Composition capable of detecting helicobacter pylori clarithromycin drug resistance gene mutation, kit and application

Abstract

The invention provides a composition capable of detecting helicobacter pylori clarithromycin drug resistance gene mutation, a kit and application, and belongs to the technical field of gene detection. The invention designs an As-RPA primer pair and crRNA capable of realizing multiple simultaneous detection of three single-base mutation A2142C, A2142G and A2143G and one double-base mutation A2142G / A2143G, and an As-RPA-CRISPER / Cas12a system established on the basis of the sequence, the system can detect four mutation types of helicobacter pylori within 1 hour under the condition of constant temperature, and the primer pair and crRNA have the advantages of high specificity, high sensitivity, high sensitivity, high sensitivity and the like. The kit has remarkable advantages in the aspects of sensitivity, specificity, detection speed, multiple detection capability, visual result interpretation, applicability and the like, provides an efficient and convenient solution for detecting the drug resistance of helicobacter pylori clarithromycin, and has important clinical application value and popularization prospect.

Description

Technical Field

[0001] The present invention relates to the technical field of gene detection, and particularly to a composition, a kit and an application for detecting clarithromycin-resistant gene mutations of Helicobacter pylori. Background Art

[0002] Helicobacter pylori (H. pylori) is a Gram-negative bacterium that can be transmitted through oral-oral, fecal-oral and other routes, and the global infection rate is about 50%. Its infection is closely related to diseases such as gastritis, peptic ulcer, gastric cancer and gastric mucosa-associated lymphoid tissue lymphoma. Eradication treatment is crucial for disease prevention and control.

[0003] The quadruple therapy with bismuth (1 proton pump inhibitor + 2 antibiotics + 1 bismuth agent) is the main method for treating Helicobacter pylori infection. However, this method mainly relies on antibiotics. With the extensive use of antibiotics, the resistance rates of Helicobacter pylori to clarithromycin (CLA), metronidazole (MTZ) and levofloxacin (LVX) all exceed 30%, resulting in a very low eradication success rate of empirical treatment for Helicobacter pylori, less than 90%. Among them, the clarithromycin resistance (Cla-res) problem is particularly prominent.

[0004] Clarithromycin resistance mainly stems from specific point mutations in the V domain of the 23S rRNA gene of Helicobacter pylori, including A2143G, A2142G and A2142C, etc. Among them, the A2143G mutation is directly related to the lowest eradication rate. When the clarithromycin resistance rate ≥ 15%, treatment needs to be guided by drug sensitivity tests. There is an urgent need for accurate and rapid drug resistance detection methods to achieve individualized treatment.

[0005] At present, the commonly used drug resistance detection methods in clinics have the following defects: (1) Traditional phenotypic methods (such as drug sensitivity tests) are time-consuming (requiring 3 - 5 days of bacterial culture) and have high requirements for sample quality, making it difficult to meet the immediate clinical needs. (2) Among molecular biology methods, PCR relies on electrophoresis or enzyme digestion analysis, with cumbersome operations and unable to distinguish single-base mutations; sequencing technology is accurate, but the equipment is expensive and the cycle is long; fluorescence in situ hybridization (FISH) can only detect a single drug resistance target; gene chip technology has strict requirements for the amount of gene template and high costs. (3) Although the emerging CRISPR technology shows potential in nucleic acid detection, its application is limited by the strict requirements of protospacer adjacent motif (PAM) sites and it is difficult to efficiently distinguish single nucleotide polymorphisms (SNPs). In addition, existing CRISPR detections mostly target a single mutation type and cannot achieve simultaneous detection of multiple targets, resulting in low detection efficiency.

[0006] Although the combination of isothermal amplification technologies (such as RPA) and CRISPR systems has been explored, traditional symmetric amplification strategies are prone to producing non-specific products, and the CRISPR system has insufficient ability to distinguish double-base mutations, which limits its application in complex mutation detection. Therefore, developing a technology that is fast, highly sensitive, low-cost, and capable of simultaneously detecting multiple mutation types has become a key requirement for clinical precision treatment and curbing the abuse of antibiotics. Summary of the Invention

[0007] The object of the present invention is to provide a combination, kit, and application for detecting Helicobacter pylori clarithromycin resistance gene mutations, which can achieve multiplex simultaneous detection of four mutation types, namely three single-base mutations A2142C, A2142G, A2143G and one double-base mutation A2142G / A2143G.

[0008] To achieve the above object of the invention, the present invention provides the following technical solutions:

[0009] The present invention provides a composition for detecting Helicobacter pylori clarithromycin resistance gene mutations, and the composition for detecting Helicobacter pylori clarithromycin resistance gene mutations includes the As-RPA primer pair shown in SEQ ID NO. 48-49 and the crRNA shown in SEQ ID NO. 47.

[0010] Preferably, the Helicobacter pylori clarithromycin resistance gene mutations include three single-base mutations A2142C, A2142G, A2143G of 23S rRNA and one double-base mutation A2142G / A2143G.

[0011] The present invention also provides an application of a composition for detecting Helicobacter pylori clarithromycin resistance gene mutations in the preparation of a product for detecting Helicobacter pylori clarithromycin resistance gene mutations.

[0012] The present invention also provides a product for detecting Helicobacter pylori clarithromycin resistance gene mutations, and the product for detecting Helicobacter pylori clarithromycin resistance gene mutations includes an As-RPA primer pair and crRNA.

[0013] The present invention also provides a method for detecting Helicobacter pylori clarithromycin resistance gene mutations, including the following steps:

[0014] (1) Extract the DNA of the sample to be tested for As-RPA amplification, and after the amplification is completed, heat to inactivate the enzyme to obtain the As-RPA amplification product;

[0015] (2) After mixing the As-RPA amplification product with crRNA, LbCAS12a protein, and the probe, determine the mutation type through fluorescence signal changes or color development of the test strip.

[0016] Preferably, in step (1), the amplification system for As-RPA amplification includes an As-RPA primer pair, an RPA dry powder ball, a PEG reaction solution, a magnesium acetate reaction solution, and a DNA sample to be tested, and the total reaction system is 25 μL; the amplification conditions are 35-42 °C for 10-20 min.

[0017] Preferably, in step (1), the As-RPA primer pair is 23S-RPA-F shown in SEQ ID NO. 48 and 23S-RPA-R shown in SEQ ID NO. 49; the concentration ratio of 23S-RPA-F to 23S-RPA-R is 50-300:1, and the Mg 2+ concentration in the As-RPA amplification system is 11.2-33.6 mM.

