LF-era detection primers and probe and detection kit for helicobacter pylori

Abstract

The present invention relates to the technical fields of medicine and biology, in particular to IPC G01N33, and more particularly to LF-ERA detection primers and a probe, and a detection kit for Helicobacter pylori. Provided are Hp-specific primers and a probe, the Hp-specific primers comprising one or more of forward primers 1F, 2F and 3F, and one or more of reverse primers 1R, 2R and 3R. Also provided is an LF-ERA kit for Helicobacter pylori, the kit comprising the Hp-specific primers and the probe, an ERA basic amplification system, a sample diluent and a detection reagent card. Further provided is a corresponding detection method. The kit uses ERA isothermal amplification in combination with nano-carbon immunochromatography technology to achieve rapid visualization of a nucleic acid amplification result, thereby greatly shortening the detection time and also avoiding using professional complex experimental operations and expensive gel imaging instruments. Therefore, the present invention solves the problem of conventional nucleic acid detection and analysis being not suitable for popularization and application in common laboratories and at-home testing.

Description

Helicobacter pylori LF-ERA detection primer, probe and detection kit Technical Field

[0001] The present invention relates to the fields of medicine and biotechnology, in particular to IPC G01N33, and more specifically to a Helicobacter pylori LF-ERA detection primer and probe and a detection kit. Background Art

[0002] Helicobacter pylori (H. pylori) is a Gram-negative, microaerophilic bacterium that parasitizes the stomach and duodenum and is one of the most common pathogens that endanger human health. In 1994, the World Health Organization defined Hp as a Class I carcinogen. In 2021, the U.S. Public Health Agency listed Hp as a newly added carcinogen. In addition, Hp infection is closely related to the occurrence of diseases such as chronic gastritis, peptic ulcer, and mucosa-associated lymphoid tissue lymphoma. Currently, Hp infection remains a major public health problem worldwide, with an infection rate of over 50% in the global natural population and an Hp infection rate of approximately 35% to 55% in the Chinese population.

[0003] Diagnostic methods for Helicobacter pylori infection are categorized into two main types based on whether the sample is invasive or not: invasive and non-invasive. Invasive methods rely on endoscopic sampling and include histological testing, bacterial culture, rapid urease test (RUT), and H. pylori nucleic acid testing. Non-invasive methods include serological (antibody) testing, stool H. pylori antigen testing or genetic testing, and urea breath testing (UBT). Each diagnostic method has its own advantages and limitations. Serological (antibody) testing and genetic testing are the most commonly used typing methods. However, conventional H. pylori genetic testing requires expensive, sophisticated equipment and specialized personnel, making it unsuitable for widespread use in remote areas or for home self-testing.

[0004] In the natural environment, humans are presumed to be infected with Helicobacter pylori through oral infection, which then colonizes the stomach and induces a series of digestive tract diseases. Oral testing can reveal bacterial infections that precede stomach infections and reveal the reasons why stomach infections are difficult to cure. Because Helicobacter pylori is in an inactive metabolic state in the mouth and the relative abundance of colonies is generally low, antigen detection is difficult. The principle of urease dry chemical detection is that urease decomposes urea to produce CO2, causing a change in pH and showing a color change through a pH indicator. This method is easily affected by the oral environment and a large number of bacteria in the mouth can produce urease, resulting in low specificity.

[0005] Existing patent CN201510748326.7 discloses a multiplex PCR detection method for oral pathogens based on melting curve analysis. This patented kit uses multiplex PCR to simultaneously amplify specific target sequences for Porphyromonas gingivalis, Helicobacter pylori, Fusobacterium nucleatum, Streptococcus mutans, and Staphylococcus aureus, followed by identification using modified Taqman probe melting curve analysis. This enables multiplex detection of five common oral pathogens. However, this kit requires a specific PCR instrument and corresponding equipment, resulting in complex identification methods, lengthy detection times, and high testing costs. Summary of the Invention

[0006] In order to solve the problems in the prior art, the first aspect of the present invention provides a Helicobacter pylori LF-ERA detection primer and probe, which are Hp-specific primers and probes. The Hp-specific primers include one or more upstream primers 1F, 2F, 3F, and one or more downstream primers 1R, 2R, 3R.

[0007] Preferably, the base sequence of the upstream primer 1F is:

[0008] SEQ ID NO: 1: 5'-ACTAATTTGAATAATCTCGGCTATCACTAGTCT-3',

[0009] The base sequence of upstream primer 2F is:

[0010] SEQ ID NO: 2: 5'-GTCTTATAAGGCGGGATTTAGAAGATAAACTA-3',

[0011] The base sequence of upstream primer 3F is:

[0012] SEQ ID NO: 3: 5'-GATTTAGAAGATAAACTAGCCACTAAAGGATT-3',

[0013] The base sequence of downstream primer 1R is:

[0014] SEQ ID NO: 4'-TTTCCTTAGGGATTTTTCAAGATCTTTCTGAG-3',

[0015] The base sequence of the downstream primer 2R is:

[0016] SEQ ID NO: 5'-TAGGGATTTTTCAAGATCTTTCTGAGCTTTTT-3';

[0017] The base sequence of the downstream primer 3R is:

[0018] SEQ ID NO: 6: 5'-CGTTTCCTTAGGGATTTTTCAAGATCTTTCT-3',

[0019] The base sequence of the probe is:

[0020] SEQ ID NO: 7: 5'-GATTTAGAAGATAAACTAGCCACTAAAGGATTTCCCTACAAGAAGC-3'.

