A kit and method for detecting helicobacter pylori drug resistance gene mutation in a sample

Abstract

This invention belongs to the field of biodetection technology, specifically disclosing a primer-probe composition and kit for detecting drug-resistant gene mutations in Helicobacter pylori. The composition comprises two sets of primers and probes, using the probe melting curve method as its principle. The composition and universal PCR reagents are used to detect multiple drug resistance mutations in the quinolone resistance gene gyrA and the clarithromycin resistance gene 23S rRNA of Helicobacter pylori in biological samples. This overcomes the limitation of existing methods that only detect single mutation sites in a single tube for Helicobacter pylori drug resistance genes, providing a new, rapid, and efficient method for detecting Helicobacter pylori drug resistance genes.

Description

Technical Field

[0001] This invention relates to the field of biological detection technology, specifically to a kit and method for detecting drug resistance gene mutations in Helicobacter pylori in samples. Background Technology

[0002] Single-nucleotide polymorphisms (SNPs) are the most common forms of genetic variation in the human genome and a major cause of individual differences. They have received widespread attention in various fields, including drug toxicity, genetic variation, and human diseases. Many destructive abnormalities, such as malignant tumors, cardiovascular diseases, inherited metabolic diseases, and autoimmune diseases, are associated with single-nucleotide variations, and SNPs are becoming important markers for clinical diagnosis and treatment. Therefore, the identification of SNPs is essential for a better understanding of an individual's gene function and health status.

[0003] Helicobacter pylori resistance to commonly used antibiotics is mainly achieved through gene mutations. These mutations reduce the ability of antibiotics to bind to their targets, thus leading to drug resistance in Helicobacter pylori.

[0004] It has been reported that SNPs are often difficult to distinguish because a single base difference in the DNA sequence corresponds to different alleles. The specificity, sensitivity, and cost-effectiveness of the method design are crucial for improving SNP detection. DNA microarrays are currently the most popular method for SNP identification. They utilize probes immobilized on a chip to hybridize with the DNA in the sample, and determine the SNP genotype by measuring the intensity of the hybridization signal. This method offers high throughput and cost-effectiveness, but is limited by its complexity and long processing time. DNA sequencing can also accurately and quickly distinguish SNPs, directly obtaining base information at the SNP site; however, the sequencing process is very expensive, especially with a large number of samples, which significantly increases economic costs. It also suffers from long sequencing runs and issues with data fidelity.

[0005] In recent years, DNA biosensors have been widely used in detecting specific gene sequences due to their high sensitivity and fast response speed. Two main types are fluorescent biosensors, such as multi-primer-mediated RCA coupled with graphene oxide-based fluorescence, universally locked nucleic acid integration X-shaped probes, core-shell gold nanocubes (AuNC) and plasma-enhanced fluorescence (PEF), and RT-PCR associated with G-quadruplex RCA. These sensors monitor changes in fluorescence signals to determine the genotype of SNPs. However, to improve sensitivity, they are often combined with nucleic acid amplification techniques or fluorescent nanomaterials, which can easily lead to background fluorescence interference during measurement. Furthermore, the measurement equipment is usually expensive, increasing the cost of SNP detection. Another type is electrochemical biosensors, such as PNA / ds-DNA triplet formation, CRISPR / dCas9-powered impedance spectroscopy, and electrochemical ligase chain reaction. These sensors immobilize DNA sequences containing SNP sites on the sensor surface, allowing hybridization reactions between the DNA in the sample and the immobilized DNA sequence. When hybridization occurs, the resulting DNA double-stranded structure causes a change in electrochemical signal. This signal change can be measured using electrochemical detection devices to detect the presence and genotype of SNPs. While easily miniaturized, electrode modification is relatively complex. However, improvements in the stability and reproducibility of the recognition element are expected.

[0006] Besides fluorescence detection, other optical methods, such as colorimetric analysis, surface plasmon resonance (SPR), and surface-enhanced Raman spectroscopy (SERS), are also used for SNP detection. Most optical methods are based on target labeling with radioactive isotopes and ultraviolet-absorbing molecules, requiring a certain level of instrument proficiency. Among these, TaqMan probe-based optical methods utilize fluorescently labeled probes to hybridize with DNA in the sample. When the probe perfectly matches the target DNA sequence, a change in fluorescence signal occurs, thus determining the SNP genotype. This method is simple to operate; however, the accuracy of fluorescence detection and color changes is easily affected by instrument and environmental factors. Therefore, there is an urgent need to develop a simple and sensitive SNP detection system that simultaneously meets the requirements of being less susceptible to external influences and having high specificity.

