A primer-probe combination, kit and method for detecting amoxicillin resistance of Helicobacter pylori

Abstract

The present invention belongs to the field of biotechnology and molecular diagnosis, and discloses a primer-probe composition, a kit and a method for detecting amoxicillin resistance of Helicobacter pylori, wherein the primer-probe composition comprises: a first upstream primer, the sequence of which is any one of SEQ ID NO.1-SEQ ID NO.6; a second upstream primer, the sequence of which is any one of SEQ ID NO.7-SEQ ID NO.12; a third upstream primer, the sequence of which is any one of SEQ ID NO.13-SEQ ID NO.18; a fourth upstream primer, the sequence of which is any one of SEQ ID NO.19-SEQ ID NO.24; a downstream primer, the sequence of which is any one of SEQ ID NO.25-SEQ ID NO.28; a probe, the sequence of which is any one of SEQ ID NO.29 and SEQ ID NO.30, the 5' end of the probe being marked with an excitation fluorescence group, and the 3' end of the probe being marked with a quenching group. The primer-probe composition of the present invention, its kit and detection method thereof, have high sensitivity, strong specificity, good anti-interference ability and repeatability, and can also detect stool samples.

Description

Technical Field

[0001] The invention relates to the fields of biotechnology and molecular diagnosis, and in particular to a primer-probe combination, a kit and a method for detecting the drug resistance of Helicobacter pylori to amoxicillin. Background Art

[0002] Helicobacter pylori (Hp) is a spiral, slightly anaerobic, gram-negative bacillus that is very demanding on growth conditions. It is the most widespread pathogen in the world. About 50% of people in the world are infected with Helicobacter pylori, and 40%-70% of people in my country are infected with Helicobacter pylori. Helicobacter pylori mainly causes gastrointestinal digestive system related diseases, such as chronic gastritis, gastric ulcer, duodenal ulcer, gastric cancer, etc. At present, the treatment of Helicobacter pylori mainly adopts triple or quadruple therapy containing antibiotics. With the extensive use of antibiotics, Helicobacter pylori has developed antibiotic resistance, which leads to the fact that the eradication rate of Helicobacter pylori cannot reach a high level due to factors such as the selection of antibacterial drugs, the compliance of patients with medication, and the patient's own medication history. Therefore, the drug resistance detection of Helicobacter pylori is of great significance to the guidance of clinical medication.

[0003] At present, the methods used to detect drug resistance of Helicobacter pylori mainly include drug sensitivity test, first-generation sequencing, melting curve method, denaturing high-performance liquid chromatography based on PCR, and PCR-FISH method. Drug sensitivity test is to determine whether drug resistance is detected by culturing Hp and different antibiotics in vitro to determine their minimum inhibitory concentration; the general steps are: taking specimens containing pathogenic bacteria from the infected part of the patient, inoculating them on appropriate culture medium, and culturing them under certain conditions; at the same time, sticking paper pieces with a certain amount of various antibiotics on the surface of the culture medium (or using stainless steel rings with a certain amount of antibiotic solution inside), and observing the results after culturing for a certain period of time. Due to the different sensitivity of pathogenic bacteria to various antibiotics, "empty circles" of different sizes appear around the drug paper pieces to inhibit the growth of pathogens, which are called inhibition circles; the size of the inhibition circle is proportional to the sensitivity of the pathogens to various antibiotics; then, antibiotics are selected in a targeted manner according to the test results; this method is complicated and time-consuming, and it is not easy to culture Helicobacter pylori. The first-generation sequencing method has a long experimental cycle and a low detection rate for samples with low mutation frequencies, and can only detect mutations above 10%. The PCR-based denaturing high-performance liquid chromatography method is simple and fast, but it can only provide information on whether the sample has mutations. It needs to be combined with sequence analysis to obtain the specific mutation type. PCR-FISH cannot determine resistance to antibiotics other than clarithromycin.