[0018] Preferably, in step (2), the concentration ratio of the LbCas12a protein to the crRNA is 1-1.5:1-2.5, and the probe includes a FAM-BHQ probe for detecting fluorescence signal changes or a FAM-Biotin probe for color development determination of a test strip.

[0019] The present invention also provides an application of a method for detecting Helicobacter pylori clarithromycin resistance gene mutations in the detection of Helicobacter pylori clarithromycin resistance gene mutations.

[0020] The beneficial effects of the present invention compared with the prior art are as follows:

[0021] The present invention designs an As-RPA primer pair (SEQ ID NOs. 48-49) and a crRNA (SEQ ID NO. 47) that can simultaneously detect three single-base mutations A2142C, A2142G, A2143G and a double-base mutation A2142G / A2143G in multiple ways, and an As-RPA-CRISPER / Cas12a system established based on this sequence. This system can detect four mutation types of Helicobacter pylori within 1 h under isothermal conditions, and has significant advantages in terms of sensitivity, specificity, detection speed, multiplex detection ability, visual result interpretation and applicability, etc., providing an efficient and convenient solution for the detection of Helicobacter pylori clarithromycin resistance, and having important clinical application value and promotion prospects. Specifically:

[0022] (1) High sensitivity and specificity: The lowest concentration of the Helicobacter pylori clarithromycin resistance gene that the present invention can detect is 10 -3ng / μL, with extremely high sensitivity. By optimizing the design of crRNA for the CRISPR / Cas12a system, strict discrimination between three single-base mutations A2142C, A2142G, A2143G and one double-base mutation A2142G / A2143G of 23S rRNA and the wild type can be achieved, significantly improving the specificity of detection and avoiding false positive results.

[0023] (2) Fast and efficient: The present invention only takes 1 h from nucleic acid extraction to result interpretation, significantly shortening the detection time compared with the traditional PCR method (usually taking 3 - 4 h), meeting the clinical demand for rapid diagnosis.

[0024] (3) Multi-target multiplex detection design: By designing a pair of universal As-RPA primers and one crRNA, the present invention can simultaneously detect three single-base mutations (A2142C, A2142G, A2143G) and one double-base mutation (A2142G / A2143G) in the 23S rRNA gene of Helicobacter pylori, significantly improving the detection efficiency and avoiding the cumbersome operations of multiple detections. By optimizing the primer concentration ratio and asymmetric amplification strategy, multiplex target amplification within a single reaction system is achieved, breaking through the dependence of traditional CRISPR technology on PAM sites.

[0025] (4) Visual result interpretation: The present invention combines lateral flow strip technology, and through the cleavage of FAM-Biotin probe, visual result interpretation with the naked eye (T line coloring indicates positive) is achieved. This method does not require complex instruments, is easy to operate, and is especially suitable for primary medical institutions and areas with limited resources.

[0026] (5) Applicability to diverse sample types: The present invention is applicable to various sample types, including gastric juice and feces, etc., broadening the application scenarios of detection. By optimizing the sample processing flow, efficient detection of low-concentration targets in different samples is ensured.

[0027] (6) Low cost and portability: The present invention does not rely on expensive instrument equipment (such as fluorescence quantitative or sequencing instruments), and only requires a constant temperature heating device and lateral flow strips to complete the detection, significantly reducing the detection cost and being suitable for large-scale popularization and use.

[0028] (7) Point-of-care testing (POCT) ability: The present invention integrates the As-RPA and CRISPR / Cas12a systems, combines lateral flow strip technology, and realizes the point-of-care testing (POCT) of clarithromycin resistance gene mutations of Helicobacter pylori, providing a rapid and accurate diagnostic tool for clinicians, helping to formulate individualized treatment plans and improve the eradication success rate. Brief Description of the Drawings

[0029] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required for use in the embodiments. Obviously, the drawings described below are only some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can also be obtained based on these drawings.

[0030] Figure 1 For the CRISOER / CAS system in Example 1 of the present invention, the tolerance to specific screening, single-base mutations, double-base mutations, and triple-base mutations is shown. Among them, A is a bar chart of the difference between the end fluorescence and the initial fluorescence of single-base mutations, and B is a bar chart of the difference between the end fluorescence and the initial fluorescence of continuous double-base mutations, spaced double-base mutations, and continuous triple-base mutations.

[0031] Figure 2 In Example 1 of the present invention, 4 crRNAs were designed with 23S rRNAA2142G and A2142G mutations simultaneously as targets, and the crRNA with the largest difference in fluorescence intensity between the mutant and the wild type was screened. A is the original fluorescence curve graph, and B is the bar chart of fluorescence difference.

[0032] Figure 3 For the effect of the As-RPA reaction time on the detection result in Example 2 of the present invention, A is the original fluorescence curve graph, and B is the bar chart of fluorescence difference.

[0033] Figure 4 For the effect of the Mg 2+ concentration on the detection result in the As-RPA reaction in Example 2 of the present invention, A is the original fluorescence curve graph, and B is the bar chart of fluorescence difference.

[0034] Figure 5 For the effect of the reaction temperature on the detection result in the As-RPA reaction in Example 2 of the present invention, A is the original fluorescence curve graph, and B is the bar chart of fluorescence difference.

[0035] Figure 6 For the effect of the primer concentration on the detection result in the As-RPA reaction in Example 2 of the present invention, A is the original fluorescence curve graph, and B is the bar chart of fluorescence difference.

[0036] Figure 7 For the effect of the CAS protein concentration on the detection result in the CRISPER / CAS reaction system in Example 2 of the present invention, A is the original fluorescence curve graph, and B is the bar chart of fluorescence difference.

[0037] Figure 8 For the effect of the concentration ratio of the CAS protein to the crRNA on the detection result in the CRISPER / CAS reaction system in Example 2 of the present invention, A is the original fluorescence curve graph, and B is the bar chart of fluorescence difference.