[0007] Chinese invention patent application CN202011013635 discloses a kit for detecting Helicobacter pylori drug resistance gene polymorphism using a multiplex fluorescent PCR melting curve method. The TaqMan probes used identify the mutation type of the target sequence by changes in the Tm value. However, TaqMan probe technology has several major problems: First, TaqMan probes have strict requirements on the position of the fluorescent and quenching groups. Probe design typically requires a length not exceeding 25 nt, which limits its application in detecting single nucleotide mutations at widely spaced sites. Especially when detecting multiple mutation sites, TaqMan probes may not be able to effectively adapt to the large distances between these sites, thus affecting the accuracy and sensitivity of the detection. Second, TaqMan probe systems usually require high-precision real-time quantitative PCR instruments and high-resolution melting curve analysis to accurately distinguish different mutation types. This requirement increases the equipment cost and operational complexity of the experiment, making it unsuitable for use in low-cost or resource-limited detection environments.

[0008] Chinese invention patent application CN202310135604 discloses a primer-probe composition and kit for detecting drug resistance gene mutations in Helicobacter pylori samples. This technical solution also employs the TaqMan probe system, which, while offering advantages in sensitivity and specificity, requires the use of locked nucleic acid modified probes to address the problem of insufficient Tm value difference to distinguish between wild-type and mutant strains. However, while improving detection performance, this modification also increases economic costs.

[0009] Therefore, it is necessary to develop efficient and convenient methods and kits for multiplex detection of Helicobacter pylori infection and detection of Helicobacter pylori drug resistance gene mutation sites. Summary of the Invention

[0010] To address the aforementioned technical shortcomings, the purpose of this invention is to provide a kit for detecting polymorphisms of Helicobacter pylori drug resistance genes (gyrA, 23S rRNA genes) using a multiplex fluorescent PCR melting curve method. This kit requires no complex design or nucleotide modification. By analyzing the probe melting curve, the differences in Tm values ​​can be used to distinguish different drug resistance mutations. It can also detect single nucleotide polymorphisms on the same target sequence for genotyping, thus achieving a sensitive, simple, and rapid SNP detection method.

[0011] In a first aspect, the present invention provides a primer-probe combination for detecting drug resistance gene mutations in Helicobacter pylori in a sample, wherein the primer-probe combination specifically binds to the following pathogen genes and is used to amplify specific amplification products corresponding to each gene: the gyrA gene and the 23S rRNA gene of Helicobacter pylori; the primer-probe combination comprises a first primer-probe combination and a second primer-probe combination:

[0012] The first primer-probe combination consists of a first primer pair and its probe corresponding to the Helicobacter pylori gyrA gene mutation, and their sequences are as follows:

[0013] The forward primer sequence is shown in SEQ ID NO:1: 5'-GCGCTAGGATCGTGGGTGATG-3'.

[0014] The reverse primer sequence is shown in SEQ ID NO:2: 5'-TCAGTGTAACGCATCGCTGCAG-3,

[0015] The detection probe sequence is shown in SEQ ID NO:3:

[0016] 5'-CCGCGGGCGATAACGCGGTTTATGATGCACTCCGCGG-3'

[0017] The second primer-probe combination consists of a second primer pair and its probe corresponding to the Helicobacter pylori 23S rRNA gene mutation, and their sequences are as follows:

[0018] The forward primer sequence is shown in SEQ ID NO:4: 5'-GCATGAATGGCGTAACGAGAT-3'.

[0019] The reverse primer sequence is shown in SEQ ID NO:5: 5'-ATAAGAGCCAAAGCCCTTACTTCAAAG-3'.

[0020] The detection probe sequence is shown in SEQ ID NO:6:

[0021] 5'-CCGCGGCGGCAAGACGGAAAGACCCCGTGGACCGCGG-3'.

[0022] In another preferred embodiment, the mutation site of the Helicobacter pylori gyrA gene is at position 691 and / or 701; and the mutation site of the Helicobacter pylori 23S rRNA gene is at position 2142 and / or 2143.

[0023] In another preferred embodiment, the Helicobacter pylori gyrA gene is mutated to the C691A and G701A drug resistance sites.

[0024] In another preferred embodiment, the Helicobacter pylori 23S rRNA gene is mutated to the A2142C, A2142G and / or A2143G resistance sites.

[0025] In another preferred embodiment, the 5' end of the detection probe is connected to a fluorescent reporter group selected from the group consisting of FAM, HEX, TET, CY3, Red-X, TAMRA, and ROX; and the fluorescent reporter group connected to the detection probe for the gyrA gene is different from that connected to the detection probe for the 23S rRNA gene.

