Mycoplasma genitalium drug resistance gene detection primer probe combination, application and product thereof

Abstract

The application discloses a primer probe combination for detecting a mycoplasma genitalium drug-resistant gene and application and products thereof, and belongs to the technical field of biological detection. The technical problem to be solved is that the existing mycoplasma genitalium parC drug-resistant detection probe cannot cover all mutation sites, low-abundance mutations cannot be detected, the system signal is unstable, and the operation process is complex. The technical solution is a primer probe combination for detecting a mycoplasma genitalium drug-resistant gene, wherein a forward primer has a nucleotide sequence as shown in SEQ ID NO. 1; a reverse primer has a nucleotide sequence as shown in SEQ ID NO. 2; and a probe has a nucleotide sequence as shown in SEQ ID NO. 3.

Description

Technical Field

[0001] This invention belongs to the field of biological detection technology, specifically relating to a primer-probe combination for detecting drug resistance genes in Mycoplasma genitalium, its application, and products. Background Technology

[0002] Mycoplasma genitalium (Mg) is a highly prevalent sexually transmitted pathogen that can induce non-gonococcal urethritis in men, cervicitis and pelvic inflammatory disease in women, and can also cause serious long-term complications such as ectopic pregnancy, tubal infertility, and premature birth. Mycoplasma genitalium lacks a cell wall, grows extremely slowly, and requires a long in vitro culture period. Drug sensitivity testing is only used for research and cannot be routinely performed clinically. Nucleic acid amplification detection (NAATs) is currently the gold standard for diagnosing Mg infection.

[0003] Azithromycin (a macrolide) is the first-line treatment in clinical practice, while moxifloxacin (a fluoroquinolone) is used as a second-line salvage therapy. However, in recent years, Mg-resistant strains have spread rapidly, making the resistance situation particularly severe, with the detection rate of dual-resistance strains to macrolides and fluoroquinolones continuing to rise. Fluoroquinolone resistance is mainly mediated by mutations in the quinolone resistance-determining region (QRDR) of the parC gene; among them, the S83I mutation is the strongest predictor of moxifloxacin treatment failure, and if the gyrA mutation is also present, the risk of treatment failure will increase significantly. Without resistance testing support, azithromycin and moxifloxacin should not be used empirically. Clinically, there is an urgent need for molecular diagnostic tools that can simultaneously detect Mg infection and resistance mutations in both classes of antibiotics to achieve personalized and precise medication.

[0004] Currently, the main technologies used for parC gene mutation detection include first-generation sequencing, high-resolution melting curve detection, and conventional fluorescent PCR probe methods, but all have significant shortcomings. Sequencing methods have low throughput, are time-consuming, and costly, making them unsuitable for large-scale clinical screening. The existing HRM detection scheme disclosed in patent CN114703306 requires two independent primer and probe systems, combined with dye and probe methods for interpretation, to complete the detection of eight mutations. This requires a large sample volume, involves a cumbersome operation process, and requires multi-level analysis of melting curve results, resulting in a high interpretation threshold.

[0005] Existing commercial and other probe systems have more prominent shortcomings: First, the probes do not cover all sites. Some probes only target the D87 region and cannot identify S83 site mutations. Even in S83I samples with 70% high mutation abundance, only wild-type peaks can be detected, which can easily lead to false negatives in drug-resistant strains. Second, the mismatch identification ability is insufficient. Conventional probes cannot distinguish mixed samples with low mutation ratios. For 20% low-abundance mutant strains, only a single melting peak or no effective melting signal appears, and it is impossible to distinguish mixed infections in which wild-type strains and drug-resistant mutant strains coexist. Third, the primer and probe compatibility is poor. Some combinations have weak amplification signals and no clear melting peaks, resulting in insufficient detection stability.

[0006] In summary, existing detection technologies suffer from multiple technical shortcomings, including incomplete coverage of mutation sites, missed detection of low-abundance mutations, complex operating procedures, and poor system stability, making it difficult to meet the clinical demand for rapid, accurate, and low-cost mass screening. Summary of the Invention

[0007] The purpose of this invention is to provide: A primer-probe combination for detecting Mycoplasma genitalium drug resistance genes, its application and products, and related technologies, to solve the technical problems of existing Mycoplasma genitalium parC drug resistance detection probes, such as incomplete coverage of mutation sites, inability to detect low-abundance mutations, unstable system signals, and complex operation procedures, or a combination thereof.