[0004] All of the above methods require obtaining gastric mucosal tissue samples from patients, and sample collection requires the use of gastroscopes to clamp the gastric mucosal tissue from the infected area of ​​the patient, which is invasive to the patient and can easily cause trauma while causing discomfort to the patient. Therefore, it is necessary to develop a rapid, sensitive, convenient sampling, and low-cost detection system for Helicobacter pylori resistance detection to meet clinical needs. Summary of the invention

[0005] In view of the existing technical problems, the present invention provides a primer-probe combination, a kit and a method for detecting the resistance of Helicobacter pylori to amoxicillin. The primer-probe combination and the kit of the present invention have high detection sensitivity, strong specificity, good anti-interference ability and good repeatability.

[0006] To achieve the above object, the present invention provides the following technical solutions:

[0007] In one aspect, the present invention provides a primer-probe combination for detecting amoxicillin resistance of Helicobacter pylori, the primer-probe combination consisting of four upstream amplification primers for four mutation sites, one downstream amplification primer and one detection probe, specifically comprising: (1) a first upstream primer for amplifying a first target sequence containing a Ser414Arg mutation of a PBP1 A gene, the sequence of which is any one of SEQ ID NO.1-SEQ ID NO.6;

[0008] (2) a second upstream primer for amplifying a second target sequence comprising the Thr556Ser mutation of the PBP1 A gene, the sequence of which is any one of SEQ ID NO.7 to SEQ ID NO.12;

[0009] (3) a third upstream primer for amplifying a third target sequence comprising the Asn562Tyr mutation of the PBP1 A gene, the sequence of which is any one of SEQ ID NO.13 to SEQ ID NO.18;

[0010] (4) a fourth upstream primer for amplifying a fourth target sequence comprising the Thr593Ala mutation of the PBP1 A gene, the sequence of which is any one of SEQ ID NO.19 to SEQ ID NO.24;

[0011] (5) a downstream primer used to cooperate with any one of the upstream primers (1) to (4) to amplify a target sequence containing the corresponding mutation site of the PBP1 A gene, the sequence of which is any one of SEQ ID NO.25 to SEQ ID NO.28;

[0012] (6) A probe for detecting any amplicon obtained by amplifying the upstream primers (1) to (4) and the downstream primer (5), wherein the sequence is any one of SEQ ID NO. 29 and SEQ ID NO. 30, wherein the 5' end of the probe is labeled with an excitation fluorescent group, and the 3' end of the probe is labeled with a quenching group.

[0013] In some specific embodiments, in the primer probe composition: the first upstream primer sequence is shown as SEQ ID NO.4 or SEQ ID NO.5; the second upstream primer sequence is shown as SEQ ID NO.7 or SEQ ID NO.8; the third upstream primer sequence is shown as SEQ ID NO.13 or SEQ ID NO.14; and the fourth upstream primer sequence is shown as SEQ ID NO.19 or SEQID NO.20.

[0014] In some other preferred embodiments, the primer-probe combination is: (1) a first upstream primer, the sequence of which is shown in SEQ ID NO.4; (2) a second upstream primer, the sequence of which is shown in SEQ ID NO.8; (3) a third upstream primer, the sequence of which is shown in SEQ ID NO.13; (4) a fourth upstream primer, the sequence of which is shown in SEQ ID NO.20; (5) a downstream primer, the sequence of which is shown in SEQ ID NO.25; (6) a probe, the sequence of which is shown in SEQ ID NO.29, the 5' end of the probe being labeled with an excitation fluorescent group, and the 3' end of the probe being labeled with a quenching group. In some specific embodiments, the excitation fluorescent group is selected from FAM, VIC, Cy5, TET, HEX, JOE, and ROX; and the quenching group is selected from BHQ series, TAMRA, and DABCYL.