[0026] In another preferred embodiment, the 3' end of the detection probe is connected to a fluorescence quenching group selected from the group consisting of: Dabcy1, BHQ1, BHQ2, and MGB.

[0027] In another preferred embodiment, the 5' end of the probe corresponding to the Helicobacter pylori gyrA gene mutation is labeled with a HEX group as a fluorescent group.

[0028] In another preferred embodiment, the 5' end of the probe corresponding to the Helicobacter pylori 23S rRNA gene mutation is labeled with a FAM group as a fluorescent group.

[0029] In another preferred embodiment, the 3' end of the probe corresponding to mutations in the Helicobacter pylori gyrA and / or 23S rRNA genes is quenched using BHQ1 as a quenching group.

[0030] In another preferred embodiment, the sample is selected from: gastric mucosal tissue and feces.

[0031] A second aspect of the present invention provides a reaction system for multiplex detection of Helicobacter pylori drug resistance gene mutations in samples, the reaction system comprising the primer-probe combination as described in the first aspect of the present invention, and a buffer, dNTP mix, and DNA polymerase for PCR amplification.

[0032] In another preferred embodiment, the concentration of the forward primer in the reaction system is 25–200 nM, the concentration of the reverse primer is 100–600 nM, and the concentration of the probe is 100–600 nM.

[0033] In another preferred embodiment, the molar ratio of the forward primer to the reverse primer in the reaction system is 1:2 to 1:10, and the molar ratio of the probe to the reverse primer is 0.4:1 to 1:1.

[0034] In another preferred embodiment, the molar ratio of the first primer-probe combination to the second primer-probe combination is 1:0.2 to 1:1.

[0035] In another preferred embodiment, the DNA polymerase concentration is 0.3–0.8 U.

[0036] A third aspect of the present invention provides a method for multiplex detection of drug resistance gene mutations in Helicobacter pylori, comprising the steps of:

[0037] (1) Provide a sample to be tested, wherein the sample to be tested contains a nucleic acid extract of a Helicobacter pylori mutant gene;

[0038] (2) The test sample is subjected to PCR amplification using the reaction system described in the second aspect of the present invention;

[0039] (3) Obtain the Tm value of the melting peak detected in each fluorescence channel of the PCR reaction system;

[0040] (4) Determine whether there is a mutation of Helicobacter pylori drug resistance gene in the sample to be tested based on the difference between the detected Tm value and the standard Tm value; wherein, the standard Tm value is the Tm value of the corresponding wild-type gene.

[0041] In another preferred embodiment, in step (4),

[0042] If the detected Tm value is within the range of "standard Tm value ± 2℃", the sample is wild-type; if the detected Tm value is less than "standard Tm value - 2℃", the sample is mutant.

[0043] In another preferred embodiment, in step (4),

[0044] If the detected Tm value is within the range of "standard Tm value ± 3℃", the sample is wild-type; if the detected Tm value is less than "standard Tm value - 3℃", the sample is mutant.

[0045] In another preferred embodiment, in step (3), the PCR amplification program is 93-95℃ for 1 min; 93-95℃ for 15 s, 55-60℃ for 30 s, for 45-60 cycles.

[0046] In another preferred embodiment, the concentration of the nucleic acid to be tested is 1×10⁻⁶. 2 Copy / μL ~ 1×10 6 copies / μL, 1×10 3 Copy / μL ~ 1×10 5 copies / μL, preferably 1×10 4 Copy / μL.

[0047] A fourth aspect of the present invention provides a method for multiplex detection of Helicobacter pylori infection and detection of Helicobacter pylori drug resistance gene mutation sites, comprising the steps of:

[0048] (1) Provide a sample to be tested, wherein the sample to be tested contains a nucleic acid extract of a Helicobacter pylori mutant gene;

[0049] (2) The test sample is subjected to PCR amplification using the reaction system described in the second aspect of the present invention;

[0050] (3) Obtain the Tm value of the melting peak detected in each fluorescence channel of the PCR reaction system;

[0051] (4) Determine whether there is Helicobacter pylori infection and Helicobacter pylori drug resistance gene mutation in the sample based on the detected Tm value.

[0052] In another preferred embodiment, in step (4),

[0053] When the fluorescence channel is FAM, if the detected Tm value is 72℃~77℃, the sample to be tested is wild-type Helicobacter pylori 23S rRNA.

[0054] When the fluorescence channel is FAM, if the detected Tm value is <72℃, the sample to be tested is a 23S rRNA mutant.