[0021] Preferably, the 5' ends of the base sequences of the downstream primers 1R, 2R and 3R are connected to biotin.

[0022] Preferably, the 5' end of the base sequence of the probe is connected to Dig (digoxigenin), the 3' end is connected to C3 Spacer, and the 32nd base is modified with tetrahydrofuran abasic site (THF);

[0023] Preferably, the concentration of the upstream primer, downstream primer and probe is 0.01 μM-10 μM; more preferably, it is 0.1 μM-10 μM; further preferably, it is 5 μM.

[0024] Preferably, the upstream primer, downstream primer and probe are synthesized by Boshang Biotechnology (Shanghai) Co., Ltd.

[0025] In the present invention, the specificity and accuracy of the kit are improved by designing specific upstream primers, downstream primers, and probes. The inventors creatively designed and optimized three upstream primers, three downstream primers, and one probe based on the conserved sequence region of the Helicobacter pylori cytotoxin-associated gene A (CagA) to ensure the specificity of the isothermal amplification reaction. Further screening yielded a primer and probe combination that specifically amplifies H. pylori DNA and H. pylori bacteria, while not amplifying oral bacteria such as Streptococcus mutans, Fusobacterium nucleatum subsp. Synechocystis, Actinomyces naeslundii, Lactobacillus acidophilus, Prevotella intermedius, Porphyromonas gingivalis, and Streptococcus salivarius, thereby improving the specificity and accuracy of the kit.

[0026] In the present invention, specific primer concentrations are used to improve the specificity and accuracy of the kit while reducing the occurrence of false positives. The inventors discovered that the concentrations of both upstream and downstream primers affect the accuracy and specificity of the LF-ERA detection method. When the concentration is below 0.1 μM, no detectable positive product is obtained, while when the concentration is too high, false positives are likely to occur. To achieve high amplification efficiency and minimize false positives, specific primer concentrations were selected.

[0027] The second aspect of the present invention provides an LF-ERA kit for Helicobacter pylori. The kit is based on ERA isothermal amplification and combined with immunochromatographic technology, and uses nanocarbon labeling sandwich method for detection. The product is easy to operate and the results can be seen in 30 minutes. It is suitable for the detection of oral samples from the general population and patients who have recovered from gastric Helicobacter pylori infection.

[0028] Preferably, the LF-ERA kit for Helicobacter pylori comprises Hp-specific primers and probes, an ERA basic amplification system, a sample diluent and a detection reagent card;

[0029] Preferably, the detection reagent card includes a sample pad, an NC membrane, a carbon pad, absorbent paper and a PVC base plate.

[0030] Preferably, the ERA basic amplification system comprises a dissolving agent, a basic amplifying agent, dd H2O and an activator.

[0031] Preferably, the ERA basic amplification system is purchased from Suzhou Xianda Gene Technology Co., Ltd.

[0032] Preferably, the sample diluent is 8.1 mM Na2HPO4, 1.9 mM NaH2PO4 and 0.85% (W / V) NaCl, pH 7.4.

[0033] Preferably, the sample pad needs to be treated with a sample pad treatment solution and dried overnight.

[0034] Preferably, the sample pad treatment solution includes NaH2PO4, Na2HPO4, NaCl, protein stabilizer 2#, Tween-20 and ProClin300;

[0035] Preferably, the sample pad treatment solution is prepared by weighing NaH2PO4, Na2HPO4, NaCl, protein stabilizer 2#, Tween-20, and ProClin300, adding pure water to prepare a solution of 0.15% (W / V) Na2HPO4, 1.45% (W / V) NaH2PO4, 0.9% (W / V) NaCl, 0.5% (W / V) protein stabilizer 2#, 1% (V / V) Tween20, and 0.1% (V / V) ProClin300, and adjusting the pH to 7.4±0.1.

[0036] Preferably, the carbon pad includes a carbon pad with nanocarbon labeled Dig antibody and a carbon pad with nanocarbon labeled mouse IgG.

[0037] Preferably, the nanocarbon-labeled Dig antibody is prepared by sequentially adding EDC and NHS coupling solution to the dispersed nanocarbon, mixing evenly, and activating at 37°C for 30 minutes; then adding digoxigenin antibody, mixing evenly, and coupling at 37°C for 45 minutes. After coupling, nanocarbon blocking solution (50mM Tris + 2% (w / v) BSA) and 10% (v / v) Tween are added, mixing evenly, and blocking at 37°C for 30 minutes. Finally, the mixture is centrifuged at 13,000 rpm for 15 minutes, washed once with nanocarbon preservation solution (50mM Tris + 0.5% BSA), and stored in 200μL of nanocarbon preservation solution for later use.