[0038] Figure 9 It is a graph showing the influence of the concentration of FAM-BHQ probe in the CRISPER / CAS reaction system of Example 2 of the present invention on the detection result. A is the original fluorescence curve graph, and B is the fluorescence difference bar graph;

[0039] Figure 10 It is the verification of the successful detection of the simultaneous mutations of A2142G, A2142C, A2143G, and A2142G / 12143G in the bacterial liquid extraction and the simultaneous mutations of A2142G, A2143G, and A2142G / 12143G in the fecal simulated specimen in Example 3 of the present invention; A is the original fluorescence curve graph, and B is the fluorescence difference bar graph;

[0040] Figure 11 It is the verification graph of the successful detection of the simultaneous mutations of A2142G, A2142C, A2143G, and A2142G / 12143G in the bacterial liquid extraction and the simultaneous mutations of A2142G, A2143G, and A2142G / 12143G in the fecal simulated specimen by the test strip method in Example 3 of the present invention. From left to right, the targets are the simultaneous mutations of A2142G, A2142C, A2143G, and A2142G / 12143G in the bacterial liquid extraction, the simultaneous mutations of A2142G, A2143G, and A2142G / 12143G in the fecal simulated specimen, and WT;

[0041] Figure 12 It is the specificity comparison of the A2143G mutation of the Helicobacter pylori 23S rRNA resistance gene with 6 other common intestinal flora and the Helicobacter pylori 23S rRNA wild type in Example 4 of the present invention; A is the original fluorescence curve graph, and B is the fluorescence difference bar graph;

[0042] Figure 13 It is the sensitivity of A2143G in the As-RPA-CRISPER / Cas12a system and the sensitivity of the test strip method in Example 4 of the present invention; A is the original fluorescence curve graph, B is the fluorescence difference bar graph, and C is the test strip method. From left to right, the target concentrations are 10 1 ng / μL, 10 0 ng / μL, 10 -1 ng / μL, 10 -2 ng / μL, 10 -3 ng / μL, 10 -4 ng / μL, 10 -5 ng / μL, WT 50 ng / μL;

[0043] Figure 14For the sensitivity of the As-RPA-CRISPER / Cas12a system A2142G and the sensitivity of the strip method in Example 4 of the present invention; A is the original fluorescence curve graph, B is the fluorescence difference histogram, and C is the test strip. From left to right, the target concentrations are 2.49*10 1 ng / μL, 2.49*10 0 ng / μL, 2.49*10 -1 ng / μL, 2.49*10 -2 ng / μL, 2.49*10 -3 ng / μL, WT 50 ng / μL;

[0044] Figure 15 For the sensitivity of the As-RPA-CRISPER / Cas12a system A2142C and the sensitivity of the strip method in Example 4 of the present invention; A is the original fluorescence curve graph, B is the fluorescence difference histogram, and C is the test strip. From left to right, the target concentrations are 2.59*10 1 ng / μL, 2.59*10 0 ng / μL, 2.59*10 -1 ng / μL, 2.59*10 -2 ng / μL, 2.59*10 -3 ng / μL, WT 50 ng / μL,;

[0045] Figure 16 For the sensitivity of the As-RPA-CRISPER / Cas12a system A2142G / A2143G and the sensitivity of the strip method in Example 4 of the present invention; A is the original fluorescence curve graph, B is the fluorescence difference histogram, and C is the test strip. From left to right, the target concentrations are 2.38*10 1 ng / μL, 2.38*10 0 ng / μL, 2.38*10 -1 ng / μL, 2.38*10 -2 ng / μL, 2.38*10 -3 ng / μL, 2.38*10 -4 ng / μL, 2.38*10 -5 ng / μL, WT 50 ng / μL;

[0046] Figure 17 For 27 gastric juice samples in Example 5 of the present invention, the PCR results of 23S rRNA were detected, and 9 positive strains were detected, which were 5, 6, 17, 21, 22, 24, 25, 26, and 27 positive respectively. There were 3 strains of the 23S rRNA mutant detected by this method, which were 6, 21, and 41;

[0047] Figure 18This is the sequencing result of the PCR product in Example 5 of the present invention. A, B, C, D, E, F, G, H, and I are 5, 6, 17, 21, 22, 24, 25, 26, and 27 in sequence. Detailed implementation manners

[0048] The various exemplary implementation manners of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, characteristics, and implementation schemes of the present invention.

[0049] It should be understood that the terms used in the present invention are only for describing specific implementation manners and are not used to limit the present invention. Additionally, for the numerical ranges in the present invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Each intermediate value within any stated value or stated range, as well as each smaller range between any other stated value or intermediate value within the stated range, is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded from the range.

[0050] Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which the present invention pertains. Although the present invention only describes preferred methods and materials, any methods and materials similar or equivalent to those described herein can also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials related to the documents. In case of conflict with any incorporated document, the content of this specification shall prevail.

[0051] Without departing from the scope or spirit of the present invention, various improvements and changes can be made to the specific implementation manners of the present invention specification, which are obvious to those skilled in the art. Other implementation manners obtained from the specification of the present invention are obvious to those skilled in the art. The specification and embodiments of the present invention are only exemplary.

[0052] Regarding the use of "comprising", "including", "having", "containing", etc. in this article, they are all open-ended terms, meaning including but not limited to.

[0053] The present invention provides a method for detecting Helicobacter pylori clarithromycin resistance gene mutations, comprising the following steps:

[0054] (1) Extract the DNA of the sample to be tested for As-RPA amplification. After the amplification is completed, heat to inactivate the enzyme to obtain the As-RPA amplification product;

[0055] Among them, the amplification system for the described As-RPA amplification includes an As-RPA primer pair, an RPA dry powder ball, a PEG reaction solution, a magnesium acetate reaction solution, and a DNA sample to be tested. The total reaction system is 25 μL; the conditions for the amplification are 35-42 °C for 10-20 min;

[0056] The As-RPA primer pair is 23S-RPA-F shown in SEQ ID NO. 48 and 23S-RPA-R shown in SEQ ID NO. 49; the final concentration of the used 23S-RPA-F is 2.0 μM and the final concentration of 23S-RPA-R is 20 nM, and the concentration ratio is 100:1. Mg 2+ The final concentration is 22.4 mM, the reaction time is 15 min, and the reaction temperature is 35 °C.

[0057] (2) After mixing the As-RPA amplification product with crRNA, LbCAS12a protein, and a probe, the mutation type is determined by fluorescence signal change or test strip color development;

[0058] Among them, the specific method for determining the mutation type by fluorescence signal change is as follows:

[0059] Add 4 μL of the amplification product to the CRISPER / CAS fluorescence detection. The reaction system includes the crRNA, LbCAS12a protein, Buffer buffer, and FAM-BHQ probe as described in claim 1, and make up the volume to 20 μL with ddH2O; the result determination is as follows: there is an amplification curve, and the difference between the end fluorescence and the initial fluorescence is significantly different from that of the wild type, which is positive;

[0060] The final concentration of the Cas12a protein is 50 nM and the final concentration of the crRNA is 100 nM, and the concentration ratio is 1:2. The final concentration of the FAM-BHQ probe is 250 nM.