[0055] When the fluorescence channel is HEX, if the detected Tm value is 63℃~69℃, the sample to be tested is gyrA wild type;

[0056] When the fluorescence channel is HEX, if the detected Tm value is <63℃, the sample to be tested is gyrA mutant.

[0057] In a fifth aspect, the present invention provides a kit for detecting Helicobacter pylori infection and detecting Helicobacter pylori drug resistance gene mutation sites, the kit comprising the primer-probe combination described in the first aspect of the present invention.

[0058] In another preferred embodiment, the Helicobacter pylori drug resistance gene includes the Helicobacter pylori gyrA gene and 23S rRNA gene.

[0059] In another preferred embodiment, the mutation site of the Helicobacter pylori gyrA gene is at position 691 and / or 701; and the mutation site of the Helicobacter pylori 23S rRNA gene is at position 2142 and / or 2143.

[0060] In another preferred embodiment, the Helicobacter pylori gyrA gene is mutated to C691A and / or G701A.

[0061] In another preferred embodiment, the Helicobacter pylori 23S rRNA gene is mutated to the A2142C, A2142G and / or A2143G resistance sites.

[0062] In another preferred embodiment, the kit further includes a buffer, dNTP mix, and DNA polymerase for PCR amplification.

[0063] It should be understood that, within the scope of this invention, the above-described technical features of this invention and the technical features specifically described below (such as in the embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be described in detail here. Attached Figure Description

[0064] Figure 1 This is a schematic diagram of the detection principle of the present invention.

[0065] Figure 2 This is a schematic diagram of the single-base mutation detection probe structure of the present invention. The left side shows the gyrA probe structure, and the right side shows the 23S rRNA probe structure.

[0066] Figure 3 This diagram illustrates the binding of the probe to the target sequence of this invention. The top image shows the gyrA gene, and the bottom image shows the 23S rRNA gene. The yellow portion represents the probe loop sequence, and the red box represents the mutation site.

[0067] Figure 4 This is a diagram showing the screening results of the gyrA gene primers and probes of this invention.

[0068] Figure 5 This is a diagram showing the screening results of the 23S rRNA gene primers and probes of this invention.

[0069] Figure 6 This is a graph showing the screening results of the gyrA and 23S rRNA gene primer-probe ratios in this invention.

[0070] Figure 7 The left image shows the detection results of the 23S rRNA wild-type and the 2142, 2143 resistance sites, and the right image shows the detection results of the gyrA wild-type and the 691, 701 resistance sites.

[0071] Figure 8 This is a diagram showing the results of dual detection of wild-type gyrA and 23S rRNA according to the present invention. Detailed Implementation

[0072] Through extensive and in-depth research, the inventors have developed a kit for detecting polymorphisms of Helicobacter pylori drug resistance genes (gyrA, 23S rRNA genes) using a multiplex fluorescent PCR melting curve method. This kit requires no complex design or procedures, nor any nucleotide modification. By analyzing the probe melting curves and utilizing the difference in Tm values ​​between wild-type and mutant genes, different types of drug resistance mutations can be distinguished in an integrated reaction system. It can also detect single nucleotide polymorphisms on the same target sequence for genotyping, thus realizing a sensitive, simple, and rapid SNP detection method.

[0073] the term

[0074] As used herein, the terms “containing” or “including (comprise)” can be open-ended, semi-closed, or closed-ended. In other words, the terms also include “consistently made of” or “made of”.

[0075] gyrA gene

[0076] The gyrA gene encodes the α subunit of DNA gyrase, an essential component of bacterial DNA replication and transcription. Certain mutations in the gyrA gene have been shown to be closely associated with resistance to fluoroquinolone antibiotics (such as levofloxacin).

[0077] 23S rRNA gene

[0078] The 23S rRNA gene is a component of the bacterial ribosome and is primarily involved in protein synthesis. Clarithromycin resistance is usually associated with specific mutations in the 23S rRNA gene.

[0079] Detection probe

[0080] The detection probe used in this invention is a molecular beacon probe. A molecular beacon is a probe with fluorescent and quenching groups, capable of complementary pairing with DNA bases. It also contains two complementary terminal sequences. When not hybridizing with the target sequence, it has a circular structure; when hybridizing with the target sequence, it has a linear structure. Different base compositions and pairing configurations result in different Tm values; the more complementary base pairings and the higher the GC content, the higher the Tm value. However, for target regions with only a single gene mutation, the difference in Tm values ​​between wild-type and mutant genes is relatively small. Therefore, it is necessary to rationally design the molecular beacon probe and reaction system to distinguish wild-type and mutant genes based on significant differences in Tm values.