[0008] Terminology Explanation: Unless otherwise defined, all technical terms in this document have the same meanings as commonly understood by one of ordinary skill in the art to which the subject matter of the claims pertains. Unless otherwise stated, all patents, patent inventions, and publications cited in this document are incorporated herein by reference in their entirety. If multiple definitions exist for terms in this document, the definitions in this chapter shall prevail.

[0009] It should be understood that the above brief description and the following detailed description are exemplary and for illustrative purposes only, and do not limit the subject matter of the invention in any way. In this invention, the singular is used in conjunction with the plural unless otherwise specifically stated. It should also be noted that, unless otherwise stated, the use of “or” or “or” means “and / or”. Furthermore, the use of the term “comprising” and other forms such as “including,” “containing,” and “contains” are not limiting.

[0010] The definition of the standard chemical term can be found in the reference "Fundamentals of Clinical Laboratory Science", Science Press, October 2022, Edition 2.

[0011] Unless otherwise specified, conventional methods within the scope of the art, such as nucleic acid extraction, shall be used.

[0012] Unless specifically defined herein, the use of all commercially available products herein employs standard techniques. For example, it may be carried out using the manufacturer's instructions for use with the kit, or in accordance with methods known in the art or the description of this invention. The techniques and methods described herein can generally be implemented according to conventional methods well known in the art, based on the descriptions in the various summary and more specific documents cited and discussed in this specification.

[0013] The term "nucleotide sequence" as used in this article refers to the sequence of nucleotides in a nucleic acid molecule. Nucleotides are composed of a phosphate group, a pentose sugar (deoxyribose in DNA and ribose in RNA), and a nitrogenous base, including adenine (A), guanine (G), cytosine (C), thymine (T, only in DNA), and uracil (U, only in RNA).

[0014] The term "primer" as used in this article refers to a short nucleic acid sequence (usually DNA or RNA), typically 15-30 nucleotides in length, which plays a crucial role as the starting point for nucleic acid synthesis in molecular biology techniques. Its core function is to provide the 3'-OH terminus for DNA polymerase to initiate synthesis, guiding the enzyme to extend the new nucleic acid chain along the template strand.

[0015] The term "upstream primer" as used in this article refers to a short oligonucleotide sequence that binds complementary to the non-coding strand (antisense strand) of the target DNA template in a PCR reaction, located at the 5' end of the target region. Its function is to guide DNA polymerase to synthesize a new DNA strand from the 5'→3' direction, starting at the upstream boundary of the target region.

[0016] The term "probe" as used in this article refers to a labeled single-stranded nucleic acid (DNA or RNA) or antibody molecule used to specifically identify and detect target molecules (such as nucleic acid sequences, proteins, cell structures, etc.) in a sample.

[0017] As used in this article, "amplification" refers to the process by which the copy number of a specific nucleic acid fragment is significantly increased through biotechnology. In molecular biology, the most common amplification method is PCR, but other techniques (such as rolling circle amplification, NASBA, etc.) are also included.

[0018] In a first aspect, the present invention provides: a primer-probe combination for detecting mycoplasma genitalium drug resistance genes, wherein the forward primer has a nucleotide sequence as shown in SEQ ID NO.1; the reverse primer has a nucleotide sequence as shown in SEQ ID NO.2; and the probe has a nucleotide sequence as shown in SEQ ID NO.3.

[0019] This includes technical features such as primers and probes.

[0020] SEQ ID NO: 1:ggagatcatggggaaataccac.

[0021] SEQ ID NO: 2: agttgttctttcagctttggga.

[0022] SEQ ID NO:3:tggtgat / +a / / +g / ttccatttat / +g / / +a / tgc.

[0023] Furthermore, the probe may be labeled with a fluorescent group or a quencher group at its 5' or 3' end.

[0024] Furthermore, the probe is labeled with a fluorescent group at the 5' end and a quenching group at the 3' end.

[0025] Furthermore, the fluorescent group is selected from one or more of ROX, VIC, TET, CAL Gold 540, JOE, HEX, TAMRA, ROX, CY3, and CY5; Preferably, the fluorescent group is ROX.