[0015] Furthermore, the primer-probe composition also includes primers and probes for internal standard genes: the upstream primer sequence of the internal standard gene is shown in SEQ ID NO.31, the downstream primer sequence of the internal standard gene is shown in SEQ ID NO.32, the probe sequence of the internal standard gene is shown in SEQ ID NO.33, the 5' end of the internal standard gene probe is labeled with an excitation fluorescent group, and the 3' end of the probe is labeled with a quenching group. In some specific embodiments, the excitation fluorescent group is selected from FAM, VIC, Cy5, TET, HEX, JOE, ROX, and is different from the excitation fluorescent group used in the probe of the drug-resistant mutation site; the quenching group is selected from BHQ series, TAMRA, DABCYL.

[0016] The second aspect of the present invention provides a use of the primer-probe combination described in the first aspect in the preparation of a reagent or a kit for detecting drug resistance of Helicobacter pylori.

[0017] The third aspect of the present invention provides a kit for detecting the resistance of Helicobacter pylori to amoxicillin, comprising the primer-probe combination described in the first aspect.

[0018] Preferably, in the primer-probe composition, the molar ratio of the first upstream primer, the second upstream primer, the third upstream primer, and the fourth upstream primer is 1:1:1:1. In some specific embodiments, for PBP1 A gene mutation detection and internal standard gene, the molar ratio of the upstream primer to the downstream primer is 1:1; the molar ratio of the upstream primer or the downstream primer to the probe is 2:1.

[0019] In some specific embodiments, the kit further comprises a PCR buffer and a PCR enzyme system, wherein the components of the PCR buffer are: 10×PCR Buffer, 1M MgCl2, 25mM dNTPs, 1M KCl and Tris-HCl; the components of the PCR enzyme system are: 1U UNG enzyme and 5U Hs Taq enzyme, and the molar ratio of Hs Taq enzyme to UNG enzyme is 4:1.

[0020] In some specific embodiments, the kit also includes a positive control and a negative control, wherein the positive control is a pseudovirus having a Helicobacter pylori amoxicillin-resistant mutant gene fragment and an internal standard fragment, and the negative control is a pseudovirus having an internal standard fragment, wherein the sequence of the amoxicillin-resistant mutant gene fragment is shown in SEQ ID NO.34, and the sequence of the internal standard fragment is shown in SEQ ID NO.35.

[0021] A fourth aspect of the present invention provides a method for detecting amoxicillin resistance of Helicobacter pylori for non-diagnostic and non-therapeutic purposes, comprising the following steps:

[0022] (1) extracting nucleic acid samples from samples to be tested;

[0023] (2) Using nucleic acid samples to prepare a PCR reaction system for real-time fluorescence PCR detection, the amoxicillin resistance of Helicobacter pylori samples to be tested is determined by different fluorescence channel curves and Ct values;

[0024] The PCR reaction system is as follows: 6.55 μL of 10×PCR Buffer, 0.05 μL of 1M MgCl2, 0.4 μL of 25 mM dNTPs, 0.5 μL of 1M KCl, 2.5 μL of Tris-HCl, 0.3 μL of each primer of the first aspect of the invention, 0.15 μL of the probe of the first aspect of the invention, 0.2 μL of each internal standard gene primer of the first aspect of the invention, 0.1 μL of the internal standard gene probe, 0.1 μL of 1U UNG enzyme, 0.4 μL of 5U Hs Taq enzyme, 5 μL of the nucleic acid sample of step (1), and sterile RNase-free water is added to make the total volume of the system 25 μL.

[0025] Furthermore, the PCR detection conditions are: 45°C for 2 min once, 95°C for 2 min once, 95°C for 15 sec and 58°C for 1 min for 40 cycles.

[0026] Furthermore, the conditions for valid interpretation of the test results are: negative control: no Ct value or Ct>35 in the FAM channel, Ct≤35 in the VIC channel; positive control: Ct≤35 in both the FAM and VIC channels, and the aforementioned interpretation conditions must be met simultaneously.