[0081] This invention designs primer pairs and probes for detecting drug resistance mutations at sites 691 and / or 701 in the gyrA gene and at sites 2142 and / or 2143 in the 23S rRNA gene of Helicobacter pylori. The molecular beacon probes of this invention require no nucleotide modification and can efficiently distinguish drug resistance gene mutations based on the significant difference in Tm values ​​between single-base mutant genes and wild-type genes.

[0082] Primer-probe combination

[0083] This invention involves multiple primer-probe combinations targeting the gyrA gene and 23S rRNA gene of Helicobacter pylori. The primer pairs in these combinations are used to specifically amplify the target genes, and the detection probes can be used to detect the amplification products with high sensitivity. Through screening different primer pair and detection probe combinations, primer-probe combinations with optimal amplification efficiency, specificity, and sensitivity were obtained.

[0084] For the detection of wild-type and mutant gyrA genes, preferred primer-probe combinations are shown in SEQ ID NO:1-3. For the detection of wild-type and mutant 23S rRNA genes, preferred primer-probe combinations are shown in SEQ ID NO:4-6.

[0085] reaction system

[0086] The reaction system of the present invention is an integrated reaction system, including the primer and probe combination for the gyrA gene 23S and rRNA gene of the present invention, as well as the buffer, dNTP mix and DNA polymerase required to realize the PCR reaction.

[0087] Detection methods

[0088] The detection method of the present invention is based on a single-tube PCR reaction system. Wild-type and mutant types can be analyzed according to different Tm values ​​of the same fluorescence channel. Multiplex PCR detection can be achieved by monitoring fluorescence changes in different fluorescence channels.

[0089] The detection method of the present invention includes the following steps:

[0090] (1) Provide a sample to be tested, wherein the sample to be tested contains a nucleic acid extract of a Helicobacter pylori mutant gene;

[0091] (2) The test sample was subjected to PCR amplification using the reaction system of the present invention;

[0092] (3) Obtain the Tm value of the melting peak detected in each fluorescence channel of the PCR reaction system;

[0093] (4) Determine whether there is a mutation of Helicobacter pylori drug resistance gene in the sample to be tested based on the difference between the detected Tm value and the standard Tm value; wherein, the standard Tm value is the Tm value of the corresponding wild-type gene.

[0094] Reagent test kit

[0095] The kit of the present invention contains the primer and probe combination described herein, which can be used for integrated detection of Helicobacter pylori infection and detection of Helicobacter pylori drug resistance gene (gyrA gene and 23S rRNA gene) mutation sites.

[0096] The main advantages of this invention include:

[0097] (1) Based on the preferred amplification reaction primers and the melting curve detection method of molecular beacons, this invention can detect multiple drug resistance sites using two fluorescent labels in a single-well reaction tube, effectively improving the detection throughput and achieving the purpose of multiple detection.

[0098] (2) Molecular beacons can be identified in multiple target regions with a single probe, which is more flexible, thus simplifying the probe requirements for multiple detection and reducing costs and operational complexity.

[0099] (3) Based on the Tm value, it is possible to effectively distinguish whether the target gene has been mutated, which can indicate whether the Helicobacter pylori in the sample has quinolone resistance and clarithromycin resistance.

[0100] (4) The detection probe of the present invention is easy to synthesize and does not require additional nucleotide modification, thus achieving efficient and highly specific detection.

[0101] (5) The present invention is simple to analyze and has a short detection time; the sample can be detected within 2 hours.

[0102] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Experimental methods in the following embodiments, unless otherwise specified, are generally performed under conventional conditions, such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or as recommended by the manufacturer. Unless otherwise stated, percentages and parts are weight percentages and parts by weight.

[0103] Experimental materials: The DNA polymerase, buffer, and dNTP mix used in this invention were purchased from Takara; the primer and probe sequences were synthesized by Shanghai Sangon Biotech.

[0104] Example 1. Primer and probe design

[0105] The nucleic acid sequences of Helicobacter pylori gyrA and 23S rRNA genes were collected from the NCBI database. Blast analysis was performed on the sequences to identify conserved sequences and design primers and probes.

[0106] Example 2. Screening of primers and probes

[0107] Primer and probe sequences were designed targeting the drug resistance sites 691 and 701 of the gyrA gene and the drug resistance sites 2142 and 2143 of the 23S rRNA gene in Helicobacter pylori, and the optimal primer sequences were screened out.