[0026] Furthermore, the quenching group is selected from one or more of DABCYL, BHQ1, BHQ2, BHQ3, and ECLIPE.

[0027] Preferably, the quenching group is BHQ1.

[0028] According to some embodiments of the present invention, the probe is labeled with a ROX fluorescent group at the 5' end and a BHQ1 quencher group at the 3' end.

[0029] Specifically, the probe contains a locked nucleic acid (LNA) modification site.

[0030] Specifically, the final concentration ratio of the forward primer, reverse primer, and probe is 1:(2-10):(1.2-5). Furthermore, the final concentration ratio of the forward primer, reverse primer, and probe is 1:3:1.5.

[0031] According to some embodiments of the present invention, the final concentration of the forward primer is 0.16 μM, the final concentration of the reverse primer is 0.48 μM, and the final concentration of the probe is 0.24 μM.

[0032] Secondly, the present invention provides the application of the above-mentioned primer-probe combination in the preparation of a kit for detecting mycoplasma genitalium drug resistance genes.

[0033] Thirdly, the present invention provides a kit for detecting drug resistance genes of Mycoplasma genitalium for non-diagnostic and therapeutic purposes, the kit comprising the primer-probe combination described above.

[0034] Specifically, the kit also includes PCR reaction solution, wild-type positive control, and negative control.

[0035] Preferably, the PCR reaction solution includes the above-mentioned primer-probe combination, PCR buffer, DNA polymerase, dNTP enzyme, dUTP enzyme, MgCl2, and water.

[0036] According to some embodiments of the present invention, the PCR reaction solution comprises 5 μL of the above primer-probe combination, 2.5 μL of PCR buffer, 0.75 μL of DNA polymerase, 0.75 μL of a mixture of dNTP and dUTP enzymes, 0.4 μL of MgCl2 and 10.6 μL of water.

[0037] Preferably, the wild-type positive control is a wild-type target gene plasmid.

[0038] Preferably, the negative control is physiological saline.

[0039] Preferably, the kit is used for the detection of fluoroquinolone and macrolide resistance in Mycoplasma genitalium.

[0040] According to some embodiments of the present invention, the kit can be a multiplex detection kit.

[0041] Furthermore, when the kit is a multiplex detection kit, it also includes primers and probes for detecting other genes and internal standard primers and probes.

[0042] According to some embodiments of the present invention, the kit further comprises a primer probe for detecting Mycoplasma genitalium 23S rRNA macrolide resistance and a human GAPDH internal standard primer probe.

[0043] Furthermore, the detection system is equipped with three fluorescence channels: the ROX channel detects parC fluoroquinolone resistance mutations, the CY5 channel detects 23S rRNA macrolide resistance mutations, and the FAM channel detects human GAPDH internal standard.

[0044] According to some embodiments of the present invention, the kit can simultaneously detect eight fluoroquinolone resistance mutations in the parC gene of Mycoplasma genitalium: A247C (S83R), A247T (S83C), G248A (S83N), G248T (S83I), G259A (D87N), G259C (D87H), G259T (D87Y), and A260G (D87G).

[0045] Specifically, the detection method of the kit is as follows: S1. Extract nucleic acid from the sample to be tested; S2. Prepare the primer and probe combination into a PCR reaction solution; S3. Perform PCR amplification detection; S4. Result Interpretation.

[0046] Furthermore, the PCR amplification conditions described in step S3 are as follows: Step 1: 37℃, 2 minutes; Step 2: 95℃, 2 minutes; Step 3: 95℃, 10s; 58℃, 20s; 72℃, 20s; 45 cycles; collect fluorescence signal at 58℃, 20s, using ROX fluorescence channel; Step 4: 95℃, 1 min; 45℃, 5 min; Step 5: Continuously melt at a heating rate of 0.05℃ / s from 45-90℃, and collect the fluorescence signal. The fluorescence channel is ROX.

[0047] Specifically, the criteria for interpreting the results in step S4 are as follows: if the wild-type positive control Tm value -2℃ < sample Tm value < wild-type positive control Tm value +2℃, it is determined to be a wild-type sample; if 55℃ < sample Tm value ≤ wild-type positive control Tm value -2℃, it is determined to be a mutant sample.

