Method for rapidly detecting clarithromycin resistance of helicobacter pylori and its detection kit

Abstract

The application discloses a method for rapidly detecting drug resistance of helicobacter pylori to clarithromycin and a detection kit thereof. The specific primer for detecting the 23S rRNA gene mutation gene of H. pylori resistant to clarithromycin comprises 23S-4F: 5'-TTGT AGTGGAGGTGAAAATTCCTC-3' and 23S-4R: 5'-ACCTTTACTACAACTTAGCACTGCT-3'. The detection scheme has stronger anti-interference ability, and even if a base mutation occurs in the amplification range of 23S rRNA gene 2099-2179 sites of a to-be-detected strain except for the 2143 site, the application can still stably detect the A2143G mutation, and the traditional probe method cannot detect the A2143G mutation in the strain.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of medical detection, and particularly relates to a method for rapidly detecting the resistance of Helicobacter pylori to clarithromycin and a detection kit thereof. BACKGROUND

[0002] Helicobacter pylori (H.pylori) is a clear carcinogenic microorganism, and once infected, the incidence of gastric cancer in patients is significantly increased. Medical institutions all over the world have pointed out that once H.pylori infection is detected, the patient should receive antibiotic eradication therapy. Clarithromycin is currently the first-line antibiotic for treating H.pylori infection. However, the global H.pylori is currently facing a serious drug resistance problem, especially the resistance rate to clarithromycin is increasing year by year, therefore, it is necessary for H.pylori infected patients to perform antibiotic susceptibility test at the time of diagnosis, and to select drugs according to the drug sensitivity test results.

[0003] The current detection methods for clarithromycin resistance of H.pylori include drug sensitivity detection, gene sequencing, real-time fluorescent PCR method using TaqMan probe, etc. Drug sensitivity detection is the gold standard method for evaluating the resistance of H.pylori to clarithromycin. This method is a detection method for judging the drug resistance of the strain by observing the change of the amount of microorganisms during the culture of H.pylori under antibiotic pressure; gene sequencing is to amplify the target gene fragment of the H.pylori to be detected by designing different sequencing primers, and to obtain the target gene sequence by Sanger sequencing, and then to judge the consistency of the strain to be tested with the known drug resistance gene mutation of H.pylori, so as to judge the drug resistance of the strain; and the real-time fluorescent PCR method using TaqMan probe uses the detection platform of fluorescent quantitative PCR, and judges whether the strain has drug resistance mutation by detecting the presence or absence of fluorescence signal of the in-target probe designed to detect the gene mutation site during PCR reaction, and then infers the resistance of the strain to antibiotics.

[0004] Although the drug sensitivity test is the current gold standard for H. pylori drug resistance detection, in actual operation, due to H. pylori is a microaerophilic difficult-to-culture microorganism, isolation and culture need special culture medium and culture equipment, experimental operation is difficult, high cost and experimental period is long up to 7-14 days, these conditions all seriously limit the gold standard method in the clinical application. The gene sequencing method although obviously improves the detection difficulty, shortens the detection time to 1 day, but due to the acquisition of gene sequence still highly depends on high-end instruments such as Sanger sequencer, capillary electrophoresis or high-throughput sequencer, therefore detection needs professional instrument equipment support, operation process is complex and cost is high. In order to simplify the detection difficulty, researchers developed the detection means for real-time observation of gene mutation such as real-time fluorescent PCR detection scheme based on TaqMan probe: commercially available The reagent kit detects 23S rRNA gene A2142G / C (2142 site A mutation to G or C) and A2143G point mutation by designing specific probe, evaluation found that the kit in gastric biopsy tissue and fecal sample detects H. pylori resistance to clarithromycin effect consistency reaches 95% with E-test. At present, the effectiveness of detection kit based on drug resistance gene mutation has been widely any in the industry, and in the 'Sixth National Helicobacter Pylori Infection Treatment Consensus Report', the Chinese Medical Association also lists gene detection as one of the recommended methods for H. pylori drug resistance detection.