[0108] For single-base mutation detection, primers are designed flanking the mutation site to ensure high-affinity binding between the primer and template. The GC content of each primer pair is controlled between 45% and 55%, and the Tm value difference is no greater than 5°C to ensure relative consistency in annealing temperature. The probe design sequence must contain the mutation site, and the mutation site should be located as close as possible to the core region of the probe. This way, when a mutation occurs, the binding force of the probe decreases significantly, thereby causing a signal change. Figure 1 Fluorescent labeling options include FAM, HEX, Texas Red, Cy3, Cy5, etc., while quenching groups can include Dabcyl, BHQ1, BHQ2, MGB, TAMRA, etc.

[0109] The primer and probe sequences designed for each site are shown in Tables 2 and 3. For example, the probe for the gyrA gene has a HEX fluorescent group attached to its 5' end, the probe for the 23S rRNA gene has a FAM fluorescent group attached to its 5' end, and the 3' end of the probe uses BHQ1 as a quenching group.

[0110] Table 2. Primer and probe screening for drug resistance sites 691 and 701 of the gyrA gene.

[0111]

[0112]

[0113] Table 3. Primer and probe screening for drug resistance sites at 2142 and 2143 of the 23S rRNA gene.

[0114]

[0115] In the reaction system for detecting drug-resistant mutation sites in Helicobacter pylori according to the present invention, based on a 25 μL reaction volume, the concentration of the forward primer is 25–200 nM, the concentration of the reverse primer is 100–600 nM, the concentration of the probe is 100–600 nM, the molar ratio of the forward primer, reverse primer, and probe is 1:2:2 to 1:10:10, the concentration of DNA polymerase is 0.3–0.8 U, the concentration of buffer is 0.5–1x, and the concentration of dNTPs is 25–100 mM. Preferably, the molar ratio of the forward primer, reverse primer, and probe is 1:4:4, the concentration of DNA polymerase is 0.4 U, the concentration of buffer is 1x, and the concentration of dNTPs is 30 mM.

[0116] Combining the peak shape and height of the melting curve, when the loop sequence is short, it cannot effectively form a single melting peak, thus affecting the specificity and sensitivity of the detection. The peak height is generally positively correlated with the detection effect; that is, the higher the peak, the stronger the detection signal, and the relatively higher the discrimination, which helps to accurately distinguish the target sequence from non-specific amplification products. Figure 4In the assay, groups 3 and 4 showed good melting peak morphology and high peak values ​​when detecting the gyrA gene, indicating better detection performance. In contrast, group 1 had a lower peak value, resulting in insufficient detection sensitivity and poor performance. Groups 2 and 5 failed to produce a single melting peak, further indicating their limited detection performance. Notably, group 4 had a relatively higher single peak value, demonstrating its superior detection performance. Figure 5 In the 23S rRNA gene detection tests, groups 6 and 9 exhibited a clear, single, and high-peak melting peak, demonstrating significantly better detection performance than other groups. In contrast, group 7 showed a lower peak and poorer detection performance, while groups 8 and 10 failed to produce a stable single peak and were unsuitable for detection. Overall, group 6 had a higher peak and demonstrated better discrimination and detection reliability. Ultimately, group 4 was selected for detecting the gyrA gene, and group 6 for detecting the 23S rRNA gene, demonstrating the best performance.

[0117] Example 3. Preparation of the reaction system

[0118] Different ratios and concentrations of primers and probes were used to prepare reaction solutions 1, 2, and 3 for detecting mutations in the Helicobacter pylori gyrA gene and 23S rRNA gene, as shown in Table 4. The differences in detection effects at different ratios and concentrations of primers and probes were compared.

[0119] Table 4 Preparation of reaction solution 1, reaction solution 2 and reaction solution 3

[0120]

[0121]

[0122] In the reaction system for detecting drug-resistant mutation sites in Helicobacter pylori of this invention, based on a reaction volume of 25 μL, the concentration of the forward primer is 25–200 nM, the concentration of the reverse primer is 100–600 nM, the concentration of the probe is 100–600 nM, the molar ratio of the forward primer to the reverse primer is 1:2–1:10, the molar ratio of the probe to the reverse primer is 0.4:1–1:1, the molar ratio of combination 1 to combination 2 is 1:0.2–1:1, the concentration of DNA polymerase is 0.3–0.8 U, the concentration of buffer is 0.5–1x, and the concentration of dNTP is 25–100 mM. Preferably, the concentration of the forward primer is 100 nM, the concentration of the reverse primer is 400 nM, the concentration range of the probe is 400 nM, the molar ratio of the forward primer to the reverse primer is 1:4, the molar ratio of the probe to the reverse primer is 1:1, the molar ratio of combination 1 to combination 2 is 1:0.5, the concentration of DNA polymerase is 0.6 U, the concentration of buffer is 1x, and the concentration of dNTP is 50 mM.