[0027] Beneficial effects of the present invention

[0028] The present invention has the following beneficial effects: the detection kit for detecting amoxicillin resistance of Helicobacter pylori provided by the present invention extracts Helicobacter pylori nucleic acid from in vitro samples, including fecal samples with low sample abundance, and adopts dual fluorescence PCR to perform drug resistance detection, which has low cost, convenient sampling, non-invasiveness, high sensitivity, and high efficiency for detecting amoxicillin resistance of Helicobacter pylori, and has great advantages in current clinical practice.

[0029] The present invention designs primer probes for the amoxicillin resistance gene mutation sites for three common mutation sites Ser414Arg, Thr556Ser, Asn562Tyr and one rare mutation site Thr593Ala of the Helicobacter pylori PBP1A gene. The resistance to amoxicillin caused by the mutations of these four sites accounts for more than 95%, making the detection rate higher. This kit is a dual fluorescence detection kit, and also includes an endogenous internal standard detection system, which is used to monitor the sample collection, nucleic acid extraction process and PCR amplification process, and can reduce the occurrence of false negative results; in terms of the detection method, an optimized PCR buffer formula and PCR program are used, and through anti-interference experiments, specificity tests, precision tests and sequencing comparison verification, the detection of the present invention has the characteristics of high sensitivity, strong specificity, no interference from other substances, good repeatability, etc. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 A primer-probe screening amplification curve graph for the primer-probe combination;

[0031] Figure 2 It is the sample amplification curve of the PCR system under different KCl added concentrations;

[0032] Figure 3 It is the amplification curve diagram under different annealing temperature gradient FAM channel;

[0033] Figure 4 It is the amplification curve diagram under different annealing temperature gradient VIC channel;

[0034] Figure 5 This is the sample amplification curve under the optimized reaction conditions. DETAILED DESCRIPTION

[0035] Unless otherwise indicated, implied from the context, or customary in the prior art, all parts and percentages in this application are based on weight, and the test and characterization methods used are all current as of the filing date of this application. Where applicable, the contents of any patent, patent application or publication referred to in this application are fully incorporated herein by reference, and their equivalent patent families are also incorporated by reference, especially the definitions of synthesis techniques, product and processing designs, polymers, comonomers, initiators or catalysts disclosed in these documents in the art. If the definition of a specific term disclosed in the prior art is inconsistent with any definition provided in this application, the definition of the term provided in this application shall prevail.

[0036] In order to make the technical problems, technical solutions and beneficial effects solved by the present invention more clearly understood, the present invention is further described in detail below in conjunction with embodiments.

[0037] Example

[0038] The following examples are used to demonstrate preferred embodiments of the present invention. It will be appreciated by those skilled in the art that the techniques disclosed in the following examples represent techniques discovered by the inventors that can be used to implement the present invention and therefore can be considered as preferred embodiments of the present invention. However, it will be appreciated by those skilled in the art based on this specification that many modifications may be made to the specific embodiments disclosed herein and still achieve the same or similar results without departing from the spirit or scope of the present invention.

[0039] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs, and the disclosure and materials cited therein are hereby incorporated by reference.

[0040] Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many technical equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the claims.

[0041] Example 1 Primer probe design

[0042] For fluorescent quantitative PCR primers, the following principles are usually followed: 1. Primers should be designed within the region and have specificity; 2. The length of the amplified product is between 80 and 150 bp, and the longest should not exceed 300 bp; 3. The product cannot form a secondary structure (free energy is less than 58.61 KJ / mol); 4. Primer length: generally between 17 and 25 bases, and the upstream and downstream primers should not differ too much; 5. The primer itself cannot have 4 consecutive bases that are complementary to avoid forming a hairpin structure; 6. There cannot be 4 consecutive bases that are complementary between primers to avoid forming primer dimers; 7. The G+C content of the primer is between 40% and 60%, and 45 to 55% is best; the bases in the primer should be randomly distributed and as uniform as possible; 8. The Tm value of the primer is between 58 and 62 degrees.