[0005] At present, the rapid detection of H. pylori drug resistance gene point mutation is the focus of the development of H. pylori drug resistance detection. With the development of molecular biology technology, the detection of H. pylori drug resistance gene mutation has become a research hotspot in recent years. Similarly, Chinese invention patent CN111850154A also discloses a method for detecting H. pylori clarithromycin-resistant mutation 23S rRNA gene A2143G mutation by designing fluorescent probes. However, the fluorescent probes used in these detection schemes are currently expensive to synthesize (about 2500 yuan for 10 nmol / L), and each detection requires 70-100 yuan per reaction on average, and the specificity of short sequence fluorescent probes is lower, while the detection of long sequence probes may be affected by nonsense mutations near the detection target. These limitations also limit the large-scale application of these kits in clinical practice. In recent years, researchers have developed rapid detection schemes for gene mutations based on saturated fluorescent dye chimeric methods such as high-resolution melting analysis (HRM): the thermal stability of double-stranded DNA is affected by its length and base composition, and sequence changes will cause changes in the melting behavior of dsDNA during heating. If a gene point mutation occurs in the target gene to be detected, the fluorescent signal produced during the RT-PCR melting process will change, and by observing the difference in melting curve melting temperature (Tm), the classification of the target fragment mutation gene point can be performed. Compared with the probe method, HRM has lower detection cost, easy result reading, and better application prospect. At present, Chinese researchers have also developed a detection scheme for detecting tetracycline resistance of strains using HRM (CN202210282373.7), but there is no similar detection scheme for H. pylori first-line treatment drug clarithromycin. The present application develops a method for detecting H. pylori resistance to clarithromycin using HRM by designing a pair of specific primers and optimizing the experimental scheme, which has the advantages of low cost, simple operation, good specificity, high sensitivity, etc. and has a huge application value for clinical diagnosis of H. pylori resistance to clarithromycin. SUMMARY

[0006] In order to overcome the shortcomings of the prior art, the present application provides a rapid, accurate, efficient and low-cost H. pylori clarithromycin resistance detection method.

[0007] The present application solves the limitations of the prior art relying on fluorescent probes, greatly reducing the cost of detection. The present application uses saturated fluorescent dyes to bind to target sites during qPCR amplification, and distinguishes H. pylori sensitive and resistant to clarithromycin according to the differences in the dissolution curve formed by different bases, thereby realizing the determination of strain resistance. The present application is based on the H. pylori strain resource library established by the invention team in the early stage to obtain the gene mutation site most related to clarithromycin resistance, and then a detection scheme is obtained through primer design, screening verification and reaction system optimization. The scheme can determine whether the strain is resistant to clarithromycin by reading the melting temperature (Tm) after detection. The present application can realize clarithromycin resistance detection of multiple H. pylori-containing samples within 2 hours, and provides a fast, efficient and low-cost detection scheme for H. pylori resistance detection in clinical work.

[0008] The first object of the present application is to provide specific primers 23S-4F: 5'-TTGTAGTGGAGGTGAAAATTCCTC-3' and 23S-4R: 5'-ACCTTTACTACAACTTAGCACTGCT-3' for detecting 23S rRNA gene mutation of H. pylori resistant to clarithromycin. The use of the primers in combination with saturated fluorescent dyes can realize faster and more accurate detection of the ability of H. pylori to resist clarithromycin.

[0009] The second object of the present application is to provide a method for rapidly detecting the resistance of H. pylori to clarithromycin, which uses the above-mentioned specific primers to perform PCR amplification on H. pylori samples, and performs melting curve analysis after amplification.

[0010] Preferably, the PCR amplification has a PCR amplification program of: 95℃ pre-denaturation for 2min; 95℃ for 10sec, 55℃-65℃ for 20sec, 72℃ for 30sec alternating cycles for 40 times; after amplification, high-resolution melting curve program is added: 95℃ for 60sec, 40℃ for 60sec, 65℃ for 1sec, 97℃ for 1sec, and when performing melting curve analysis, set 0.02-0.1℃ to collect fluorescence once.