[0061] The specific method for determining the mutation type by test strip color development is as follows:

[0062] Add 4 μL of the amplification product to the CRISPER / CAS test strip detection. The reaction system includes the crRNA, LbCAS12a protein, Buffer buffer, and FAM-Biotion probe as described in claim 1, and make up the volume to 20 μL with ddH2O; the result determination is as follows: the appearance of a T line on the test strip is positive;

[0063] The final concentration of the FAM-Biotion probe is 50 nM.

[0064] The present invention also provides a kit for detecting Helicobacter pylori clarithromycin resistance gene mutations, including As-RPA primer pairs and crRNA, RPA dry powder balls, PEG reaction solution, magnesium acetate reaction solution, Cas12a protein, reaction buffer, positive / negative controls, fluorescent / biotin probes, and lateral flow test strips.

[0065] Example 1

[0066] In Example 1 of the present invention, sequences capable of detecting Helicobacter pylori clarithromycin resistance gene mutations were designed and screened, and the specific steps are as follows:

[0067] (1) Sequence design

[0068] Log in to the NCBI website and search for and download the H. pylori 23S rRNA gene sequence in GeneBanK. Use the DNAMAN software for sequence alignment to find the A2142 and A2143 regions, and for the A2142G and A2143G mutation forms, use the Primer6 software to design RPA primers (SEQ ID NO.48 - SEQ ID NO.49) and a set of PCR primers (SEQ ID NO.50 - SEQ ID NO.51) for the conserved region; design 4 crRNA sequences (SEQ ID NO.44 - SEQ ID NO.47) for the A2142G and A2143G simultaneous mutation sequences; randomly design crRNA1 (where the sequence UCCGUCUUGCCGCGGGUA is the spacer, i.e., the region binding to the target) for the pre-experiment of evaluating the tolerance of the mutation site of the CRISPER / CAS system; design the primers shown in SEQ ID NO.2 - SEQ ID NO.43 for crRNA1 for the pre-experiment of evaluating the tolerance of the mutation site of the CRISPER / CAS system, as shown in Table 1 specifically:

[0069] Table 1 LAMP primer sequence list

[0070]

[0071]

[0072]

[0073]

[0074] Note: The underlined part is the mutation site and the mutated base.

[0075] (2) Establishment of the As-RPA reaction system

[0076] 1) Inoculate the bacterial strain sample provided by the First Hospital of Nanjing on a Columbia blood agar plate and place it in an anaerobic jar with 85% N2, 10% CO2, and 5% O2, and culture it at 37°C for 2 - 3 days. Sequence the cultured bacterial strain, and the identification results are strains of H. pylori with single-base mutations (A2142C, A2142G, or A2143G) in 23S rRNA, and strains of H. pylori with double-base mutations (A2142G / A2143G). Preserve them at -80°C.

[0077] 2) Extract H. pylori DNA: Take 2 mL of the bacterial liquid culture medium, centrifuge it at 12000 g for 2 min, discard the supernatant, add 100 μL of 0.4% TritonX - 100, shake it for 2 min, then add all the liquid to the extraction plate, and extract the H. pylori DNA of different mutant strains according to the extraction instructions of the magnetic bead method extraction kit, and place it in a -20°C refrigerator for later use.

[0078] 3) Establishment of the As - RPA reaction system

[0079] Using the extracted H. pylori DNA as a template, the whole process is completed in a 25 μL reaction system. The reaction system is as follows:

[0080] Add 30 μL of the PEG reaction solution to a dry RPA powder ball, then add 2 μL of 23S - RPA - F (50 μm) and 2 μL of 23S - RPA - R (0.5 μm), shake and mix well. After a brief centrifugation, divide the reaction system equally into 2 tubes of 200 μL PCR reaction tubes, 18 μL in each tube, add 5 μL of the extracted H. pylori DNA, and add 2 μ μL of the magnesium acetate reaction solution to make the final concentration of Mg 2+ 22.4 mM. Cover the tube lid, invert and mix well. Set the PCR instrument to a constant temperature of 35°C with a 30 s cycle for 30 cycles. After the reaction is completed, heat it at 85°C for 5 min, take out the As - RPA reaction product, and centrifuge it for later use.

[0081] (3) Establishment of the As - RPA - CRISPER / Cas12a fluorescence reaction system

[0082] Take 4 μL of the As - RPA reaction product, 1 μL of LbCas12a (1 μm), 2 μL of crRNA (1 μm), 0.5 μL of FAM - BHQ (10 μm), and 3 μL of buffer and add them to a 200 μL PCR reaction tube, add 9.5 μL of ddH2O to make the volume up to 20 μL. Put the reaction tube into the PCR instrument, set the program to a constant temperature of 40°C with a 30 s cycle for 60 cycles, and set the FAM channel to read the fluorescence curve.

[0083] (4) Establishment of the As-RPA-CRISPER / Cas12a strip reaction system

[0084] Add 1 μL of LbCas12a (1 μm), 2 μL of crRNA (1 μm), 2 μL of FAM-Biotin (1 μm), 3 μL of buffer, and 4 μL of the As-RPA reaction product into a 200 μL PCR reaction tube, add 8 μL of ddH2O, and make up the volume to 20 μL. Put the reaction tube into a PCR instrument, set the program to cycle at 40 °C for 30 s per cycle for 60 cycles. After the reaction, centrifuge the product briefly and then pipette 5 μL into 45 μL of ddH2O and mix well. Insert the test strip and read the result of the test strip after 5 min.

[0085] (5) Preliminary experiment for evaluating the tolerance of mutation sites in the CRISPER / CAS system

[0086] Add 1 μL of LbCas12a (1 μm), 2 μL of crRNA (1 μm) (SEQ ID NO.1), 0.5 μL of FAM-BHQ (10 μm), 3 μL of buffer, and 4 μL of the synthesized single-stranded target (SEQ ID NO.2 - 43) into a 200 μL PCR reaction tube, add 9.5 μL of ddH2O, and make up the volume to 20 μL. Set the fully complementary single-stranded as the positive control and ddH2O as the negative control, and repeat 3 times. Put the reaction tube into a PCR instrument, set the program to cycle at 40 °C for 30 s per cycle for 60 cycles, set to read the fluorescence curve in the FAM channel, and use the ratio of the end fluorescence to the initial fluorescence to find the positions where Cas12a is tolerant to single-site mutations and intolerant to double-site mutations. The results are as Figure 1 shown.