[0123] exist Figure 6In the assay, reaction solutions 1, 2, and 3 all produced double peaks during the detection process, and no interference was observed between the FAM and HEX channels, demonstrating good channel separation and non-interference performance. Furthermore, compared to reaction solutions 1 and 2, reaction solution 3 exhibited significantly higher double peak values ​​under the same experimental conditions, indicating a stronger signal response or higher sensitivity. Preferably, reaction solution 3 was selected as the single-tube PCR reaction system.

[0124] Example 4. Reaction program screening

[0125] The reaction procedure for detecting drug-resistant mutation sites in Helicobacter pylori according to this invention is shown in Table 5.

[0126] Table 5

[0127]

[0128]

[0129] The preferred reaction procedure is shown in Table 6.

[0130] Table 6

[0131]

[0132] Example 5. Identification of Helicobacter pylori infection and detection of Helicobacter pylori drug resistance gene mutation sites

[0133] The use of kits to identify Helicobacter pylori infection and detect Helicobacter pylori drug resistance gene mutation sites includes the following steps:

[0134] (1) Nucleic acid extraction from the sample

[0135] After pretreatment, fecal samples were used to extract nucleic acids using a commercial fecal genomic DNA extraction kit. The elution buffer was used as a PCR template and stored at -20°C for later use.

[0136] (2) Preparation of PCR reaction system

[0137] Prepare 25 μL of PCR amplification reaction solution by mixing DNA polymerase (including DNA polymerase, buffer, and dNTP) and primers and probes according to the ratios shown in Table 7.

[0138] (3) PCR amplification

[0139] Add the PCR template (1×10⁻⁶) obtained in step one to the PCR amplification reaction solution prepared in step two. 3 Copy / μL ~ 1×10 5 copies / μL, preferably 1×10 4PCR amplification was performed using copies / μL, with nuclease-free water added as a negative control.

[0140] (4) Melting curve detection

[0141] The difference in the Tm value of the melting peak detected in each channel of the PCR reaction system is used to determine whether there is a mutation of the Helicobacter pylori drug resistance gene in the sample to be tested.

[0142] In the result interpretation, when the "test sample Tm value" is within "wild-type control Tm value ± 3℃", the test sample is wild-type; when the "test sample Tm value" is less than "wild-type control Tm value - 3℃", the test sample is mutant.

[0143] Table 7 PCR reaction system

[0144] Composition Dosage DNA polymerase (5 U / μL) 0.125μL 10x Buffer 2.5μL dNTP 2μL SEQ ID NO:1 (10μM) 0.25μL SEQ ID NO:2 (10μM) 1μL SEQ ID NO:3 (10μM) 1μL SEQ ID NO:4 (10μM) 0.125μL SEQ ID NO:5 (10μM) 0.5μL SEQ ID NO:6 (10μM) 0.5μL template 1μL water 16μL

[0145] The results of wild-type and mutant 23S rRNA gene and wild-type and mutant gyrA gene are as follows: Figure 7 And as shown in Table 8.

[0146] Table 8

[0147]

[0148]

[0149] like Figure 7 The results show that this invention can simultaneously detect multiple mutations in the Helicobacter pylori drug resistance gene gyrA and the 23S rRNA gene using a single reaction system. Specifically, when the fluorescence channel is HEX, the Tm of the wild-type gyrA gene is 63–69℃ (approximately 66℃), the Tm of gyrA gene mutant 1 (C691A) is approximately 57–60℃ (approximately 58.5℃), and the Tm of gyrA gene mutant 2 (G701A) is approximately 60–63℃ (approximately 61.5℃); when the fluorescence channel is FAM, the Tm of the wild-type 23S rRNA gene is 73–74℃ (approximately 74℃), the Tm of 23S rRNA gene mutant 1 (A2142C) is 70–72℃ (approximately 71℃), the Tm of 23S rRNA gene mutant 2 (2142G) is approximately 69℃, and the Tm of 23S rRNA gene mutant 3 (A2143G) is approximately 68℃.

[0150] All documents mentioned in this invention are incorporated herein by reference as if each document were individually incorporated by reference. Furthermore, it should be understood that after reading the foregoing teachings of this invention, those skilled in the art can make various alterations or modifications to this invention, and these equivalent forms also fall within the scope defined by the appended claims.