[0048] The present invention has at least the following beneficial effects: This invention provides a primer-probe combination comprising primers and probes for Mg parC; the primers have nucleotide sequences as shown in SEQ ID NO. 1-2; and the probes have nucleotide sequences as shown in SEQ ID NO. 3. The kit prepared using the primer-probe combination provided by this invention can accurately distinguish between mutant and wild-type samples. It can be used to detect Mg parC gene mutations, establishing a new method for detecting fluoroquinolone resistance in Mycoplasma genitalium. A single tube with a single ROX fluorescence channel can accurately genotype all eight mainstream clinical fluoroquinolone resistance mutations at the S83 and D87 sites of the parC gene. It can detect mixed samples with low mutation abundance as low as 20%. Low-copy templates of 1 cp / μL can still be stably amplified without non-specific interference, and there is no amplification competition inhibition when wild-type and mutant templates coexist. A single tube can simultaneously complete the combined detection of 23S rRNA macrolide resistance and parC fluoroquinolone resistance, effectively avoiding false negatives. It has high sensitivity, broad mutation coverage, and low-abundance mutation identification, and can quickly provide a basis for drug resistance genotyping for Mycoplasma genitalium infection.

[0049] Considering the possibility of this invention entering other countries, this invention also provides the following technical solutions: A method for detecting drug resistance genes in Mycoplasma genitalium includes detection using the primer-probe combination described above or the kit described above.

[0050] Specifically, the detection includes the following steps: S1. Extract nucleic acid from the sample to be tested; S2. Prepare the primer and probe combination into a PCR reaction solution; S3. Perform PCR amplification detection; S4. Result Interpretation. Attached Figure Description

[0051] Figure 1 This is a schematic diagram of the linear detection amplification curve for wild-type samples.

[0052] Figure 2 This is a schematic diagram of the linear detection melting peak for wild-type samples.

[0053] Figure 3 This is a schematic diagram of the linear detection amplification curve for the G248T (S83I) mutant sample.

[0054] Figure 4 This is a schematic diagram of the melting peak in the linear detection of the G248T (S83I) mutant sample.

[0055] Figure 5 This is a schematic diagram of the linear detection amplification curve for a mixed sample with a mutation frequency of 50% G248T (S83I).

[0056] Figure 6 This is a schematic diagram of the melting peak in the linear detection of a mixed sample with a mutation frequency of 50% for G248T (S83I).

[0057] Figure 7 This is a schematic diagram of the A247C (S83R) results (50% mutant).

[0058] Figure 8 This is a schematic diagram of the A247T (S83C) results (50% mutant).

[0059] Figure 9 This is a schematic diagram of the results for G248A (S83N) (50% mutant).

[0060] Figure 10 This is a schematic diagram of the G248T (S83I) results (50% mutant).

[0061] Figure 11 This is a schematic diagram of the results for G259A (D87N) (50% mutant).

[0062] Figure 12 This is a schematic diagram of the results for G259C (D87H) (50% mutant).

[0063] Figure 13 This is a schematic diagram of the G259T (D87Y) results (50% mutant).

[0064] Figure 14 This is a schematic diagram of the A260G (D87G) results (50% mutant).

[0065] Figure 15 This is a schematic diagram of the linear detection amplification curve for a mixed sample with a mutation frequency of 20%-80% G248T (S83I).

[0066] Figure 16 This is a schematic diagram of the melting peak in the linear detection of a mixed sample of G248T (S83I) with a mutation frequency of 20%-80%.

[0067] Figure 17 This is a schematic diagram of the amplification curve of the 23S rRNA mutant gene in the joint detection of multiple mutations.

[0068] Figure 18 This is a schematic diagram of the melting peak of the 23S rRNA mutant gene in the joint detection of multiple mutations.

[0069] Figure 19 This is a schematic diagram of the amplification curve of the parC mutant gene in the joint detection of multiple mutations.

[0070] Figure 20 This is a schematic diagram of the melting peak of the parC mutant gene in the joint detection of multiple mutations.

[0071] Figure 21 This is a schematic diagram of the amplification curve of human internal quality control in the joint detection of multiple mutations.

[0072] Figure 22 This is a schematic diagram of the melting peak of the sample in Comparative Example 1.