[0043] For fluorescent quantitative PCR probes, the following principles are usually followed: 1. Design the probe first, and then design the primers to make them as close to the probe as possible; 2. The Tm value of the probe should be between 68 and 70. If it is a visual probe, the GC-rich region should be carefully examined; 3. Select a chain with more C than G as the probe. If the content of G is more than C, the reaction efficiency will be reduced. At this time, the other paired chain should be selected as the probe; 4. The probe should be as short as possible, generally not exceeding 30 bp; 5. Detect the DNA folding and secondary structure of the probe.

[0044] Based on the above principles, we designed the following primers and probes. For the probe detecting PBP1 A gene, the reporter group at the 5' end is set to FAM, the reporter group at the 5' end of the internal standard gene probe is VIC, and there is a quenching group BHQ1 at the 3' end of the probe.

[0045] (1) The first upstream primer was designed for the Ser414Arg mutation of the PBP1 A gene, and the nucleotide sequence is shown in Table 1 below.

[0046] Table 1 Nucleotide sequence of the first upstream primer

[0047]

[0048] (2) A second upstream primer was designed for the Thr556Ser mutation of the PBP1 A gene, and the nucleotide sequence is shown in Table 2 below.

[0049] Table 2 Nucleotide sequence of the second upstream primer

[0050]

[0051] (3) A third upstream primer was designed for the Asn562Tyr mutation of the PBP1 A gene, and the nucleotide sequence is shown in Table 3 below.

[0052] Table 3 Nucleotide sequence of the third upstream primer

[0053]

[0054] (4) A fourth upstream primer was designed for the Thr593Ala mutation of the PBP1 A gene, and the nucleotide sequence is shown in Table 4 below:

[0055] Table 4 Nucleotide sequence of the fourth upstream primer

[0056]

[0057] (5) Design downstream primers that cooperate with the first upstream primer, the second upstream primer, the third upstream primer, and the fourth upstream primer to amplify the corresponding target sequence. The nucleotide sequence is shown in Table 5 below:

[0058] Table 5 Nucleotide sequences of downstream primers

[0059]

[0060] (6) The nucleotide sequence of the probe used to detect the amplified product is shown in Table 6 below:

[0061] Table 6 Probe nucleotide sequence

[0062]

[0063] The primers and probes designed above were combined and amplified for detection experiments. The detection results of each combination are shown in Table 7 below. The primer and probe screening amplification curves are shown in Figure 1 shown.

[0064] Table 7 Ct values ​​obtained by primer and probe combination

[0065]

[0066]

[0067] Note: In the above table, 1-24 represent the first upstream primer, the second upstream primer, the third upstream primer, and the fourth upstream primer sequences SEQ ID NO.1-SEQ ID NO.24; 25-28 represent the downstream primer sequences SEQ ID NO.25-SEQ ID NO.28; 29 and 30 represent the probe sequences SEQ ID NO.29 and SEQ ID NO.30.

[0068] Table 7 and Figure 1The results showed that the combination of upstream primer SEQ ID NO.4 for Ser414Arg mutation site, upstream primer SEQ ID NO.8 for Thr556Ser mutation site, upstream primer SEQ ID NO.13 for Asn562Tyr mutation site, and upstream primer SEQ ID NO.20 for Thr593Ala mutation site, downstream primer SEQ ID NO.25 and probe SEQ ID NO.29 was the best.

[0069] (7) The nucleotide sequences of the internal standard gene primers and probes are designed as shown in Table 8 below:

[0070] Table 8 Nucleotide sequences of internal standard gene primers and probes

[0071]

[0072] (8) The sequence of the amoxicillin-resistant mutant gene fragment in the positive control is shown in SEQ ID NO.34, and the sequence of the internal standard fragment is shown in SEQ ID NO.35.