[0087] Figure 1 shown that: (1) Single-site mutations at positions 8, 9, 10, 15 (SNV8, SNV9, SNV10, SNV15) have the same activation effect as the positive control (pc) and are tolerant to single-site mutations. (2) Among the double-site mutations, 4,5 - 8,9 (DNV4,6,7,9) has better specificity and is intolerant. Therefore, consider using positions 8 and 9 with single-site mutation tolerance and double-site mutation intolerance as the mutation sites.

[0088] (6) crRNA design

[0089] According to the results of step (5), four crRNAs (SEQ ID NO.44 - 47) were designed. Using the As-RPA-CRISPER / Cas12a fluorescence reaction system in step (3), simultaneously detect the A2143G nucleic acid target and the WT nucleic acid target. The results are as Figure 2 shown.

[0090] Figure 2 It shows that crRNA(8,9)-20(SEQ ID NO.47) has a good amplification effect, and the difference between A2143G and WTΔF is obvious.

[0091] Example 2

[0092] In Example 2 of the present invention, the effects of different conditions on the detection results were detected, and the specific steps are as follows:

[0093] (1) As-RPA reaction time

[0094] Using the 23S rRNA As-RPA primer set (SEQ ID NO.48-49) designed in Example 1 and crRNA (SEQ ID NO.47) for reaction. Dilute the forward primer (23S-RPA-F) to 50 μmol / L and the reverse primer (23S-RPA-R) to 0.5 μmol / L. Mix 2 μL each of the forward primer and the reverse primer, with the final concentration of 23S-RPA-F being 2 μM and 23S-RPA-R being 20 nM, and 30 μL of the PEG reaction solution. After mixing evenly, dispense into 2 tubes, add 5 μL each of different target A2143G mutant and WT type, and 2 μL of the magnesium acetate reaction solution on the tube cap. Cover the tube cap and invert to mix evenly. After putting the reaction system into the PCR amplifier, keep the temperature constant at 35 °C, set 30 s as a cycle, and set 20 cycles, 30 cycles, 40 cycles, 50 cycles, and 60 cycles respectively. After the reaction is completed, raise the temperature to 85 °C and inactivate the enzyme for 5 min. Each optimized condition is tested with A2143G mutant and WT type targets. Add the reaction product to the CRISPER / Cas12a system, repeat each reaction 3 times, and read the fluorescence on the machine. The results are as Figure 3 shown.

[0095] Figure 3 It shows that the As-RPA reaction for 15 min has a good amplification effect, and the difference between 2143 and wtΔF is the most obvious.

[0096] (2) As-RPA reaction Mg 2+ concentration

[0097] Using the 23S rRNA As-RPA primer set (SEQ ID NO.48-49) designed in Example 1 and crRNA (SEQ ID NO.47), carry out the reaction using the As-RPA-CRISPER / Cas12a fluorescence reaction system in step (3) of Example 1. The reaction time is 15 min. For the magnesium acetate reaction solution (280 mM Mg 2+) Add 1 μL, 1.5 μL, 2 μL, 2.5 μL, and 3 μL to the tube caps respectively, and perform the A2143G mutant and WT targets for each optimization condition. Add the reaction products to the CRISPER / Cas12a system, repeat each reaction 3 times, and read the fluorescence on the machine.

[0098] The results are as Figure 4 shown. The As-RPA reaction with Mg 2+ (280 mM) has a better amplification effect at 2 μL, and the gap between 2143 and wtΔF is the most obvious.

[0099] (3) As-RPA reaction temperature

[0100] Using the 23S rRNA As-RPA primer set (SEQ ID NO.48 - 49) designed in Example 1 and crRNA (SEQ ID NO.47), perform the reaction in the As-RPA-CRISPER / Cas12a fluorescence reaction system of step (3) in Example 1. The reaction time is 15 min. Add 2 μL of the magnesium acetate reaction solution (280 mM Mg 2+ ) to the tube cap, put the reaction system into a PCR instrument, set different reaction temperatures of 35 °C, 37 °C, 40 °C, and 42 °C, and perform the A2143G mutant and WT targets for each optimization condition. Add the reaction products to the CRISPER / Cas12a system, repeat each reaction 3 times, and read the fluorescence on the machine.

[0101] The results are as Figure 5 shown. The As-RPA reaction has a better amplification effect at 35 °C.

[0102] (4) Optimization of the As-RPA reaction primer concentration ratio

[0103] Using the 23S rRNA As-RPA primer set (SEQ ID NO.48 - 49) designed in Example 1, the reaction system of Example 3, and the crRNA (SEQ ID NO.47) designed in Example 6 to perform the reaction. The reaction time is 15 min. Add 2 μL of the magnesium acetate reaction solution (280 mM Mg 2+ ) to the tube cap, the reaction temperature is 35 °C, add 3 μL, 2 μL, 1 μL, and 0.5 μL of 23S-RPA-F (50 μmol / L) respectively, and always add 1 μL of 23S-RPA-R (0.5 μmol / L). Perform the A2143G mutant and WT targets for each optimization condition. Add the reaction products to the CRISPER / Cas12a system, repeat each reaction 3 times, and read the fluorescence on the machine.

[0104] The results are as Figure 6As shown, when the primer concentration is 100:1, the amplification effect is better, and the gap between 2143 and wtΔF is the most obvious.

[0105] (5) Optimization of Cas protein concentration in the CRISPER / Cas12a reaction system

[0106] Using the 23S rRNA As-RPA primer set (SEQ ID NO.48 - 49) designed in Example 1 and crRNA (SEQ ID NO.47), perform the reaction using the As-RPA-CRISPER / Cas12a fluorescence reaction system in step (3) of Example 1. The reaction time is 15 min. Add 2 μL of the magnesium acetate reaction solution (280 mM Mg 2+ ) to the tube cap, the reaction temperature is 35 °C, add 1 μL of 23S-RPA-F (50 μmol / L), add 1 μL of 23S-RPA-R (0.5 μmol / L), and add 4 μL of A2143G mutant and WT targets. Add the reaction product to the CRISPER / Cas12a system, and add 1 μL, 1.5 μL, 2 μL, 2.5 μL of LbCas12a protein (1 μm) respectively. For each optimization condition, both A2143G mutant and WT targets are used, and each reaction is repeated 3 times, then read the fluorescence on the machine.

[0107] The results are as Figure 7 shown, and 1 μL is the optimal addition amount of LbCas12a protein.