[0073] Figure 23 This is a schematic diagram of the melting peak of the sample in Comparative Example 2. Detailed Implementation

[0074] Unless otherwise specified, all raw materials and reagents used in this invention were purchased from commercial suppliers, and experiments were conducted in accordance with the operating instructions. Unless otherwise specified, all instruments, equipment, and apparatus used in this invention are conventional instruments, equipment, and apparatus, and experiments were conducted in accordance with the operating instructions and the accompanying reagents.

[0075] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to embodiments. Unless otherwise specified in the embodiments, conditions are performed under conventional conditions or conditions recommended by the manufacturer. All reagents or instruments without specified manufacturers are commercially available conventional products. Numerous specific details are provided in the following detailed embodiments to better illustrate the invention. The specific embodiments described herein are for illustrative purposes only and are not intended to constitute any limitation on the invention.

[0076] Example 1: Primer and probe composition for detecting fluoroquinolone resistance genes in Mycoplasma genitalium The sequence information of the primers and probes is shown in Table 1: Table 1 Primer and probe sequence information

[0077] Note: "+" in the table represents locked nucleic acid (LNA) modification.

[0078] Example 2: Kit and Usage Method for Detecting Fluoroquinolone Resistance Genes in Mycoplasma Reproductive Tract. 1. Reagent preparation (solution preparation area) (1) First, remove all reagents from the refrigerator and allow them to equilibrate to room temperature.

[0079] (2) Preparation of primer and probe mixture: Prepare primer and probe mixture according to the composition in Table 2.

[0080] Table 2 Composition of primer-probe mixture

[0081] (3) Prepare the PCR reaction solution according to the composition in Table 3, add it to the PCR reaction tube, shake to mix for a few seconds, centrifuge at 3000 rpm for a few seconds. The prepared PCR reaction solution can be used within 1 hour at 4℃ or within 4 hours at -20℃.

[0082] Table 3 Composition of PCR reaction solution

[0083] (4) Dispensing of PCR reaction solution: Dispense PCR reaction solution into PCR thin-walled reaction tubes in 20 μL per tube.

[0084] (5) Transfer the prepared PCR reaction tubes to the extraction room and store them at 4℃ or -20℃ until the sample extraction process is completed.

[0085] 2. Sample extraction (extraction area) (1) DNA extraction from Mycoplasma genitalium samples Nucleic acid extraction, enrichment, and purification steps are performed using nucleic acid extraction or purification reagents. In this embodiment of the invention, the MagaBio plus Viral DNA / RNA Purification Kit III (purchased from Borui Technology, catalog number BSC86S1B) is used, wherein the nucleic acid enrichment method is magnetic bead capture method.

[0086] (2) Add 5 μL of the corresponding DNA extraction sample, negative control or wild-type positive control to each PCR thin-walled reaction tube using a micro-pipette, and immediately close the tube cap tightly; (3) Transfer the PCR thin-walled reaction tube containing the template to the PCR amplification area.

[0087] 3. PCR amplification (PCR amplification region) (1) The PCR amplification program is set as follows: Step 1: 37℃, 2 minutes; Step 2: 95℃, 2 minutes; Step 3: 95℃, 10s; 58℃, 20s; 72℃, 20s; 45 cycles; collect fluorescence signal at 58℃, 20s, using ROX fluorescence channel; Step 4: 95℃, 1 min; 45℃, 5 min; Step 5: Continuously melt at a heating rate of 0.05℃ / s from 45-90℃, and collect the fluorescence signal. The fluorescence channel is ROX.

[0088] (2) After the program is completed, take out the PCR thin-walled reaction tube and put it into the embossed bag. Seal the bag tightly and treat it as a source of contamination.

[0089] 4. Result Determination Determination of fluoroquinolone resistance mutations in Mycoplasma genitalium: After the PCR reaction is completed, the products are analyzed by melting curves. Based on the differences in melting point and shape of the melting peaks, wild-type and mutant types are distinguished, thus completing the detection of fluoroquinolone resistance mutations in Mycoplasma genitalium.

[0090] The specific methods for distinguishing between wild type and mutant type are as follows: If the wild-type positive control Tm value -2℃ < the sample Tm value < the wild-type positive control Tm value +2℃, it is determined to be a wild-type sample; if the sample Tm value is 55℃ < the wild-type positive control Tm value ≤ the wild-type positive control Tm value -2℃, it is determined to be a mutant sample.