Claims

1. A primer-probe combination for detecting drug resistance gene mutations in Helicobacter pylori in samples, characterized in that, The primer-probe combination specifically binds to the following pathogen genes and is used to amplify the specific amplification products corresponding to each gene: the gyrA gene and the 23S rRNA gene of Helicobacter pylori; the primer-probe combination comprises a first primer-probe combination and a second primer-probe combination: The first primer-probe combination consists of a first primer pair and its probe corresponding to the Helicobacter pylori gyrA gene mutation, and their sequences are as follows: The forward primer sequence is shown in SEQ ID NO:1: 5'-GCGCTAGGATCGTGGGTGATG-3', and the reverse primer sequence is shown in SEQ ID NO:2: 5'-TCAGTGTAACGCATCGCTGCAG-3'. The detection probe sequence is shown in SEQ ID NO:3: 5'-CCGCGGGCGATAACGCGGTTTATGATGCACTCCGCGG-3'; The second primer-probe combination consists of a second primer pair and its probe corresponding to the Helicobacter pylori 23S rRNA gene mutation, and their sequences are as follows: The forward primer sequence is shown in SEQ ID NO:4: 5'-GCATGAATGGCGTAACGAGAT-3'. The reverse primer sequence is shown in SEQ ID NO:5: 5'-ATAAGAGCCAAAGCCCTTACTTCAAAG-3', The detection probe sequence is shown in SEQ ID NO:6: 5'-CCGCGGCGGCAAGACGGAAAGACCCCGTGGACCGCGG-3'.

2. The primer-probe combination as described in claim 1, characterized in that, The mutation sites of the Helicobacter pylori gyrA gene are at positions 691 and / or 701; the mutation sites of the Helicobacter pylori 23S rRNA gene are at positions 2142 and / or 2143.

3. The primer-probe combination as described in claim 1, characterized in that, The 5' end of the detection probe is connected to a fluorescent reporter group selected from the group consisting of: FAM, HEX, TET, CY3, Red-X, TAMRA, and ROX; and the fluorescent reporter groups of the two detection probes are different.

4. A reaction system for multiplex detection of Helicobacter pylori drug resistance gene mutations in samples, characterized in that, The reaction system includes the primer-probe combination as described in claim 1, and a buffer, dNTP mix, and DNA polymerase for PCR amplification.

5. A method for multiplex detection of drug resistance gene mutations in Helicobacter pylori, characterized in that, Including the following steps: (1) Provide a sample to be tested, wherein the sample to be tested contains a nucleic acid extract of a Helicobacter pylori mutant gene; (2) Perform PCR amplification on the test sample using the reaction system described in claim 3; (3) Obtain the Tm value of the melting peak detected in each fluorescence channel of the PCR reaction system; (4) Determine whether there is a mutation of Helicobacter pylori drug resistance gene in the sample to be tested based on the difference between the detected Tm value and the standard Tm value; wherein, the standard Tm value is the Tm value of the corresponding wild-type gene.

6. The method as described in claim 5, characterized in that, In step (4), If the detected Tm value is within the range of "standard Tm value ± 3℃", the sample is wild-type; if the detected Tm value is less than "standard Tm value - 3℃", the sample is mutant.

7. A method for multiplex detection of Helicobacter pylori infection and detection of Helicobacter pylori drug resistance gene mutation sites, characterized in that, Including the following steps: (1) Provide a sample to be tested, wherein the sample to be tested contains a nucleic acid extract of a Helicobacter pylori mutant gene; (2) Perform PCR amplification on the test sample using the reaction system described in claim 3; (3) Obtain the Tm value of the melting peak detected in each fluorescence channel of the PCR reaction system; (4) Determine whether there is Helicobacter pylori infection and Helicobacter pylori drug resistance gene mutation in the sample based on the detected Tm value.

8. The method as described in claim 7, characterized in that, In step (4), When the fluorescence channel is FAM, if the detected Tm value is 72℃~77℃, the sample to be tested is wild-type Helicobacter pylori 23S rRNA. When the fluorescence channel is FAM, if the detected Tm value is <72℃, the sample to be tested is a 23S rRNA mutant. When the fluorescence channel is HEX, if the detected Tm value is 63℃~69℃, the sample to be tested is gyrA wild type; When the fluorescence channel is HEX, if the detected Tm value is <63℃, the sample to be tested is gyrA mutant.

9. A kit for detecting Helicobacter pylori infection and detecting Helicobacter pylori drug resistance gene mutation sites, characterized in that, The kit comprises the primer-probe combination as described in claim 1.

10. The kit according to claim 9, characterized in that, The Helicobacter pylori drug resistance genes include the gyrA gene and the 23S rRNA gene of Helicobacter pylori.

Images