[0108] (6) Optimization of the concentration ratio of LbCas12a protein to crRNA in the CRISPER / Cas12a reaction system

[0109] Using the 23S rRNA As-RPA primer set (SEQ ID NO.48 - 49) designed in Example 1 and crRNA (SEQ ID NO.47), perform the reaction using the As-RPA-CRISPER / Cas12a fluorescence reaction system in step (3) of Example 1. The reaction time is 15 min. Add 2 μL of the magnesium acetate reaction solution (280 mM Mg 2+) Add 2 μL to the tube cap, the reaction temperature is 35°C, add 1 μL of 23S-RPA-F (50 μmol / L), add 1 μL of 23S-RPA-R (0.5 μmol / L), and add 4 μL of A2143G mutant and WT targets. Add the reaction product to the CRISPER / Cas12a system, add 1.5 μL of Cas protein (1 μm), add 1 μL of crRNA (1 μm), add 1 μL of Cas protein (1 μm), and add 1 μL, 2 μL, and 2.5 μL of crRNA (1 μm) respectively. For each optimized condition, both A2143G mutant and WT targets are used, and each reaction is repeated 3 times, then read the fluorescence on the machine.

[0110] The results are as Figure 8 shown, the optimal concentration ratio of LbCas12a protein to crRNA is 1:2.

[0111] (7) Optimization of the concentration of ssReporter (FAM-BHQ) in the CRISPER / Cas12a reaction system

[0112] Using the 23S rRNA As-RPA primer set (SEQ ID NO.48 - 49) designed in Example 1 and crRNA (SEQ ID NO.47), carry out the reaction using the As-RPA-CRISPER / Cas12a fluorescence reaction system in step (3) of Example 1. The reaction time is 15 min, and add the magnesium acetate reaction solution (280 mM Mg 2+ ) Add 2 μL to the tube cap, the reaction temperature is 35°C, add 1 μL of 23S-RPA-F (50 μmol / L), add 1 μL of 23S-RPA-R (0.5 μmol / L), and add 5 μL of A2143G mutant and WT targets. Add the reaction product to the CRISPER / Cas12a system, add 1 μL of Cas protein (1 μm), add 2 μL of crRNA (1 μm), and add 0.3 μL, 0.5 μL, 0.8 μL, and 1.2 μL of ssReporter (FAM-BHQ) (10 μm) respectively. For each optimized condition, both A2143G mutant and WT targets are used, and each reaction is repeated 3 times, then read the fluorescence on the machine.

[0113] The results are as Figure 9 shown, the optimal addition amount of ssReporter (FAM-BHQ) (10 μm) is 0.5 μL.

[0114] Example 3

[0115] Example 3 of the present invention detected the effects of the 23S rRNA As-RPA primer set (SEQ ID NO. 48-49) and crRNA (SEQ ID NO. 47). The specific steps are as follows:

[0116] (1) According to the established As-RPA-CRISPER / Cas12a system, verify the feasibility of detecting three single-base mutations (A2142C, A2142G, A2143G) and one double-base mutation (A2142G / A2143G).

[0117] Using the 23S rRNA As-RPA primer set (SEQ ID NO. 48-49) designed in Example 1 and crRNA (SEQ ID NO. 47), perform the reaction using the As-RPA-CRISPER / Cas12a fluorescence reaction system in step (3) of Example 1. The reaction time is 15 min. Add 2 μL of magnesium acetate reaction solution (280 mM Mg 2+ ) to the tube cap, the reaction temperature is 35°C, add 1 μL of 23S-RPA-F (50 μmol / L), add 1 μL of 23S-RPA-R (0.5 μmol / L), add 5 μL of bacterial liquid to extract the A2142C, A2142G, A2143G, and A2142G / A2143G mutants, and simulate fecal specimens to extract the A2142G, A2143G, A2142G / A2143G mutants and the WT target. Add the reaction product to the CRISPER / Cas12a system, add 1 μL of Cas protein (1 μm), add 2 μL of crRNA (1 μm), add 0.5 μL of ssReporter (FAM-BHQ) (10 μm), repeat each reaction 3 times, and read the fluorescence on the machine.

[0118] The results are as Figure 10 shown. The established As-RPA-CRISPER / Cas12a system of the present invention can well detect three single-base mutations (A2142C, A2142G, A2143G) and one double-base mutation (A2142G / A2143G), and the fluorescence differences are all significantly different from the WT type.

[0119] (2) According to the established As-RPA-CRISPER / Cas12a system combined with a lateral flow test strip, verify the feasibility of detecting three single-base mutations (A2142C, A2142G, A2143G) and one double-base mutation (A2142G / A2143G). The specimen sources are cultured bacterial liquid and fecal simulated specimens.

[0120] The 23S rRNA As-RPA primer set (SEQ ID NO.48-49) designed in Example 1 and the crRNA (SEQ ID NO.47) were used to react in the As-RPA-CRISPER / Cas12a lateral flow strip reaction system of step (4) in Example 1. The reaction time was 15 min. 2 μL of the magnesium acetate reaction solution (280 mM Mg 2+ ) was added to the tube cap. The reaction temperature was 35 °C. 1 μL of 23S-RPA-F (50 μmol / L) was added, 1 μL of 23S-RPA-R (0.5 μmol / L) was added, and 5 μL of bacterial liquid was added to extract the A2142C, A2142G, A2143G, and A2142G / A2143G mutants. A2142G, A2143G, A2142G / A2143G mutants and WT type targets were simulated and extracted from fecal specimens. The reaction product was added to the CRISPER / Cas12a system. 1 μL of Cas protein (1 μm) was added, 2 μL of crRNA (1 μm) was added, and 2 μL of ssReporter (FAM-Biotin) (1 μm) was added. Each reaction was repeated 3 times. 5 μL of the reaction product was aspirated and added to 45 μL of ddH2O, and after mixing, it was inserted into the test strip.

[0121] The results are as Figure 11 shown. The As-RPA-CRISPER / Cas12a system established by the present invention combined with the test strip can well detect three single-base mutations (A2142C, A2142G, A2143G) and one double-base mutation (A2142G / A2143G). Among them, obvious T lines can be seen for the mutants, while only C lines can be seen for the WT type.