[0038] Preferably, the nanocarbon blocking solution comprises 40-60 mM Tris and 1-3% (W / V) BSA; further preferably, it is 50 mM Tris and 2% (W / V) BSA.

[0039] Preferably, the preparation method of the carbon pad of the nanocarbon-labeled Dig antibody is as follows: Dig antibody labeled with nanocarbon is diluted 10 to 15 times with 40 to 50% nanocarbon labeled diluent, and then the pad is laid and dried overnight.

[0040] Preferably, the preparation method of the nanocarbon-labeled mouse IgG carbon pad is as follows: nanocarbon-labeled mouse IgG is diluted 10 to 15 times with 40 to 50% nanocarbon labeling diluent, and then used as a pad, and then dried overnight.

[0041] Preferably, the nanocarbon label diluent is 70mM Tris, 1% (W / V) BSA, 10% (W / V) sucrose, 5% (W / V) trehalose and 0.4% (V / V) TritonX-100, pH=8.2.

[0042] Preferably, the NC membrane is coated with parallel detection lines T and quality control lines C, with a spacing of 5 to 10 mm, and is dried overnight after coating.

[0043] The assembly process of the detection reagent card is as follows: the sample pad and the carbon pad are sequentially pasted on the coated NC film to form a large plate, which is then cut into 3-4mm wide test strips, mounted on a plastic part, and sealed and stored at room temperature with a desiccant.

[0044] It should be noted that the percentage content (% W / V) of each substance in the present invention is the ratio of mass to volume, specifically 1 g / 100 mL. The percentage content (% V / V) is the ratio of volume to volume, specifically 1 mL / 100 mL.

[0045] The inventors discovered that by using specific primers and probes labeled with biotin and digoxigenin, and by labeling the downstream primer chain and one end of the probe with biotin and digoxigenin, respectively, the target amplified product of Helicobacter pylori can bind to the corresponding nanocarbon-labeled digoxigenin antibody (DIG antibody) particles to form a complex, which can then be bound by the capture antibody streptavidin (SA) conjugate on the NC membrane to form visible lines, achieving visualization. Furthermore, the present invention utilizes a nanocarbon labeling method, which offers significant advantages over colloidal gold. Nanocarbon has higher detection sensitivity and better stability, and the carbon material is environmentally friendly. The cost of nanocarbon raw materials is lower than that of colloidal gold, and the black and white color contrast between nanocarbon and NC membrane is easier to distinguish. This solves the problems of instability and low sensitivity of colloidal gold detection products currently on the market, while also improving the clarity of visual resolution.

[0046] The third aspect of the present invention provides a method for LF-ERA of Helicobacter pylori, comprising the following steps:

[0047] S1: Mix the lytic agent, upstream primer, downstream primer, probe, sample, and dd H2O to prepare 48 μL of premix, and centrifuge by vortexing.

[0048] S2: Transfer 48 μL of the premix to the amplification reagent tube, resuspend and mix thoroughly, and centrifuge briefly.

[0049] S3: Add activator to the amplification reagent tube to a total volume of 50 μL; shake to mix, and incubate at 37°C for 15-25 minutes;

[0050] S4: Take 100 μL of the diluted amplification product and add it dropwise to the sample well of the detection reagent card. Observe the results after 10 to 15 minutes.

[0051] Preferably, the reaction is carried out specifically according to the system and operation process in Table 1.

[0052] Table 1

[0053]

[0054] The kit of the present invention utilizes ERA isothermal amplification (ERA) combined with nanocarbon immunochromatography and a double-antibody sandwich method to detect Helicobacter pylori DNA in oral specimens. The inventors discovered that the principle of ERA isothermal amplification is that a recombinase binds to a primer and probe to form a complex, which searches for sequences complementary to the primer and probe within double-stranded DNA. The strand displacement activity of the recombinase then inserts the primer and probe into homologous sites, and a single-stranded binding protein stabilizes the displaced DNA strand. This process replaces the DNA melting and annealing process. In this double-stranded environment, the THF site on the probe is cleaved by endonuclease IV to create a new 3'-hydroxyl group (effectively removing the C3 spacer blocking the probe), which serves as a primer site for polymerase extension, thereby converting the probe into a primer. DNA polymerase then replicates and extends the target DNA fragment on the template, and this process is repeated cyclically to exponentially amplify the target DNA fragment. The resulting amplification product, labeled with digoxigenin and biotin, can be used in downstream test strips. In this system, a pair of primers and a probe jointly initiate a synthesis process. The entire amplification reaction proceeds very quickly, and detectable levels of amplified DNA products can generally be obtained within 20 minutes.

