Combined with positive and negative controls, this effectively monitors the sample nucleic acid pretreatment process and the performance of the detection reagents. The primer set provided by this invention exhibits no nonspecific amplification, no diffuse bands, and good repeatability stability. Its amplification product length is 127 bp, facilitating detection.
[0040] The 16S rDNA probe of this invention has a fluorescent reporter group modified with thymine at a distance of 32 nt from the 5' end and a fluorescent quencher group modified with thymine at a distance of 14 nt from the 3' end; the bases between the fluorescent reporter group and the fluorescent quencher group are replaced with THF as the exonuclease cleavage site; at the same time, the 3' end is labeled with the modification group.
[0041] In the β-Globin probe sequence of the internal reference gene of this invention, a fluorescent reporter group is modified with thymine at a distance of 34 nt from the 5' end, and a fluorescent quencher group is modified with thymine at a distance of 13 nt from the 3' end; any base between the fluorescent reporter group and the fluorescent quencher group is replaced with THF as the exonuclease cleavage site; the 3' end of the probe is labeled with the modification group.
[0042] The fluorescent reporter group of this invention is selected from ALEX-350, FAM, VIC, TET, CAL Fluor Gold540, JOE, HEX, CAL Flour Orange 560, TAMRA, Cal Fluor Red 590, ROX, CAL Fluor 20Red 610, TEXASRED, CAL Flour Red 635, Quasar 670, Cy3, Cy5, Cy5.5, or Quasar 705; the fluorescent quencher group is selected from TAMRA, BHQ1, BHQ2, or MGB; and the modifying group is selected from amino groups, phosphate groups, biotin, biotin-TEG, or C3-spacer.
[0043] This invention also discloses a Helicobacter pylori detection kit, comprising:
[0044] The Helicobacter pylori detection composition described above;
[0045] In addition, nucleic acid release agents, positive and negative control groups, RPA basic reagents and activators.
[0046] The nucleic acid releasing agents include: guanidine salt 0.05-2M; Tris 25-75mM; DMSO 1-2wt%; polyethylene glycol 0.5-1wt%; trehalose 0.1-0.5wt%; Tween 0.1-0.5wt%; and proteinase K 0.06-0.12mg / mL.
[0047] The positive and negative control groups include positive and negative controls; the positive control includes the pUC57 vector plasmid of the 16S rDNA gene and the pUC57 vector plasmid of the human β-Globin internal reference gene; the negative control includes the pUC57 empty vector plasmid of any gene.
[0048] The RPA basic reagent contains an enzyme mixture; the enzyme mixture includes exonuclease III, DNA polymerase, single-stranded DNA binding protein, recombinase, and phosphokinase.
[0049] The activators include: magnesium acetate 10-20 mM, trehalose 0.5-1.5 wt%, sucrose 2-4 wt%, glycine 0.5-1.5 wt%, sorbitol 0.05-1 wt%, and polyethylene glycol 1-3 wt%.
[0050] In the above Helicobacter pylori detection composition, the final concentrations of the forward primer F and the reverse primer R are both 400 nM, and the final concentration of the probe P is 120 nM.
[0051] The Helicobacter pylori detection composition, nucleic acid release agent, positive and negative control groups, and RPA basic reagents of this invention are all lyophilized reagents. The microfluidic chip of this invention does not require the extraction of genomic DNA from the sample. The collected sample can be directly added to the sample injection well of the microfluidic chip for instrument detection to obtain amplification products. The presence of Helicobacter pylori in the sample can be determined based on the detection results.
[0052] Example 1
[0053] Based on the published Hp16S rDNA gene sequence in GenBank, and by reviewing literature and analyzing pathogen genome sequences, conserved sequences of specific target genes of each pathogen were determined. Then, using Primer Premier 5, DNAMAN software, and Primer-BLAST (NCBI), different primers were designed for Hp 16S rDNA gene and human β-Globin gene, respectively. There are a large number of primers, and only some of the relevant primers are shown in Table 1 below.