[0122] Example 4

[0123] In Example 4 of the present invention, the accuracy and sensitivity of the 23S rRNA As-RPA primer set (SEQ ID NO.48-49) and the crRNA (SEQ ID NO.47) were detected. The specific steps are as follows:

[0124] (1) Specificity of the As-RPA-CRISPER / Cas12a fluorescence reaction system

[0125] The 23S rRNA As-RPA primer set (SEQ ID NO.48-49) designed in Example 1 and the crRNA (SEQ ID NO.47) were used to react in the As-RPA-CRISPER / Cas12a fluorescence reaction system of step (3) in Example 1. The reaction time was 15 min. 2 μL of the magnesium acetate reaction solution (280 mM Mg 2+) Add 2 μL to the tube cap, the reaction temperature is 35°C, add 1 μL of 23S-RPA-F (50 μmol / L), add 1 μL of 23S-RPA-R (0.5 μmol / L), add 5 μL of bacterial liquid to extract A2143G, including Bacillus subtilis, Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa, Klebsiella pneumoniae, Proteus vulgaris, and WT type targets. Add the reaction product to the CRISPER / Cas12a system, add 1 μL of Cas protein (1 μm), add 2 μL of crRNA (1 μm), add 0.5 μL of ssReporter (FAM-BHQ) (10 μm), repeat each reaction 3 times, and read the fluorescence curve on the machine,

[0126] The results are as Figure 12 shown. The As-RPA-CRISPER / Cas12a system established in the present invention has good specificity. Only the A2143G mutant has a fluorescence curve, and the fluorescence difference is significantly different from that of other strains.

[0127] (2) According to the established As-RPA-CRISPER / Cas12a system and combined with a lateral flow test strip to detect the sensitivity of A2143G.

[0128] Using the 23S rRNA As-RPA primer set (SEQ ID NO.48-49) designed in Example 1 and crRNA (SEQ ID NO.47), carry out the reaction using the As-RPA-CRISPER / Cas12a lateral flow test strip reaction system in step (4) of Example 1. The reaction time is 15 min. Add the magnesium acetate reaction solution (280 mM Mg 2+ ) Add 2 μL to the tube cap, the reaction temperature is 35°C, add 1 μL of 23S-RPA-F (50 μmol / L), add 1 μL of 23S-RPA-R (0.5 μmol / L), add 5 μL of the A2143G gene extracted from the bacterial liquid and the genomic DNA serially diluted 10-fold as targets. Add the amplified product to the CRISPER / Cas12a system, add 1 μL of Cas protein (1 μm), add 2 μL of crRNA (1 μm), add 0.5 μL of ssReporter (FAM-BHQ) (10 μm) or 2 μL of ssReporter (FAM-Biotin) (1 μm) respectively. Repeat each reaction 3 times and read the fluorescence curve on the machine. Add 5 μL of the test strip reaction product, mix it with 45 μL of ddH2O and insert the test strip.

[0129] The results are as Figure 13 shown. The sensitivity of the As-RPA-CRISPER / Cas12a system established in the present invention to detect A2143G is 10 -2 ng / μL, and the sensitivity of the test strip method to detect A2143G is 10-2 ng / μL.

[0130] (3) Detection of A2142C sensitivity

[0131] According to the established As-RPA-CRISPER / Cas12a system and combined with a lateral flow test strip to detect the sensitivity of A2142C.

[0132] Using the 23S rRNA As-RPA primer set (SEQ ID NO.48-49) designed in Example 1 and crRNA (SEQ ID NO.47), carry out the reaction using the As-RPA-CRISPER / Cas12a lateral flow test strip reaction system in step (4) of Example 1. The reaction time is 15 min. Add 2 μL of the magnesium acetate reaction solution (280 mM Mg 2+ 2+) to the tube cap, the reaction temperature is 35 °C, add 1 μL of 23S-RPA-F (50 μmol / L), add 1 μL of 23S-RPA-R (0.5 μmol / L), add 5 μL of the bacterial liquid to extract A2142C, and continuously dilute the extracted bacterial liquid 10-fold as the target. Add the amplified product to the CRISPER / Cas12a system, add 1 μL of Cas protein (1 μm), add 2 μL of crRNA (1 μm), and add 0.5 μL of ssReporter (FAM-BHQ) (10 μm) or 2 μL of ssReporter (FAM-Biotin) (1 μm) respectively. Each reaction is repeated 3 times, read the fluorescence curve on the machine, add 5 μL of the test strip reaction product, mix it with 45 μL of ddH2O and then insert the test strip.

[0133] The results are as Figure 14 shown. The sensitivity of the As-RPA-CRISPER / Cas12a system established by the present invention for detecting A2142C is 2.49×10 -3 ng / μL, and the sensitivity of the test strip method for detecting A2142C is 2.49×10 -2 ng / μL.

[0134] (3) A2142G sensitivity

[0135] According to the established As-RPA-CRISPER / Cas12a system and combined with a lateral flow test strip to detect the sensitivity of A2142G.

[0136] Using the 23S rRNA As-RPA primer set (SEQ ID NO. 48-49) designed in Example 1 and crRNA (SEQ ID NO. 47), perform the reaction using the As-RPA-CRISPER / Cas12a lateral flow test strip reaction system in step (4) of Example 1. The reaction time is 15 min. Add 2 μL of the magnesium acetate reaction solution (280 mM Mg 2+ 2) to the tube cap. The reaction temperature is 35°C. Add 1 μL of 23S-RPA-F (50 μmol / L), 1 μL of 23S-RPA-R (0.5 μmol / L), add 5 μL of the bacterial liquid extract A2142G, and use the extracted bacterial liquid diluted 10-fold continuously as the target. Add the amplified product to the CRISPER / Cas12a system, add 1 μL of Cas protein (1 μm), 2 μL of crRNA (1 μm), add 0.5 μL of ssReporter (FAM-BHQ) (10 μm) or 2 μL of ssReporter (FAM-Biotin) (1 μm) respectively. Repeat each reaction 3 times, read the fluorescence curve on the machine, add 5 μL of the test strip reaction product, mix well with 45 μL of ddH2O and then insert the test strip.

[0137] The results are as Figure 15 shown. The sensitivity of the As-RPA-CRISPER / Cas12a system established by the present invention for detecting A2142G is 2.58×10 -3 ng / μL, and the sensitivity of the test strip method for detecting A2142G is 2.58×10 -2 ng / μL.

[0138] (4) Sensitivity of A2142G / A2143G

[0139] According to the established As-RPA-CRISPER / Cas12a system and combined with the lateral flow test strip to detect the sensitivity of A2142G / A2143G.