[0011] The third object of the present application is to provide a method for rapidly determining the resistance of H. pylori to clarithromycin, characterized in that the above-mentioned primers are used for fluorescent quantitative PCR detection, when the detected Tm value is 79.43±0.2℃, the base at position 2143 is A, and the detected strain is sensitive to clarithromycin; when the detected Tm value is 80.11±0.2℃, the base at position 2143 is G, and the detected strain is resistant to clarithromycin.

[0012] The fourth object of the present application is to provide a rapid detection kit for detecting the drug resistance of Helicobacter pylori to clarithromycin, characterized in that the kit comprises the specific primers.

[0013] Compared with the existing detection of drug resistance of H. pylori, the present application has the following advantages:

[0014] (1) The difference is obvious after the differential mapping algorithm is used to process the melting curve;

[0015] (2) The wild type A base of the 23S rRNA gene can be accurately distinguished from the mutant G base;

[0016] (3) The detection sensitivity is high, and the DNA concentration range in the detection system can be as low as 0.005 ng / ul from 50 ng / ul;

[0017] (4) The detection specificity is high, and the interference caused by the rest of the stomach microorganisms in the sample can be excluded;

[0018] (5) The detection time is short, and the detection time is only 2 hours;

[0019] (6) Compared with the fluorescent probe detection method which needs 70-100 yuan per reaction, the high-resolution melting curve method detection is low in price, and the average cost of each reaction is only 8 yuan.

[0020] (7) Compared with the existing commercial kit designed by the fluorescent probe method, the anti-interference ability of the present application is stronger, that is, even if there is a base mutation in the 23S rRNA gene 2099-2179 site amplification range except for the 2143 site, the present application can still stably detect the A2143G mutation, while the traditional probe method cannot detect the A2143G mutation in such strains. BRIEF DESCRIPTION OF DRAWINGS

[0021] Figure 1 Primer optimization and screening experiment for the embodiment of the present application

[0022] Figure 2 Amplification reaction condition optimization and screening for the embodiment of the present application

[0023] Figure 3 Verification of the detection limit result of the high-resolution melting curve method for the embodiment of the present application

[0024] Figure 4 Verification of the primer specificity result of the high-resolution melting curve method for the embodiment of the present application

[0025] Figure 5 A is the melting curve map of the high-resolution melting curve method for the 23S rRNA A2143G site for drug resistance molecular typing in the embodiment of the present application, Figure 5B is the melting curve profile of 35 clinical isolates of H. pylori using high resolution melting curve method for detection of 23S rRNA A2143G site for molecular typing of drug resistance by the Difference Plot algorithm of Roche LightCycler 480 PCR instrument 480 PCR instrument using the Difference Plot algorithm of Roche LightCycler 480 PCR instrument for molecular typing of drug resistance after the melting curve profile is obtained

[0026] Figure 6 A is the melting curve profile of 35 clinical isolates of H. pylori using high resolution melting curve method for detection of 23S rRNA A2143G site for molecular typing of drug resistance by the Difference Plot algorithm of Roche LightCycler 480 PCR instrument Figure 6 B is the melting curve profile of 35 clinical isolates of H. pylori using high resolution melting curve method for detection of 23S rRNA A2143G site for molecular typing of drug resistance by the Difference Plot algorithm of Roche LightCycler 480 PCR instrument 480 PCR instrument using the Difference Plot algorithm of Roche LightCycler 480 PCR instrument for molecular typing of drug resistance after the melting curve profile is obtained. DETAILED DESCRIPTION

[0027] To make the technical solutions of the present application clearer, the following specific examples are provided to further illustrate the present application.

[0028] The culture medium involved in the following examples is as follows:

[0029] Preparation of antibiotic suspension: vancomycin 10 mg / mL, amphotericin B 5 mg / mL, trimethoprim 20 mg / mL, cefsulfidine 10 mg / mL, and the above antibiotics are dissolved in sterilized water.

[0030] H. pylori isolation medium (g / L): yeast extract powder 2.02 g / L, tryptone 10.52 g / L, glucose 1.56 g / L, sodium bisulfite 0.10 g / L, soluble starch 10.0 g / L, sodium chloride 5.0 g / L, agar 13.5 g / L, the above ingredients are dissolved in sterilized water according to their contents, sterilized at 121°C for 20 min, then 100 mL / L of calf serum and 10 mL / L of antibiotic suspension are added, and mixed uniformly to prepare a plate.