[0091] 5. Reference values ​​for drug resistance gene mutation detection The Tm value range of the wild type in the above reaction system and detection channel is as follows: The Tm value of the wild type positive control peak in the detection channel is 67.8℃ ( Figures 1-2 ).

[0092] The Tm values ​​are common values ​​obtained on a specific Macrostone SLAN-96S instrument and are used as a reference. When using other instruments, the Tm values ​​may vary slightly. The Tm values ​​obtained from the wild-type positive control in this test shall prevail. The Tm value should be obtained automatically by the instrument. When the instrument gives more than one Tm value, please refer to the peak shape of the wild-type positive control to select the effective Tm value. When the instrument cannot automatically give the Tm value, obtain the Tm value by adjusting the baseline or by direct manual interpretation.

[0093] Example 3: Use of a kit for linear or sensitivity detection of wild-type, mutant, and mixed-type samples of Mycoplasma genitalium The samples were measured using the kit described in Example 2, following the method described in Example 2.

[0094] Wild-type samples were synthesized using the Mg parC wild-type plasmid, manufactured by Shanghai Jereh Biotechnology Co., Ltd. Mutant samples were synthesized using the Mg parC G248T(S83I) mutant plasmid, also manufactured by Shanghai Jereh Biotechnology Co., Ltd. Each plasmid was synthesized and quantified independently. Wild-type and mutant plasmids were serially diluted 10-fold using TE buffer to concentrations ranging from 1 cp / μL to 1E6 cp / μL.

[0095] The samples were analyzed using the kit described in Example 2, following the method described in Example 2. The 50% mutation frequency mixed sample was a 1:1 mixture of wild-type and mutant samples, i.e., 2.5 μL of each was added before detection. The volume of either wild-type or mutant sample added was 5 μL.

[0096] The results are as follows Figures 3-6 As shown, the results indicate that the kit of the present invention has good sensitivity, detecting wild-type, mutant, and mixed samples as low as 1 cp / μL. Pure mutant samples show only a low-temperature melting peak, pure wild-type samples show only a high-temperature melting peak, and mixed samples show both peaks. The interpretation is intuitive, with values ​​ranging from 1 to 10. 6 The amplification and melting peak positions within the cp / μL range are stable, making it suitable for clinical samples with different Mg loads.

[0097] Example 4: Use of a kit for detecting different drug resistance mutation types of Mycoplasma genitalium Wild-type samples, consisting of the Mg parC wild-type plasmid, were synthesized by Shanghai Jierui Biotechnology Co., Ltd. Mutant samples, consisting of the Mg parC mutant plasmid, were also synthesized by Shanghai Jierui Biotechnology Co., Ltd., including eight mutant plasmids: A247C (S83R), A247T (S83C), G248A (S83N), G248T (S83I), G259A (D87N), G259C (D87H), G259T (D87Y), and A260G (D87G). Each mutant plasmid was synthesized and quantified independently.

[0098] Each plasmid was serially diluted 10-fold to 10 cp / μL using TE buffer. The samples were analyzed using the kit described in Example 2, following the method described in Example 2.

[0099] The results are as follows Figures 7-14 As shown, the results indicate that the kit of the present invention can simultaneously identify eight clinically mainstream fluoroquinolone resistance mutations, including four types of S83 and four types of D87, and can achieve clear typing even under conditions where wild-type and mutant strains coexist in a 50% mixed environment.

[0100] Example 5: Use of a kit for detecting different drug resistance mutation frequencies in Mycoplasma genitalium The samples were measured using the kit described in Example 2, following the method described in Example 2.

[0101] Wild-type samples were synthesized using the Mg parC wild-type plasmid, manufactured by Shanghai Jereh Biotechnology Co., Ltd. Mutant samples were synthesized using the Mg parC G248T(S83I) mutant plasmid, also manufactured by Shanghai Jereh Biotechnology Co., Ltd. Each plasmid was synthesized and quantified independently. Wild-type and mutant plasmids were serially diluted 10-fold using TE buffer to a concentration of 1E2cp / μL.

[0102] Subsequently, wild-type and mutant plasmids were mixed according to Table 4 to prepare test samples with different mutation ratios. The sample mixing methods are shown in Table 4 below: Table 4 Sample preparation at different mutation frequencies

[0103] The samples were measured using the kit described in Example 2, following the method described in Example 2.