[0140] Using the 23S rRNA As-RPA primer set (SEQ ID NO. 48-49) designed in Example 1 and crRNA (SEQ ID NO. 47), perform the reaction using the As-RPA-CRISPER / Cas12a lateral flow test strip reaction system in step (4) of Example 1. The reaction time is 15 min. Add 2 μL of the magnesium acetate reaction solution (280 mM Mg 2+) Add 2 μL to the tube cap, with the reaction temperature at 35°C. Add 1 μL of 23S-RPA-F (50 μmol / L) and 1 μL of 23S-RPA-R (0.5 μmol / L). Add 5 μL of bacterial liquid to extract A2142G / A2143G, and serially dilute the extracted bacterial liquid 10-fold as the target. Add the amplified product to the CRISPER / Cas12a system, add 1 μL of Cas protein (1 μm) and 2 μL of crRNA (1 μm), and add 0.5 μL of ssReporter (FAM-BHQ) (10 μm) or 2 μL of ssReporter (FAM-Biotion) (1 μm) respectively. Repeat each reaction 3 times, read the fluorescence curve on the machine, add 5 μL of the test strip reaction product, mix it with 45 μL of ddH2O and then insert the test strip.

[0141] The results are as Figure 16 shown. The sensitivity of the As-RPA-CRISPER / Cas12a system established by the present invention for detecting A2142G / A2143G is 2.39×10 -3 ng / μL, and the sensitivity of the test strip method for detecting A2142G / A2143G is 2.39×10 -3 ng / μL.

[0142] Example 5

[0143] In Example 5 of the present invention, clinical gastric juice specimens were detected, and the specific steps are as follows:

[0144] Take 27 gastric juice specimens (from Jiangning Traditional Chinese Medicine Hospital, Nanjing), treat them with 0.4% Trtion, and extract them using the Tianlong magnetic bead method kit. First, use PCR amplification to read and find 23S rRNA positive samples, and then detect the positive samples using the established As-RPA-CRISPER / Cas12a system. Read the results using the amplification curve. Samples with obvious amplification curves and relatively high fluorescence differences are positive.

[0145] The results are as Figure 17 shown. Among the 27 gastric juice samples, through the PCR results of 23S rRNA, 9 positive samples were detected, which are samples No. 5, 6, 17, 21, 22, 24, 25, 26, and 27 respectively. The As-RPA-CRISPER / Cas12a system established by the present invention detected 3 mutations in the above 9 23S rRNA positives, which are samples No. 6, 21, and 24 respectively.

[0146] Send the above PCR products and PCR primers to Sangon for sequencing. The sequencing results of the clinical gastric juice PCR products are as Figure 18As shown, the sequencing results of Sangon Biotech showed that No. 6, No. 21, and No. 24 had the A2143G mutation, and the rest of the strains were all WT without mutation. This result was consistent with the detection results of the As-RPA-CRISPER / Cas12a system established in the present invention.

[0147] The experimental results of the above examples showed that the As-RPA-CRISPER / Cas12a system established in the present invention could detect Helicobacter pylori clarithromycin resistance within 1 h under isothermal conditions, and only used a set of primers and one crRNA to achieve multiplex simultaneous detection of four mutation types, namely three single-base mutations (A2142C, A2142G, A2143G) and one double-base mutation (A2142G / A2143G) of 23S rRNA. This combined detection greatly increased the detection rate, and the results could be observed with the naked eye within 1 h, laying a foundation for POCT detection. The present invention had significant advantages in terms of sensitivity, specificity, detection speed, multiplex detection ability, visual result interpretation, and applicability, providing an efficient and convenient solution for the detection of Helicobacter pylori clarithromycin resistance, and having important clinical application value and popularization prospects.

[0148] The above are only the preferred embodiments of the present invention. It should be pointed out that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and refinements can still be made, and these improvements and refinements should also be regarded as the protection scope of the present invention.

Claims

1. A composition capable of detecting Helicobacter pylori clarithromycin resistance gene mutations, characterized in that, The composition capable of detecting Helicobacter pylori clarithromycin resistance gene mutations includes the As-RPA primer pair shown in SEQ ID NO. 48-49 and the crRNA shown in SEQ ID NO.

47.

2. The composition for detecting Helicobacter pylori clarithromycin resistance gene mutation according to claim 1, characterized in that, The Helicobacter pylori clarithromycin resistance gene mutations include three single-base mutations of A2142C, A2142G, and A2143G in 23S rRNA and one double-base mutation of A2142G / A2143G.

3. Use of the composition capable of detecting Helicobacter pylori clarithromycin resistance gene mutations according to claim 1 or 2 in the preparation of a product for detecting Helicobacter pylori clarithromycin resistance gene mutations.

4. A product for detecting Helicobacter pylori clarithromycin resistance gene mutations, characterized in that The product for detecting Helicobacter pylori clarithromycin resistance gene mutations includes the As-RPA primer pair and crRNA according to claim 1.

5. A method for detecting Helicobacter pylori clarithromycin resistance gene mutations, characterized in that, It includes the following steps: (1) Extract the DNA of the sample to be tested for As-RPA amplification. After the amplification is completed, heat to inactivate the enzyme to obtain the As-RPA amplification product; (2) After mixing the As-RPA amplification product with crRNA, LbCAS12a protein, and the probe, determine the mutation type through fluorescence signal changes or test strip color development.

6. The Helicobacter pylori clarithromycin resistance gene mutation detection method according to claim 5, characterized in that, In step (1), the amplification system for As-RPA amplification includes the As-RPA primer pair, RPA dry powder ball, PEG reaction solution, magnesium acetate reaction solution, and the DNA of the sample to be tested. The total reaction system is 25 μL; the amplification conditions are 35-42 °C for 10-20 min.

7. The Helicobacter pylori clarithromycin resistance gene mutation detection method according to claim 6, wherein In step (1), the As-RPA primer pair is 23S-RPA-F shown in SEQ ID NO.48 and 23S-RPA-R shown in SEQ ID NO.49; the concentration ratio of 23S-RPA-F to 23S-RPA-R is 50-300:1, and the Mg 2+ concentration in the As-RPA amplification system is 11.2-33.6 mM.

8. The Helicobacter pylori clarithromycin resistance gene mutation detection method according to claim 5, wherein, In step (2), the concentration ratio of LbCas12a protein to crRNA is 1-1.5:1-2.5, and the probe includes a FAM-BHQ probe for detecting fluorescence signal changes or a FAM-Biotion probe for determining test strip color development.

9. Use of the method for detecting Helicobacter pylori clarithromycin resistance gene mutations according to any one of claims 5-8 in the detection of Helicobacter pylori clarithromycin resistance gene mutations.

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