[0031] H. pylori proliferation medium (g / L): yeast extract powder 2.02 g / L, tryptone 10.52 g / L, glucose 1.56 g / L, sodium bisulfite 0.10 g / L, soluble starch 10.0 g / L, sodium chloride 5.0 g / L, fetal bovine serum 70 ml / L.

[0032] Example 1 Isolation, identification, drug resistance detection and whole genome sequencing of H. pylori

[0033] (1) Isolation and culture of H. pylori

[0034] ① Isolation of H. pylori: Under aseptic environment, gastric mucosa tissue of patients with chronic gastritis was taken and coated on H. pylori isolation medium, and then placed in a microaerobic environment (5% O2, 10% CO2, 85% N2) at 37°C for 5-7 days. Morphologically typical colonies on the plate were picked and inoculated on H. pylori isolation medium for streaking and purification, and the purified H. pylori isolates were obtained after two times of purification.

[0035] ② Culture of H. pylori: Single colonies were picked from the plate containing H. pylori and inoculated in H. pylori proliferation medium, and cultured in a microaerobic environment at 37°C for 72-96 hours to obtain H. pylori isolate liquid.

[0036] (2) Identification of H. pylori

[0037] The isolated strains were identified by a method based on 16S rRNA gene sequence: the DNA of H. pylori was extracted by a bacterial DNA extraction kit (Ringkai Bio, Guangdong, China), and then amplified by 2x PCR mix (Ringkai Bio). The PCR amplification primers were 16S rRNA gene universal primers, the upstream primer sequence was 27F: 5'-AGA GTT TGA TCC TGG CTC AG-3', and the downstream primer sequence was 1492R: 5'-CTA CGG CTA CCT TGT TAC G A-3'. The PCR reaction conditions were as follows: pre-denaturation at 95°C for 5 min; 95°C for 30 s, 56°C for 30 s, 72°C for 1 min 30 s for 35 cycles, and 72°C for 10 min for annealing extension. The PCR product was recovered by gel cutting, and then subjected to Sanger sequencing. The obtained 16S rRNA sequence was compared with the NCBI database (https: / / blast.ncbi.nlm.nih.gov), and the results showed that it had the highest homology with 16S rRNA, indicating that the isolated strain was H. pylori.

[0038] (3) Drug resistance detection of H. pylori

[0039] Agar dilution method was used for antibiotic susceptibility testing of H. pylori, and H. pylori ATCC 43504 was used as a quality control strain. The method and standard were determined according to the method and standard developed by the American Clinical and Laboratory Standards Institute (The Clinical & Laboratory Standards Institute, CLSI).

[0040] The powder of clarithromycin was dissolved in the corresponding solute according to the CLSI recommendation, configured into an antibiotic stock solution, and then added to the H. pylori isolated culture medium according to the required detection concentration of drug sensitivity, to prepare H. pylori drug sensitivity detection plates containing the corresponding concentration of antibiotics. The concentration of H. pylori bacterial suspension was adjusted to 1×10 7 cfu / mL, and then inoculated into the detection plate containing the corresponding concentration of antibiotics, and cultured at 37°C in a micro-aerobic environment for 72 h. The presence or absence of H. pylori colony formation on the detection plate containing the corresponding concentration of antibiotics was observed. The lowest concentration of drug in the drug plate that could not form a colony was the minimum inhibitory concentration (MIC) of the corresponding H. pylori to the antibiotic. Each drug concentration was operated in parallel for 3 times, and the average value of the minimum inhibitory concentration was obtained. The evaluation criteria for H. pylori resistance to clarithromycin (Resistant, R) was MIC≥0.25mg / L.