[0055] LF-ERA detection technology combines ERA technology with lateral flow immunoassays, enabling rapid visualization of nucleic acid detection. Key features include biotin-labeled primers and digoxigenin-labeled probes. The lateral flow immunoassay strip utilizes a nitrocellulose (NC) membrane matrix, immobilized with a streptavidin (SA) conjugate (test line T) and goat anti-mouse IgG antibody (control line C). The conjugate pad is labeled with digoxigenin antibody (DIG antibody) and mouse IgG nanocarbon. During testing, the nucleic acid product from an oral sample, after isothermal ERA amplification, is dripped onto the sample loading area of ​​the strip. The sample flows forward due to capillary action, dissolving the colloidal carbon on the carbon pad and migrating with the sample. Because the isothermally amplified H. pylori DNA is labeled with digoxigenin and biotin, upon reaching the test zone, the colloidal carbon-labeled digoxigenin antibody, the H. pylori DNA in the sample amplification product, and the streptavidin conjugate coated on the test line form a complex, resulting in the test line appearing gray or black. The H. pylori DNA content in the sample is positively correlated with the intensity of the color developed on the test line. After a 10-15 minute reaction at room temperature, the color development is visually observed. If both the control line and the test line appear on the test strip, the sample is positive for H. pylori infection. The intensity of the colloidal carbon color development on the test line can be used to determine the H. pylori DNA content. The control line should appear black regardless of the test line's color intensity. The absence of color on the control line invalidates the test, and the sample must be retested.

[0056] Beneficial effects

[0057] 1. The kit of this invention utilizes ERA isothermal amplification combined with nanocarbon immunochromatography technology and a double-antibody sandwich method to detect Helicobacter pylori DNA in oral samples. ERA is a method that amplifies DNA under a single temperature. Compared with conventional PCR technology, ERA eliminates the limitations of expensive precision instruments and specialized technicians, significantly shortens the nucleic acid amplification time, and offers advantages such as rapidity and high specificity. This method helps improve the accuracy and sensitivity of oral Helicobacter pylori detection.

[0058] 2. In the present invention, the specificity and accuracy of the kit are improved by designing specific upstream primers, downstream primers and probes.

[0059] 3. In the present invention, the specificity and accuracy of the kit are improved by using a specific primer concentration, while the occurrence of false positives is reduced.

[0060] 4. In the present invention, by using biotin and digoxigenin to label specific downstream primers and probes respectively, combined with nanocarbon immunochromatography technology, the operation process is simplified and time is saved.

[0061] 5. The kit of the present invention uses ERA isothermal amplification combined with nanocarbon immunochromatography technology to achieve rapid visualization of nucleic acid amplification results. Compared with conventional nucleic acid agarose gel electrophoresis, it has the same sensitivity and can obtain results within 15 minutes, greatly shortening the detection time. At the same time, it avoids professional and complex experimental operations and expensive gel imaging instruments, solving the problem that conventional nucleic acid detection and analysis are not suitable for general laboratories and home self-testing promotion and application. BRIEF DESCRIPTION OF THE DRAWINGS

[0062] Figure 1 is a graph showing the results of conventional PCR-agarose gel electrophoresis using the primer pairs in Examples 2, 3, 4, 1, and 5; from left to right are the electrophoretic amplification bands of the primer pairs in Examples 2, 3, 4, 1, and 5, respectively.

[0063] Figure 2 is a graph showing the results of conventional PCR-agarose gel electrophoresis using the primer pairs in Examples 6, 7, 8, and 9; from left to right are the electrophoretic amplification bands of the primer pairs in Examples 6, 7, 8, and 9, respectively.

[0064] Figure 3 shows the results of screening using the LF-ERA detection method of Example 1 after combining the primer pairs in Examples 1, 5, and 6 with the probes; from left to right, they are the test results after detecting the Helicobacter pylori standard strain (HP), Streptococcus mutans DNA (SM), and negative control (water) using the primer and probe combinations in Examples 1, 5, and 6.

[0065] FIG4 is a graph showing the electrophoresis results of conventional PCR screening using primer pairs of different concentrations of 10-18 in Example 1.

[0066] FIG5 is a diagram showing the results of kit screening after detecting Hp DNA using primer pairs of different concentrations of 10-18 in Example 1.

[0067] FIG6 is a diagram showing the results of kit screening after testing the negative control (water) with primer pairs of different concentrations from Example 1, 10 to 18.

[0068] FIG7 is a graph showing the results of a cross-test of seven common oral bacteria using the kit in Example 1.

[0069] Figure 8 is a graph showing the LF-ERA test results of four real samples using the kit in Example 1; the samples from left to right are the negative control (water), Li, He, and Qu, among which water is sterile water and the result is negative; the result of volunteer Li is negative; the results of volunteers He and Qu are positive.

[0070] DETAILED DESCRIPTION Example

[0071] In a first aspect, this embodiment provides a LF-ERA primer and probe for Helicobacter pylori, which are Hp-specific primers and probes, namely, an upstream primer 2F and a downstream primer 1R.

[0072] The base sequence of the upstream primer 2F is: SEQ ID NO: 2;

[0073] The base sequence of the downstream primer 1R is: SEQ ID NO: 4;

[0074] The base sequence of the probe is: SEQ ID NO: 7.