[0054] Table 1
[0055]
[0056] The specific primers designed in this invention are single-stranded DNA sequences targeting the Helicobacter pylori 16S rDNA gene. Each primer pair consists of two single-stranded DNA sequences that specifically recognize the upstream and downstream nucleotide sequences of a nucleic acid target. The primers are 30-35 nucleotides (nt) in length and contain no palindromic structures, continuous repeats, or internal secondary structure regions. The primer pairs provided by this invention were optimized and screened through extensive experiments, and their amplification products are single bands, without non-specific amplification or obvious primer dimers. They can accurately amplify the Helicobacter pylori 16S rDNA gene under micro-scale conditions, thereby detecting whether a sample is infected with Helicobacter pylori.
[0057] In the probe sequence of this invention, THF serves as the recognition site for exonuclease. Upstream of the THF site, a fluorescent reporter group is modified with thymine at a distance of 32 nt from the 5' end of the sequence; a fluorescent quencher group is modified with thymine at a distance of 14 nt from the 3' end, with a spacing of 1-4 nt between the two groups; a modifying group, such as an amino group, a phosphate group, biotin, biotin-TEG, or C3-spacer, is labeled at the 3' end to inhibit DNA polymerase extension from the 3' end; the fluorescent reporter group includes, but is not limited to, ALEX-350, FAM, VIC, TET, CAL Fluor Gold540, JOE, HEX, CAL Flour Orange 560, TAMRA, Cal Fluor Red 590, ROX, CAL Fluor 20Red 610, TEXASRED, CAL Flour Red635, Quasar 670, Cy3, Cy5, Cy5.5, or Quasar. For 705, the quenching groups can be TAMRA, BHQ1, BHQ2, or MGB.
[0058] Preparation of primer-probe mixture
[0059] The synthesized primers and probes were dissolved in DEPC water to prepare a 100 μmol / L stock solution, which was then prepared according to the proportions shown in Table 2.
[0060] Table 2 Primer and probe assembly preparation system
[0061]
[0062] Nine combinations of the designed Helicobacter pylori 16S rDNA primers and human internal reference gene β-Globin primers were paired. The corresponding primers were added to the system in the prescribed amounts to prepare the reaction solution and establish the reaction system. The reaction tubes were placed in a real-time fluorescence quantitative PCR instrument and incubated at 40°C for 20 min.
[0063] like Figure 1 As shown in the results of Helicobacter pylori primer screening, the fluorescence signal of the 16S rDNA-P-F1R3 primer probe set increased the fastest, indicating that its amplification efficiency was better. Figure 2 As shown in the results of primer screening for the human internal reference gene β-Globin, the primer probe group β-Globin-F2R3 has the highest peak CT value and the fastest fluorescence signal growth rate.
[0064] In summary, after multiple verifications, the results of combining 16SrDNA-P-F1R3 and β-Globin-F2R3 remained consistent. No obvious competition or interference was observed between the two primer-probe pairs, and the amplification results showed good repeatability and stability.
[0065] Example 2
[0066] This invention also discloses a Helicobacter pylori detection kit, comprising a microfluidic chip. The microfluidic chip consists of an inlet port, a mixing chamber, multiple amplification chambers, and a control valve. The different chambers are connected by microfluidic paths and driven by gas pressure. The chip substrate is sealed with a polypropylene film using thermo-pressing technology to form a closed chamber. The mixing chambers are pre-embedded with RPA basic reagents containing an enzyme mixture, and the amplification chambers are pre-embedded with lyophilized primer and probe sets capable of amplifying specific target gene sequences and magnesium acetate as an activator.
[0067] After the nucleic acid release agent containing the sample is added to the sample chamber, it flows into the mixing chamber under air pressure for sample pretreatment. After pretreatment, the control valve opens, and the pretreated sample flows into different amplification chambers to dissolve the primer-probe set and lyophilized activator, which are then mixed by aspiration. After mixing, the reagents begin an isothermal amplification reaction in the amplification chamber. After the reaction, the software automatically interprets the positive and negative results.