[0041] (4) Whole genome sequencing of H. pylori

[0042] The DNA of H. pylori was extracted by the above method, and the AMT Rapid DNA-Seq Kit for Illumina (CISTRO, China) was used to build a library, and the High Output v2.5 kit (Illumina, USA) kit was used for sequencing. The data was quality controlled by Trimmomatic (v0.39) software, and then assembled by SPAdes (v3.13.1) software. The assembled H. pylori genome was evaluated by Quast (v5.0.2) software for assembly quality control. For the qualified genome, Prokka (v1.13) software was used for annotation. After annotation, the 23S rRNA gene sequence of each strain was obtained, and the sequence alignment analysis showed that the high correlation drug resistance mutation site 2143 site from A to G (A2143G) was closely related to clarithromycin resistance (p<0.001). Tshibangu et al. pointed out that this mutation would cause the conformational change of the V domain of 23S rRNA molecule in the 50S subunit of ribosome, leading to the loose binding of macrolide antibiotics such as clarithromycin and peptidyl transferase ring, thereby leading to drug failure (doi:10.1038 / s41575-021-00449-x).

[0043] (5) Analysis of common gene sites of H. pylori resistance to clarithromycin

[0044] The inventors' team previously isolated 35 strains of H. pylori from a hospital in Guangzhou. Through drug sensitivity experiment, 15 strains were resistant to clarithromycin, and 20 strains were sensitive to clarithromycin. Genome analysis showed that the 23S rRNA A2143G of all H. pylori was the highest clarithromycin resistance related site. Among the 15 clarithromycin resistant strains, 11 strains had 23S rRNA A2143G mutation, and 4 strains had no mutation; 20 wild type sensitive strains had no 23S rRNA A2143G mutation.

[0045] Example 2 HRM primer design for clarithromycin-resistant H. pylori

[0046] The present application designs a pair of primers for HRM detection according to the position of 23S rRNA gene A2143G mutation of H. pylori. The length of the amplicon is a key factor affecting HRM analysis. Shorter PCR products can improve the resolution of HRM; but when the primer is too short, the specificity will be reduced and non-specific amplification will occur. Therefore, in the design of PCR primers, in addition to following the general principles, the product length is kept between 50-120, the SNP site is as close to the middle of the PCR product sequence as possible, the primer is required to have no dimer, the primer itself has no hairpin structure, and the length of the extended product is moderate, which can ensure the amplification efficiency and exclude the interference of dimer. Therefore, the primer of the present application can not only be used for detecting H. pylori strains, but also can be directly used for actual samples, such as gastric mucosa detection.

[0047] In the embodiment of the present application, 8 pairs of primers are designed according to the size gradient of the amplified fragments by Primer Premier 6 software, and the resolution difference caused by the size of the 50-120 bp amplicon is compared by melting temperature (Tm) (see Table 1). Figure 1 The primer sequence with the highest resolution is 23S-4, and the specific sequence is:

[0048] 23S-4F: 5'-TTGTAGTGGAGGTGAAAATTCCTC-3'

[0049] 23S-4R: 5'-ACCTTTACTACAACTTAGCACTGCT-3'

[0050] Table 1 HRM primer specific conditions

[0051]

[0052]

[0053] Example 3 HRM detection amplification condition optimization for clarithromycin-resistant H. pylori

[0054] Annealing temperature refers to the temperature parameter when the primer and the template are combined, the temperature when 50% of the primer and the complementary sequence exhibit a double-stranded DNA molecule. It is a more important factor affecting the specificity of PCR. In an ideal state, the annealing temperature is kept low enough to ensure that the primer is effectively annealed to the target sequence, and is kept high enough to reduce non-specific binding. In order to further explore the optimal amplification reaction condition, the present application selects the optimal reaction condition by designing 5 gradients of annealing temperature of 55, 58, 60, 63 and 65℃. The PCR amplification reaction of the present application adopts a 20μL system, which includes: 2×HRMAnalysis PreMix (With Dye), 0.6μL forward primer (concentration 10μmol / L) and 0.6μL reverse primer (concentration 10μmol / L), 1μL template DNA, and finally RNase-Free ddH2O is used to make the system constant volume at 20μL.

[0055] Table 2 HRM reaction program

[0056]

[0057] Table 3 HRM reaction system

[0058]

[0059] Experimental results Figure 2 A-E) show that the reaction annealing temperature from 55℃ to 65℃ can be used as the annealing temperature of the amplification reaction.