[0075] The 5' end of the base sequence of the downstream primer 1R is connected to Biotin;

[0076] The 5' end of the base sequence of the probe is connected to Dig, the 3' end is connected to C3 Spacer, and the 32nd base is modified with tetrahydrofuran abasic site (THF);

[0077] The concentration of the upstream primer, downstream primer and probe was 5 μM.

[0078] The upstream primer, downstream primer and probe were synthesized by Boshang Biotechnology (Shanghai) Co., Ltd.

[0079] The second aspect of this embodiment provides an LF-ERA kit for Helicobacter pylori. The kit is based on ERA isothermal amplification and combined with immunochromatographic technology, and uses a nanocarbon labeling sandwich method for detection. The product is easy to operate and the results can be seen in 30 minutes. It is suitable for the detection of oral samples from the general population and patients who have recovered from gastric Helicobacter pylori infection.

[0080] The LF-ERA kit for Helicobacter pylori comprises Hp-specific primers and probes, an ERA basic amplification system, a sample diluent, a detection reagent card, and a desiccant;

[0081] The detection reagent card comprises a sample pad, an NC film, a carbon pad, absorbent paper, a PVC bottom plate and plastic parts.

[0082] The ERA basic amplification system includes a dissolving agent, a basic amplifying agent, dd H2O and an activator.

[0083] The ERA basic amplification system was purchased from Suzhou Xianda Gene Technology Co., Ltd.

[0084] The sample diluent is 8.1 mM Na2HPO4, 1.9 mM NaH2PO4 and 0.85% (W / V) NaCl, pH 7.4.

[0085] The sample pad was treated with a sample pad treatment solution to form glass fiber 8964 (8964 purchased from Shanghai Hanyi Biotechnology Co., Ltd.) and then dried at 37° C. overnight.

[0086] The sample pad treatment solution includes NaH2PO4, Na2HPO4, NaCl, protein stabilizer 2#, Tween-20 and ProClin300;

[0087] The sample pad treatment solution was prepared by weighing NaH2PO4, Na2HPO4, NaCl, protein stabilizer 2#, Tween-20, and ProClin300, adding pure water to prepare a solution of 0.15% (W / V) Na2HPO4, 1.45% (W / V) NaH2PO4, 0.9% (W / V) NaCl, 0.5% (W / V) protein stabilizer 2#, 1% (V / V) Tween20, and 0.1% (V / V) ProClin300, and adjusting the pH to 7.4±0.1.

[0088] The carbon pad includes a carbon pad with nano-carbon labeled Dig antibody and a carbon pad with nano-carbon labeled mouse IgG.

[0089] The nanocarbon-labeled Dig antibody was prepared as follows: 50 μL each of 0.8 mg / mL EDC and NHS were added to 300 μL of ultrasonically dispersed nanocarbon, mixed evenly, and activated at 37°C for 30 minutes. Then, 40 μg of digoxigenin antibody was added, mixed evenly, and coupled at 37°C for 45 minutes. After coupling, nanocarbon blocking solution (50 mM Tris + 2% (w / v) BSA) and 10% (v / v) Tween were added, mixed evenly, and blocked at 37°C for 30 minutes. Finally, the solution was centrifuged at 13,000 rpm for 15 minutes, washed once with nanocarbon preservation solution (50 mM Tris + 0.5% BSA), and stored in 200 μL of nanocarbon preservation solution until ready for use.

[0090] The preparation method of the nanocarbon-labeled mouse IgG is the same as the preparation method of the nanocarbon-labeled Dig antibody.

[0091] The preparation method of the carbon pad of the nanocarbon-labeled Dig antibody is as follows: Dig antibody labeled with nanocarbon is diluted 10 times with 40% nanocarbon labeling diluent and then used as padding, and then dried at 37° C. overnight.

[0092] The preparation method of the nanocarbon-labeled mouse IgG carbon pad is as follows: nanocarbon-labeled mouse IgG is diluted 10 times with 40% nanocarbon labeling diluent and then used as a pad, and then dried at 37° C. overnight.

[0093] The nanocarbon standard diluent is 70mM Tris, 1% (W / V) BSA, 10% (W / V) sucrose, 5% (W / V) trehalose and 0.4% (V / V) TritonX-100, pH=8.2.

[0094] The NC membrane is coated with a detection line T line and a quality control line C line parallel to each other, with a spacing of 5 mm. After coating, the membrane is dried at 37° C. overnight.

[0095] The specific preparation process of the NC membrane is as follows: the NC membrane is adhered to a PVC base plate, 1.0 mg / mL goat anti-mouse polyclonal antibody (C line) and 0.1 mg / mL streptavidin conjugate (T line) are coated on the NC membrane using a gold spray film streak apparatus, the coated NC membrane is dried at 37°C overnight, and then a desiccant is added to seal it for future use.

[0096] The assembly process of the detection reagent card is as follows: on a PVC base plate, the components are alternately pasted in the order of absorbent paper, NC membrane, carbon pad (4 mm) for nanocarbon-labeled mouse IgG, carbon pad (5 mm) for nanocarbon-labeled Dig antibody, and sample pad, with the absorbent paper located away from the sample well. After forming a large plate, the test strips are cut into 4 mm wide strips, mounted on a plastic housing, and sealed and stored at room temperature with a desiccant. This is the detection card.