[0068] The entire reaction process after sample addition in this invention is a fully automated, closed-loop reaction, which is simple to operate and greatly reduces the professional requirements for laboratory personnel. At the same time, this application offers rapid diagnostic speed, is portable, and can quickly diagnose pathogens in the field or clinical setting, which is crucial for real-time health management and emergency response.
[0069] The primer and probe kit of this invention includes primers and probes for detecting the 16S rDNA gene of Helicobacter pylori, and primers and probes for the human β-Globin gene, requiring only a single amplification chamber to meet detection needs. The remaining amplification chambers can support the combined detection of more pathogens in the future. The kit also includes positive and negative controls. The positive control mainly consists of a pUC57 vector plasmid containing the 16S rDNA gene and a pUC57 vector plasmid containing the human β-Globin internal reference gene. The negative control mainly consists of an empty pUC57 vector plasmid containing no genes.
[0070] Example 3
[0071] The method of using the Helicobacter pylori detection kit of the present invention is as follows:
[0072] (1) Taking fecal samples as an example: Take 200 μL of freshly collected fecal samples, add 3 mL of one-step nucleic acid release agent provided by the kit, mix well and use it as a sample to be tested.
[0073] (2) Taking gastric mucosa sample as an example: Take an appropriate amount of gastric mucosa tissue sample, add 3 mL of the one-step nucleic acid release agent provided by the kit, mix well and use it as a sample to be tested.
[0074] Remove the microfluidic chip from the kit, open the well cap, and pipette 65 μL of the sample to be tested (or positive / negative control) into the well and close the cap. Place the microfluidic chip into the accompanying nucleic acid amplification analyzer for amplification; after amplification, the data is processed and analyzed by the analyzer's built-in software. The amplification program for the isothermal reaction is: 40℃ for 20 min.
[0075] This invention is applicable to various sample types, including gastric mucosal biopsy tissue samples and feces. After sampling, the samples are placed in the nucleic acid release agent provided with the kit and can be directly added to a microfluidic chip for one-step detection. This eliminates the need for laborious and time-consuming manual operations such as nucleic acid extraction. The entire process is a closed reaction, reducing the possibility of contamination.
[0076] Example 4
[0077] Clinical samples were tested using the Helicobacter pylori detection kit of this invention, and the results of the kit were compared with those of the fluorescent PCR nucleic acid detection kit. The reference kits were selected from Jiangsu Kangwei Century Biotechnology Co., Ltd.'s Helicobacter pylori nucleic acid detection kit (PCR fluorescent probe method, used as a reference kit for fecal samples) and Helicobacter pylori nucleic acid detection kit (fluorescent PCR method, used as a reference kit for gastric mucosal samples).
[0078] This invention selected 200 fecal samples and 200 gastric mucosa samples for testing, with a positive rate of at least 60%. All case diagnoses were determined based on a comprehensive consideration of the patient's clinical data and the test results of the reference kit. 200 fecal samples and 200 gastric mucosa samples were collected and tested for Helicobacter pylori using the kit of this invention, as described in Example 3. A reference nucleic acid detection kit was also used for testing according to the instructions. The results of both tests were compared.
[0079] The test results for fecal samples showed 131 positive cases of Helicobacter pylori and 69 negative cases, with a positive concordance rate of 97.76%. The test results for gastric mucosa samples showed 133 positive cases of Helicobacter pylori and 67 negative cases, with a positive concordance rate of 99.25%. The results were largely consistent with those of PCR nucleic acid detection, indicating that the test results of this kit have high reliability.
[0080] Forty samples (including 3 differentially expressed samples) were tested using this product and compared with Sanger sequencing results. Except for the 3 differentially expressed samples, the concordance rate with Sanger sequencing results was 100%. The nucleic acid detection CT values of the 3 differentially expressed samples were all above 30 CT. After nucleic acid extraction from these differentially expressed samples, they were retested using the kit of this invention, and all 3 samples were detectable. This indicates that although the inhibitors in the samples slightly reduced the sensitivity of the kit of this invention, it can still be well applied to the clinical detection and auxiliary diagnosis of Helicobacter pylori. Correspondingly, the advantages of this invention in point-of-care testing (POCT) environments—extraction-free and ultra-fast detection—can be used for the rapid diagnosis of one or even multiple diseases, which is of great significance for public health and individual health management.