[0060] The PCR amplification program of the present application is: 95℃ pre-denaturation for 2min; 95℃ for 10sec, 60℃ for 20sec, 72℃ for 30sec alternating cycle for 40 times; after the amplification is completed, the high-resolution melting curve program is added: 95℃ for 60sec, 40℃ for 60sec, 55℃-65℃ for 1sec, 97℃ for 1sec, and when the melting curve analysis is performed, set to collect fluorescence once at 0.02-0.10℃.

[0061] Example 4 HRM detection sensitivity evaluation of H. pylori clarithromycin-resistant

[0062] Take one portion of the DNA sample of the drug-resistant and sensitive strain, dilute the sample to the corresponding content (50ng / μL, 5ng / μL, 0.5ng / μL, 0.05ng / μL, 0.005ng / μL, 0.0005ng / μL) with ultrapure water, and detect the detection limit of the present application method by the PCR amplification system and program of Example 3 to verify the detection sensitivity.

[0063] The experimental results are as follows Figure 3As shown, the DNA in the drug-resistant and sensitive experimental groups can be detected in the range of 50 ng / μL to 0.005 ng / μL, and no amplification occurs at the concentration gradient of 0.0005 ng / μL, and the obtained melting curve has good differentiation, proving that the high-resolution melting curve method proposed in the application can detect DNA concentrations as low as 0.005 ng / μL in the system, and has a lower detection limit and high detection sensitivity.

[0064] Example 5 Evaluation of HRM detection specificity of H. pylori resistant to clarithromycin

[0065] To verify the HRM detection method and the specificity of the primers proposed in the application, the specificity of the system was verified by using the remaining gastric strains. Cross-reaction mainly tests whether other gastric microorganisms that may produce cross-reaction produce false positive results, so as to effectively reduce the probability of false positives in the clinical detection process. The present method tests 8 kinds of gastric microorganisms, and the specific information is shown in the following table.

[0066] Table 4 Tm of different gastric microorganisms in the HRM system of the application

[0067]

[0068] The specific primers used in the present method were used to detect H. pylori clarithromycin-resistant positive samples (isolates), negative samples (standard strain ATCC 43504) and the remaining 8 kinds of gastric microorganisms, and the results are as follows Figure 4 The melting curve temperature analysis of each sample shows that the detection results of each sample are normal, the H. pylori positive sample is detected as positive, the sensitive negative sample is detected as negative, and the remaining 8 kinds of gastric microorganisms are amplified by the specific primers of the application to produce different melting curves, which do not interfere with the detection results, indicating that the present method has normal detection results for each sample, has high analysis specificity, and has good applicability for the detection of clinical samples.

[0069] Example 6 Application of HRM detection of H. pylori resistant to clarithromycin

[0070] (1) Sample preparation

[0071] Gastric mucosa tissue samples to be detected were collected from a clinical unit, and the sample was extracted using a tissue sample microbial DNA extraction kit, and then HRM detection was performed according to the above scheme, and H. pylori ATCC 43504 standard strain DNA was prepared as a negative control.

[0072] (2) qPCR amplification detection

[0073] Put the reaction tube into Roche Amplification and detection were performed using a 480 PCR instrument, and the high-resolution melting curve detection method employed the primers and amplification reaction conditions selected in Examples 2 and 3 of this invention.

[0074] (3) Results Analysis and Interpretation

[0075] Using Roche The high-resolution melting module software of the 480 PCR instrument was used to analyze the results of each sample. The results of the clinical samples were used for diagnosis, and the peak values ​​of the melting curves were statistically analyzed. If the peak temperature of the melting curve of the tested strain was 79.64℃... Figure 5 A), then the tested strain is sensitive to clarithromycin; if the peak temperature of the melting curve of the tested strain is 80.12℃ ( Figure 5 B) indicates that the tested strain is resistant to clarithromycin.