[0073] Example 2 Sample Detection

[0074] During the sample testing process, we conducted an optimization study on the KCl concentration and annealing temperature in the PCR buffer:

[0075] (1) Investigation of KCl concentration in PCR buffer

[0076] In order to obtain the best amplification efficiency and the best curve line shape, two Helicobacter pylori-positive amoxicillin-resistant stool samples were collected and extracted: Sample 1 and Sample 2. The present invention uses the stool genomic DNA extraction kit (Cat. No.: DP328) produced by Tiangen Biochemical Technology (Beijing) Co., Ltd. to extract nucleic acid from the stool samples to be tested; and adds different concentrations of potassium ions to the PCR system to detect the effect of different KCl concentrations on the amplification results.

[0077] Prepare the PCR reaction system according to the ratio in Table 9 below.

[0078] Table 9 PCR reaction system

[0079]

[0080] The reaction and detection were performed according to the cycle conditions in Table 10 (FAM channel and VIC channel were selected for fluorescence acquisition).

[0081] Table 10 Cycle conditions

[0082]

[0083] During the test, the results are automatically saved after the reaction is completed, and the amplification curves of the detection target and the internal standard are analyzed separately. According to the image after analysis, adjust the Start value, End value and Threshold value of the Baseline (the user can adjust it according to the actual situation, the Start value can be set between 3 and 15, the End value can be set between 5 and 20, and adjust the amplification curve of the negative control to make it flat or below the threshold line), click Analyze to analyze, so that each parameter meets the requirements of the following "Quality Control", and then record the qualitative results in the Plate window. Among them, the positive control is a pseudovirus with Helicobacter pylori amoxicillin resistance and internal standard gene fragments, and the negative control is a pseudovirus with internal standard fragments.

[0084] Quality control: Negative control: FAM channel has no Ct value or Ct>35, VIC channel Ct≤35;

[0085] Positive control: Ct≤35 for both FAM and VIC channels. The above requirements must be met in the same experiment, otherwise the experiment will be invalid and must be repeated.

[0086] The effects of different KCl concentrations on amplification results are shown in Table 11 and Figure 2 .

[0087] Table 11 Different K + Ct value of PCR system under ion concentration

[0088]

[0089] Results: From Table 11 and Figure 2 From the sample amplification curve and Ct value, it can be seen that under the condition of exogenous addition of potassium ions, the addition of potassium ions at various concentrations can improve the fluorescence value and Ct value of the system, which indicates that the amplification efficiency of the system has been improved. The amplification effect of potassium ions at a concentration of 0.5 μL of 1M KCl per person is the best and there is no non-specific amplification. Therefore, this kit uses 0.5 μL of 1M KCl per person as the optimal potassium ion dosage for PCR buffer.

[0090] (2) Optimization of annealing temperature in cycling conditions

[0091] Sample 3 was selected and the PCR system and cycle conditions selected above were used. The annealing temperatures were set to 66°C, 65.5°C, 64.3°C, 62.4°C, 60.0°C, 58°C, 56.7°C, and 56°C, respectively, to conduct an annealing temperature gradient test. The test results are shown in Table 12. Figure 3 and Figure 4 .

[0092] Table 12 Ct values ​​of PCR system at different annealing temperatures

[0093]

[0094] Results: From Table 12 and Figure 3 , Figure 4 It can be seen that as the annealing temperature decreases, the fluorescence amplification curves of the two channels rise. However, if the annealing temperature is too low, it is easy to cause non-specific amplification; if the temperature is too high, it is easy to cause amplification failure. The 58°C annealing temperature used in this product can make the Rn value of the amplification curve higher and the non-specific amplification is minimized. Therefore, 58°C is the optimal annealing temperature of the present invention.