[0097] The size of the PVC bottom plate is 60 mm×280 mm (MT101200507 purchased from Quzhou Maike New Materials Co., Ltd.).

[0098] The NC membrane is 1UN14ER100020NT (purchased from Sartorius).

[0099] The absorbent paper was purchased from 21050901 produced by Shanghai Pharmaceuticals Co., Ltd.

[0100] The third aspect of this embodiment provides a method for LF-ERA of Helicobacter pylori, comprising the following steps: performing a reaction according to the system and operation flow in Table 1.

[0101] Table 1

[0102]

[0103] The sample is an oral sample to be tested.

[0104] Example 2

[0105] The specific implementation of Example 2 is the same as that of Example 1, except that the Hp-specific primers are upstream primer 1F and downstream primer 1R.

[0106] Example 3

[0107] The specific implementation of Example 3 is the same as that of Example 1, except that the Hp-specific primers are upstream primer 1F and downstream primer 2R.

[0108] Example 4

[0109] The specific implementation of Example 4 is the same as that of Example 1, except that the Hp-specific primers are upstream primer 1F and downstream primer 3R.

[0110] Example 5

[0111] The specific implementation of Example 5 is the same as that of Example 1, except that the Hp-specific primers are upstream primer 2F and downstream primer 2R.

[0112] Example 6

[0113] The specific implementation of Example 6 is the same as that of Example 1, except that the Hp-specific primers are upstream primer 2F and downstream primer 3R.

[0114] Example 7

[0115] The specific implementation of Example 7 is the same as that of Example 1, except that the Hp-specific primers are upstream primer 3F and downstream primer 1R.

[0116] Example 8

[0117] The specific implementation of Example 8 is the same as that of Example 1, except that the Hp-specific primers are upstream primer 3F and downstream primer 2R.

[0118] Example 9

[0119] The specific implementation of Example 9 is the same as that of Example 1, except that the Hp-specific primers are upstream primer 3F and downstream primer 3R.

[0120] Example 10

[0121] The specific implementation of Example 10 is the same as that of Example 1, except that the concentrations of the upstream primer and the downstream primer are 10 μM.

[0122] Example 11

[0123] The specific implementation of Example 11 is the same as that of Example 1, except that the concentrations of the upstream primer and the downstream primer are 2.5 μM.

[0124] Example 12

[0125] The specific implementation of Example 12 is the same as that of Example 1, except that the concentrations of the upstream primer and the downstream primer are 1 μM.

[0126] Example 13

[0127] The specific implementation of Example 13 is the same as that of Example 1, except that the concentration of the upstream primer and the downstream primer is 0.5 μM.

[0128] Example 14

[0129] The specific implementation of Example 14 is the same as that of Example 1, except that the concentration of the upstream primer and the downstream primer is 0.25 μM.

[0130] Example 15

[0131] The specific implementation of Example 15 is the same as that of Example 1, except that the concentration of the upstream primer and the downstream primer is 0.1 μM.

[0132] Example 16

[0133] The specific implementation of Example 16 is the same as that of Example 1, except that the concentration of the upstream primer and the downstream primer is 0.05 μM.

[0134] Example 17

[0135] The specific implementation of Example 17 is the same as that of Example 1, except that the concentration of the upstream primer and the downstream primer is 0.025 μM.

[0136] Example 18

[0137] The specific implementation of Example 18 is the same as that of Example 1, except that the concentration of the upstream primer and the downstream primer is 0.01 μM.

[0138] 1. Primer screening: Use conventional PCR-agarose gel electrophoresis to screen specific primers.

[0139] Specific primers and probes were designed and optimized based on the conserved regions of the cytotoxin-associated gene A (CagA) sequence of Helicobacter pylori to ensure the specificity of the isothermal amplification reaction. The primer pair combinations of Examples 1 to 9 were screened using three samples: a standard H. pylori strain (H. pylori), H. pylori genomic DNA extracted using a bacterial genomic DNA extraction kit, and a negative control (water). (The H. pylori strain was purchased from BNCC339501, a commercial product of Beina Chuanglian Biotechnology Co., Ltd.; the bacterial genomic DNA extraction kit was purchased from DP302, a commercial product of Tiangen Biochemical Technology (Beijing) Co., Ltd.) The reaction solutions listed in Table 2 were added, mixed thoroughly, and then amplified in a PCR instrument. The reaction procedure was 94°C pre-denaturation for 5 minutes, followed by denaturation at 94°C for 30 seconds, annealing at 57°C for 30 seconds, and extension at 72°C for 40 seconds. Amplification was repeated for 35 cycles, followed by extension at 72°C for 10 minutes. The amplified product was subjected to gel electrophoresis to observe the appearance of target bands, nonspecific bands, and the brightness of the target bands, and the optimal primers were screened based on amplification efficiency and specificity.

[0140] The results are shown in Table 3 and Figures 1 and 2.