[0076] Example 7: Evaluation of the actual effect and anti-interference ability of H. pylori clarithromycin-resistant HRM detection

[0077] In this embodiment of the invention, an optimized high-resolution melting curve detection system for H. pylori resistance to clarithromycin was used to detect 35 clinically isolated Helicobacter pylori strains, including 15 clarithromycin-resistant strains (11 of which had 23S rRNA A2143G mutations) and 20 clarithromycin-sensitive strains. The results showed that... Figure 6 In both A and B, 11 mutant strains had a Tm value of 79.66±0.1℃; when the Tm value was detected, it was 80.11±0.10℃, and genomic analysis indicated that the detection site was G. The Tm values ​​of 4 non-mutant drug-resistant strains and 20 wild-type sensitive strains were all 80.11±0.10℃, and genomic analysis indicated that the detection site was G. These results indicate that the detection effect is excellent.

[0078] Simultaneously, whole-genome sequencing was performed on the aforementioned 35 Helicobacter pylori strains. After annotation, the 23S rRNA gene sequence of each strain was obtained. Using the preferred primers from Example 2, the gene was amplified to obtain an 80bp amplicon fragment. This fragment corresponds to the base position range of positions 2099 to 2179 on the genome. Alignment analysis of the gene sequence within this range for each sample was performed using CLC Genomics Workbench software. The mutation status of the 23S rRNA gene at positions 2099 to 2179 in the 35 clinical H. pylori strains and the HRM detection results were statistically analyzed (Table 5).

[0079] Table 5. 23S rRNA mutation status and HRM detection of 35 clinical isolates of Helicobacter pylori.

[0080]

[0081]

[0082] In the published review, Alireza et al. evaluated the defects of probe method in detecting SNP mutations. If there are other mutations in the amplicon except the detection point, the sequence-specific probe will not be able to bind to the target sequence smoothly and then emit a fluorescence signal (doi: 10.1021 / acssensors.8b01604). The detection method based on HRM is not affected by mutations at other positions in the amplicon, and it does not need to design a series of allele-specific primers and probes to overcome these mutations. As shown in Table 5, it is a common phenomenon that other base mutations occur at positions 2099-2179 of the 23S rRNA gene, and for base mutations occurring in the PCR amplification range except for position 2143, the detection of the present application can still stably detect A2143G mutation through Tm change. In the detection of drug-resistant molecules based on the probe method, if other mutation sites in the probe detection range are mutated, the fluorescence signal in the probe cannot be successfully quenched, affecting the detection accuracy. Therefore, it can be known that the detection scheme of the present application has stronger anti-interference ability.

Claims

1. A specific primer for detecting a mutation of 23S rRNA gene of H. pylori resistant to clarithromycin, characterized in that, Comprise: 23S-4F: 5'-TTGTAGTGGAGGTGAAAATTCCTC-3' and 23S-4R: 5'-ACCTTTACTACAACTTAGCACTGCT-3'.

2. A method for rapidly detecting the resistance of Helicobacter pylori to clarithromycin, characterized by, The PCR amplification is carried out on the H. pylori sample by using the specific primer pair of claim 1, and after the amplification, the melting curve analysis is carried out.

3. The method of claim 2, wherein, The PCR amplification procedure is as follows: 95℃ pre-denaturation for 2 min; 95℃ for 10 sec, 55-65℃ for 20 sec, 72℃ for 30 sec, alternating circulation for 40 times; after the amplification, the high-resolution melting curve procedure is added: 95℃ for 60 sec, 40℃ for 60 sec, 65℃ for 1 sec, 97℃ for 1 sec, and when the melting curve analysis is carried out, the setting is 0.02-0.1℃ collection of fluorescence once.

4. A method for rapidly determining the resistance of H. pylori to clarithromycin, characterized in that, When the detected Tm value is 79.43±0.2℃, the base at 2143 site is A, and the detected strain is sensitive to clarithromycin; when the detected Tm value is 80.11±0.2℃, the base at 2143 site is G, and the detected strain is resistant to clarithromycin.

5. A test kit for rapidly detecting the resistance of Helicobacter pylori to clarithromycin, characterized by comprising the polynucleotide of claim 1 or 2. The specific primer of claim 1 is contained.

Images