[0081] Finally, it should be noted that the above preferred embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit it. Although the present invention has been described in detail through the above preferred embodiments, those skilled in the art should understand that various changes can be made to it in form and detail without departing from the scope defined by the claims of the present invention.
Claims
1. A Helicobacter pylori detection composition, characterized in that, include: Helicobacter pylori primer and probe set and internal reference gene β-Globin primer and probe set; The Helicobacter pylori primer and probe set includes a 16S rDNA forward primer, a 16S rDNA reverse primer, and a 16S rDNA probe; the sequence of the 16S rDNA forward primer is shown in SEQ ID NO.1, the sequence of the 16S rDNA reverse primer is shown in SEQ ID NO.6, and the sequence of the 16S rDNA probe is shown in SEQ ID NO.
7. The internal reference gene β-Globin primer and probe set includes a β-Globin forward primer, a β-Globin reverse primer, and a β-Globin probe; the sequence of the β-Globin forward primer is shown in SEQ ID NO.9, the sequence of the β-Globin reverse primer is shown in SEQ ID NO.13, and the sequence of the β-Globin probe is shown in SEQ ID NO.
14.
2. The Helicobacter pylori detection composition according to claim 1, characterized in that, The 16S rDNA probe has a thymine-modified fluorescent reporter group at 32 nt from the 5' end and a thymine-modified fluorescent quencher group at 14 nt from the 3' end; the bases between the fluorescent reporter group and the fluorescent quencher group are replaced with THF as exonuclease cleavage sites; the 3' end of the probe is labeled with the modification group.
3. The Helicobacter pylori detection composition according to claim 2, characterized in that, The fluorescent reporter group is selected from ALEX-350, FAM, VIC, TET, CAL Fluor Gold540, JOE, HEX, CAL Flour Orange 560, TAMRA, CalFluor Red 590, ROX, CAL Fluor 20Red 610, TEXASRED, CAL Flour Red 635, Quasar 670, Cy3, Cy5, Cy5.5, or Quasar 705; the fluorescent quencher group is selected from TAMRA, BHQ1, BHQ2, or MGB; the modifying group is selected from amino groups, phosphate groups, biotin, biotin-TEG, or C3-spacer.
4. The Helicobacter pylori detection composition according to claim 1, characterized in that, It also includes positive and negative control groups; the positive and negative control groups include a positive control and a negative control; the positive control includes the pUC57 vector plasmid of the 16S rDNA gene and the pUC57 vector plasmid of the human β-Globin internal reference gene; the negative control includes the pUC57 empty vector plasmid of any gene.
5. A Helicobacter pylori detection kit, characterized in that, include: The detection composition according to any one of claims 1-4; In addition, nucleic acid release agents, RPA basic reagents and activators.
6. The Helicobacter pylori detection kit according to claim 5, characterized in that, The nucleic acid releasing agent comprises: guanidine salt 0.05-2M; Tris 25-75mM; DMSO 1-2wt%; polyethylene glycol 0.5-1wt%; trehalose 0.1-0.5wt%; Tween 0.1-0.5wt%; and proteinase K 0.06-0.12mg / mL.
7. The Helicobacter pylori detection kit according to claim 5, characterized in that, The RPA basic reagent contains an enzyme mixture; the enzyme mixture includes exonuclease III, DNA polymerase, single-stranded DNA binding protein, recombinase and phosphokinase.
8. The Helicobacter pylori detection kit according to claim 5, characterized in that, The activator comprises: 10-20 mM magnesium acetate, 0.5-1.5 wt% trehalose, 2-4 wt% sucrose, 0.5-1.5 wt% glycine, 0.05-1 wt% sorbitol, and 1-3 wt% polyethylene glycol.
9. The Helicobacter pylori detection kit according to claim 5, characterized in that, The final concentrations of both the forward and reverse primers in the detection composition are 400 nM, and the final concentration of the probe is 120 nM.