[0141] The experimental results in Table 3 show that only Examples 1-6 were able to simultaneously amplify H. pylori bacteria and H. pylori DNA using a conventional PCR amplification procedure. The three primer pairs 3F+1R, 3F+2R, and 3F+3R in Examples 7-9 were able to amplify only H. pylori DNA samples. Further analysis, combined with gel electrophoresis Figures 1 and 2, revealed that the presence of primer dimers in Examples 2-4 affected amplification efficiency. Therefore, the three primer pairs 2F+1R, 2F+2R, and 2F+3R were selected and paired with probes to construct the LF-ERA detection system for further screening.

[0142] Table 2

[0143]

[0144] Table 3

[0145]

[0146] 2. Establishment of LF-ERA detection system:

[0147] Using the LF-ERA method for Helicobacter pylori (HP) in Example 1, three samples—the H. pylori standard strain (HP), the Streptococcus mutans genome (SM) extracted using a bacterial genomic DNA extraction kit, and a negative control (water)—were used as templates to screen the 2F+1R, 2F+2R, and 2F+3R primer pairs described in Examples 1, 5, and 6. Detection results were observed according to the protocol in Table 1. (The Streptococcus mutans strain was purchased from Shangcheng Beina Chuanglian Biotechnology Co., Ltd. as BNCC336931, and the bacterial genomic DNA extraction kit was purchased from Tiangen Biochemical Technology (Beijing) Co., Ltd. as DP302.) If both the control line (C) and the test line (T) appear on the test strip, it indicates that the sample contains H. pylori. If only the control line (C) appears on the test strip, the sample does not contain H. pylori. Results are shown in Table 4 and Figure 3. Note to Table 4: In the LF-ERA test results, a T line intensity ≥ C3.5 indicates a positive test, while < C3.5 indicates a negative test.

[0148] The results in Table 4 show that the 2F+1R primer in Example 1 does not cross with the Streptococcus mutans DNA to produce nonspecific detection signals, while the 2F+2R in Example 5 and the 2F+3R in Example 6 cross with the Streptococcus mutans DNA and the negative control produces a false positive signal.

[0149] Therefore, the primer pair in Example 1 has the best specific amplification effect in the LF-ERA detection system.

[0150] Table 4

[0151]

[0152] 3. Primer concentration exploration:

[0153] Under the premise of consistent H. pylori DNA concentration and dosage, different concentrations of the 2F+1R primer pair were prepared and tested using the H. pylori LF-ERA method described in Examples 1 and 10-18, respectively, using three samples as templates: a H. pylori standard strain (H. pylori), the H. pylori standard strain genome (H. pylori DNA) extracted from the kit, and a negative control (water). If both the control line (C) and the test line (T) appeared on the test strip, it indicated that the sample contained H. pylori. If only the control line (C) appeared on the test strip, the sample did not contain H. pylori.

[0154] Add the reaction solution as indicated in Table 2, mix, and perform amplification in a PCR instrument. The reaction procedure was 94°C pre-denaturation for 5 minutes, 94°C denaturation for 30 seconds, 57°C annealing for 30 seconds, 72°C extension for 40 seconds, 35 cycles of amplification, and 72°C extension for 10 minutes. The amplified product was subjected to gel electrophoresis. ERA isothermal amplification was also performed according to the experimental method in Table 1, and the results were observed using immunochromatographic strips. The results are shown in Table 5 and Figures 4-6. Note to Table 5: In the LF-ERA test results, a T-line intensity ≥ C3.5 indicates a positive test, and < C3.5 indicates a negative test.

[0155] The results in Table 5 demonstrate that as the primer working concentration decreases, the brightness of the electrophoretic bands gradually decreases, and the PCR amplification efficiency weakens. The bands are darkest when the primer working concentration is reduced to 0.1 μM. When the primer working concentration is below 0.1 μM, no detectable positive product is obtained. The same conclusion can be drawn based on the positive (Hp DNA) results of the LF-ERA assay. However, it should be noted that when the LF-ERA assay is performed using 10 μM primers, the negative control (water) produces a false positive C4. Therefore, the 5 μM primer pair in Example 1 demonstrates high amplification efficiency and prevents false positives.

[0156] Table 5

[0157]

[0158] 4. Cross-test of common oral bacteria:

[0159] Using the LF-ERA method for Helicobacter pylori described in Example 1, cross-reactivity testing was performed on the seven common oral bacteria strains listed in the table (all strains were purchased from Beina Chuanglian Biotechnology Co., Ltd.; detailed strain names and numbers are shown in Table 6). Using the H. pylori standard strain (HP) as a positive control and sterile water (water) as a negative control, the assay was performed according to the protocol in Table 1. Results are shown in Table 6 and Figure 7. Note to Table 6: In the LF-ERA test results, a T-line intensity ≥ C3.5 indicates a positive test, while < C3.5 indicates a negative test.

[0160] The results in Table 6 show that the 2F+1R primer in Example 1 does not cross-react with Streptococcus mutans, Fusobacterium nucleatum subsp. polynucleatum, Actinomyces naeslundii, Lactobacillus acidophilus, Prevotella intermedia, Porphyromonas gingivalis, and Streptococcus salivarius to produce nonspecific detection signals, and has no cross-reaction with the seven common oral bacteria.