[0095] (3) Sample test results

[0096] The fecal genomic DNA extraction kit (Cat. No.: DP328) produced by Tiangen Biochemical Technology (Beijing) Co., Ltd. was used to extract nucleic acid from samples 1, 2, and 3 mentioned above; the following PCR system and reaction conditions were used for detection: the PCR reaction system was prepared according to the proportions in Table 9, wherein KCL (1M) was 0.5 μL. The reaction and detection were performed according to the cycle conditions in Table 10 (FAM channel and VIC channel were selected for fluorescence acquisition), wherein 95°C 15sec 58°C 1min, 40 cycles.

[0097] The test results of samples 1, 2 and 3 are shown in Tables 13 and Figure 5 shown.

[0098] Table 13 Ct values ​​of sample 1, sample 2 and sample 3

[0099]

[0100] Example 3 Anti-interference test

[0101] The stool samples with interfering substances were tested using the method described in Example 2.

[0102] The detection results of different interfering substances are shown in Table 14 below.

[0103] Table 14 Detection results of different interfering substances

[0104]

[0105] Note: In the above table, 0 means that in the three experiments, the Ct value results in the test results were all No Ct.

[0106] The results showed that the interfering substances had no obvious interference with the reaction system of the present invention.

[0107] Example 4 Specificity Detection

[0108] Positive samples infected with different pathogens were tested using the method described in Example 2. The test results are shown in Table 15.

[0109] Table 15 Detection results of positive samples of different pathogens

[0110]

[0111] The results showed that there was no cross-reaction to the positive samples infected with different pathogens, showing specificity for the Helicobacter pylori PBP1A gene.

[0112] Example 5 Precision Testing

[0113] The nucleic acid of stool samples with positive Helicobacter pylori resistance mutation was diluted to intermediate positive concentration (target FAM Ct value 24-25) and critical positive concentration (target FAM Ct value 32-34), and the negative samples of Helicobacter pylori amoxicillin resistance mutation and normal saline were used as the samples to be tested for intra-batch precision determination.

[0114] Each precision sample was analyzed for one experimental batch per day, and each precision sample was repeated 5 times for 5 consecutive days. The kit of the present invention that passed the quality inspection was tested on the same ABI7500 instrument, and the positive detection rate and negative detection rate were analyzed to examine the precision performance of the kit. The results are shown in Table 16 below:

[0115] Table 16 Results of intra-batch precision test of reagents

[0116]

[0117]

[0118] The results show that the detection results of the probe primer combination of the present invention are consistent between each batch, showing good precision.

[0119] Example 6 Detection Results Sequencing Verification

[0120] (1) Extraction of nucleic acid from samples to be tested

[0121] Fecal samples from 23 Helicobacter pylori-positive patients were collected, and the fecal genomic DNA extraction kit (Cat. No. DP328) produced by Tiangen Biochemical Technology (Beijing) Co., Ltd. was used to extract the test samples. Positive quality control products and negative quality control products were extracted simultaneously to obtain nucleic acid samples 1 to sample 23, positive quality control products and negative quality control products respectively;

[0122] (2) Take 5 μL of nucleic acid sample, prepare PCR reaction system for each sample, and perform amplification reaction in real-time fluorescence PCR instrument. After PCR, the amoxicillin resistance of Helicobacter pylori in patients is determined by different fluorescence channel curves and Ct values.

[0123] (3) Sequencing to verify sample sequence and mutation results.

[0124] The test results are shown in Table 17;

[0125] Table 17 Test results of 20 stool samples

[0126]

[0127]

[0128] The sequencing verification results show that the qualitative detection accuracy of the detection system of the present invention reaches 100%.

[0129] All documents mentioned in the present invention are cited as references in this application, just as each document is cited as reference individually. In addition, it should be understood that after reading the above teachings of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the claims attached to this application.