[0161] Table 6

[0162]

[0163] 5. Practical analysis of LF-ERA testing of oral samples:

[0164] (1) Sample collection: Gum, dental plaque, and tartar samples were collected from the oral cavity to ensure comprehensive sample collection. DNA extracted from a standard H. pylori strain and sterile water were used as positive and negative controls, respectively.

[0165] (2) Conventional PCR amplification test: Add the reaction solution according to Table 2, mix, and perform amplification in a PCR instrument. The reaction procedure is 94℃ pre-denaturation for 5 minutes, 94℃ denaturation for 30 seconds, 57℃ annealing for 30 seconds, 72℃ extension for 40 seconds, amplification for 35 cycles, and then extension at 72℃ for 10 minutes. The amplified product is subjected to gel electrophoresis. If a clearly bright band appears, the sample is tested positive; otherwise, it is tested negative.

[0166] (3) LF-ERA detection: Add reagents in sequence as shown in Table 1 for reaction. After completion, take the amplified product and combine it with lateral flow immunoassay strips to observe the results.

[0167] (4) The results are shown in Table 7 and Figure 8.

[0168] The present invention provides a detection method and kit for detecting Helicobacter pylori by LF-ERA. The method of the present invention is a simple, accurate and economical on-site rapid detection method, which is suitable for high-risk screening of Helicobacter pylori infection at home.

[0169] Table 7

[0170] .

Claims

1. A primer and probe for detecting Helicobacter pylori LF-ERA, characterized in that: Hp-specific primers and probes, wherein the Hp-specific primers include one or more of upstream primers 1F, 2F, and 3F and one or more of downstream primers 1R, 2R, and 3R; The base sequence of the upstream primer 1F is: SEQ ID NO: 1, The base sequence of the upstream primer 2F is: SEQ ID NO: 2, The base sequence of the upstream primer 3F is: SEQ ID NO: 3, The base sequence of the downstream primer 1R is: SEQ ID NO: 4, The base sequence of the downstream primer 2R is: SEQ ID NO: 5, The base sequence of the downstream primer 3R is: SEQ ID NO: 6, The base sequence of the probe is: SEQ ID NO:

7.

2. The Helicobacter pylori LF-ERA detection primer and probe according to claim 1, characterized in that: The 5' ends of the base sequences of the downstream primers 1R, 2R, and 3R are connected to Biotin; The 5' end of the base sequence of the probe is connected to Dig, the 3' end is connected to C3 Spacer, and the 32nd base is modified with tetrahydrofuran abasic site (THF).

3. The Helicobacter pylori LF-ERA detection primer and probe according to claim 1, characterized in that: The concentrations of the upstream primer, downstream primer and probe are 0.01 μM-10 μM.

4. The Helicobacter pylori LF-ERA detection primer and probe according to claim 3, characterized in that: The concentrations of the upstream primer, downstream primer and probe are 0.1 μM-10 μM.

5. An LF-ERA kit for Helicobacter pylori comprising the LF-ERA primers and probes according to any one of claims 1 to 4, characterized in that: It includes Hp-specific primers and probes, ERA basic amplification system, sample diluent and detection reagent card.

6. The LF-ERA kit for Helicobacter pylori according to claim 5, characterized in that: The ERA basic amplification system comprises a dissolving agent, a basic amplifying agent, dd H2O and an activating agent.

7. The LF-ERA kit for Helicobacter pylori according to claim 5, characterized in that: The sample diluent includes Na2HPO4, NaH2PO4 and NaCl.

8. The LF-ERA kit for Helicobacter pylori according to claim 5, characterized in that: The detection reagent card comprises a sample pad, an NC film, a carbon pad, absorbent paper and a PVC bottom plate.

9. The LF-ERA kit for Helicobacter pylori according to claim 8, characterized in that: The sample pad needs to be treated with a sample pad treatment solution and dried overnight; the sample pad treatment solution includes NaH2PO4, Na2HPO4, NaCl, protein stabilizer 2#, Tween-20 and ProClin300; the carbon pad includes a carbon pad of nanocarbon labeled Dig antibody and a carbon pad of nanocarbon labeled mouse IgG; the NC membrane is coated with a detection line T line and a quality control line C line parallel to each other, with an interval of 5 to 10 mm, and is dried overnight after coating.

10. The LF-ERA kit for Helicobacter pylori according to claim 9, characterized in that: The preparation method of the carbon pad for the nanocarbon-labeled Dig antibody is as follows: the Dig antibody labeled with nanocarbon is diluted 10 to 15 times with 40 to 50% nanocarbon-labeled diluent, and then used as a pad, and then dried overnight; the preparation method of the carbon pad for the nanocarbon-labeled mouse IgG is as follows: the mouse IgG labeled with nanocarbon is diluted 10 to 15 times with 40 to 50% nanocarbon-labeled diluent, and then used as a pad, and then dried overnight.

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