Claims

1. Use of a primer-probe combination in preparing a kit for detecting the resistance of Helicobacter pylori to amoxicillin, characterized in that: The test sample of the kit is a stool sample, and the primer-probe combination comprises: (1) a first upstream primer for amplifying a first target sequence comprising a Ser414Arg mutation in the PBP1 A gene, wherein the sequence of the first upstream primer is shown in SEQ ID NO. 4; (2) a second upstream primer for amplifying a second target sequence comprising the Thr556Ser mutation of the PBP1 A gene, wherein the sequence of the second upstream primer is shown in SEQ ID NO.8; (3) a third upstream primer for amplifying a third target sequence comprising the Asn562Tyr mutation of the PBP1 A gene, wherein the sequence of the third upstream primer is shown in SEQ ID NO.13; (4) a fourth upstream primer for amplifying a fourth target sequence comprising the Thr593Ala mutation of the PBP1 A gene, wherein the sequence of the fourth upstream primer is shown in SEQ ID NO. 20; (5) a downstream primer used to cooperate with any of the upstream primers in (1) to (4) to amplify a target sequence containing a corresponding mutation site of the PBP1 A gene, wherein the sequence of the downstream primer is SEQ ID NO. 25; (6) A probe for detecting any amplicon obtained by amplifying any upstream primer in (1) to (4) and the downstream primer in (5), wherein the sequence of the probe is SEQ ID NO. 29, the 5' end of the probe is labeled with an excitation fluorescent group, and the 3' end of the probe is labeled with a quenching group.

2. The use according to claim 1, characterized in that: The primer-probe composition also includes a primer and a probe for an internal standard gene: the upstream primer sequence of the internal standard gene is shown in SEQ ID NO.31, the downstream primer sequence of the internal standard gene is shown in SEQ ID NO.32, the internal standard gene probe sequence is shown in SEQ ID NO.33, the 5' end of the internal standard gene probe is labeled with an excitation fluorescent group, and the 3' end of the probe is labeled with a quenching group.

3. The use according to claim 1, characterized in that: In the primer-probe composition, the molar ratio of the first upstream primer, the second upstream primer, the third upstream primer and the fourth upstream primer is 1:1:1:

1.

4. The use according to claim 3, characterized in that: The kit also includes a PCR buffer and a PCR enzyme system. The components of the PCR buffer are: 10×PCR Buffer, 1M MgCl2, 25mM dNTPs, 1M KCl and Tris-HCl; the components of the PCR enzyme system are: 1U UNG enzyme and 5U Hs Taq enzyme.

5. The use according to claim 4, characterized in that: The kit also includes a positive control and a negative control. The positive control is a pseudovirus having a Helicobacter pylori amoxicillin-resistant mutant gene fragment and an internal standard fragment, and the negative control is a pseudovirus having an internal standard fragment, wherein the sequence of the amoxicillin-resistant mutant gene fragment is shown in SEQ ID NO.34, and the sequence of the internal standard fragment is shown in SEQ ID NO.

35.

6. The use according to any one of claims 1 to 5, characterized in that: The use of the kit comprises the following steps: (1) Extracting nucleic acid samples from samples to be tested; (2) Use nucleic acid samples to prepare a PCR reaction system for real-time fluorescence PCR detection, and interpret the results of Helicobacter pylori amoxicillin resistance detection of the tested samples through different fluorescence channel curves and Ct values; The PCR reaction system is: 6.55 μL of 10×PCR Buffer, 0.05 μL of 1M MgCl2, 0.4 μL of 25mM dNTPs, 0.5 μL of 1M KCl, 2.5 μL of Tris-HCl, 0.3 μL of each primer described in claim 1, 0.15 μL of the probe described in claim 1, 0.2 μL of each internal standard gene primer described in claim 2, 0.1 μL of the internal standard gene probe, 0.1 μL of 1U UNG enzyme, 0.4 μL of 5U Hs Taq enzyme, 5 μL of the nucleic acid sample of step (1), and sterile RNase-free water is added to a total volume of 25 μL. The PCR detection conditions are: 45°C 2 min once, 95°C 2 min once, 95°C 15 sec, 58°C 1 min, and 40 cycles.

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