[0104] The results are as follows Figures 15-16As shown, the results indicate that the kit of the present invention can amplify normally in mixed templates with a mutation ratio of 20%-80% across the entire range. There is no competitive inhibition between wild-type and mutant templates, and the amplification efficiency is stable. Even in samples with a low mutation frequency of 20%, it can still stably distinguish between wild-type and mutant bimodal peaks. It can detect a small number of clinically resistant mutant subclones and avoid missed diagnosis of mixed drug-resistant infections.

[0105] Example 6: Use of a combined detection kit for macrolide and fluoroquinolone resistance genes in Mycoplasma genitalium Clinically, macrolide antibiotics (such as azithromycin) and fluoroquinolone antibiotics (such as moxifloxacin) are the first-line and second-line drugs recommended for the treatment of Mycoplasma genitalium, respectively. Due to the wide range of applications and high dosages of azithromycin, the resistance rate in many Mycoplasma genitalium infections has reached as high as 50%. Macrolide resistance in Mycoplasma genitalium is caused by mutations in the Mg23S rRNA gene. Combined detection of macrolide and fluoroquinolone resistance gene mutations is of great significance for clinical diagnosis and treatment.

[0106] 1. Reagent preparation (solution preparation area) (1) First, remove all reagents from the refrigerator and allow them to equilibrate to room temperature.

[0107] (2) Preparation of primer and probe mixture: Prepare primer and probe mixture according to the composition in Table 5.

[0108] Table 5 Composition of primer-probe mixture

[0109] (3) Prepare the PCR reaction solution according to the composition in Table 6, add it to the PCR reaction tube, shake to mix for a few seconds, centrifuge at 3000 rpm for a few seconds. The prepared PCR reaction solution can be used within 1 hour at 4℃ or within 4 hours at -20℃.

[0110] Table 6 Composition of PCR reaction solution

[0111] (4) Dispensing of PCR reaction solution: Dispense PCR reaction solution into PCR thin-walled reaction tubes at 12 μL per tube.

[0112] (5) Transfer the prepared PCR reaction tubes to the extraction room and store them at 4℃ or -20℃ until the sample extraction process is completed.

[0113] 2. Sample preparation In this experiment, wild-type samples were inactivated Mycoplasma genitalium control samples, purchased from Nanjing Kebai Biotechnology Co., Ltd., and quantified by the manufacturer using dPCR. Mutant samples were Mg 23S rRNA A2059G and Mg parCG248A (S83N) mutant plasmids, synthesized by Shanghai Jierui Biotechnology Co., Ltd. Each plasmid was synthesized and quantified independently.

[0114] The Mg inactivated control and mutant plasmid were serially diluted 10-fold using TE buffer to 1E1cp / μL, 1E2cp / μL, and 1E3cp / μL, respectively. Subsequently, samples with different mutation ratios were prepared by mixing the same concentration of Mg inactivated control with Mg 23S rRNA A2059G and Mg parCG248A (S83N) mutant plasmids according to Table 7. Table 7. Preparation of samples with 30% mutation frequency at different concentrations

[0115] (2) Add 13 μL of the corresponding sample to each PCR thin-walled reaction tube using a micro-pipette, and immediately close the tube cap tightly. The negative control and wild-type control should be added in a volume of 10 μL. Use 3 μL of TE buffer to make up the reaction volume. (3) Transfer the PCR thin-walled reaction tube containing the template to the PCR amplification area.

[0116] 3. PCR amplification (PCR amplification region) (1) The PCR amplification program is set as follows: Step 1: 37℃, 2 minutes; Step 2: 95℃, 2 minutes; Step 3: 95℃, 10s; 58℃, 20s; 72℃, 20s; 45 cycles; collect fluorescence signals at 58℃, 20s, using FAM, ROX and CY5 fluorescence channels; Step 4: 95℃, 1 min; 45℃, 5 min; Step 5: Continuous melting at a heating rate of 0.05℃ / s from 45-90℃, collecting fluorescence signals. The fluorescence channels are FAM, ROX, and CY5.

[0117] (2) After the program is completed, take out the PCR thin-walled reaction tube and put it into the embossed bag. Seal the bag tightly and treat it as a source of contamination.

[0118] 4. Result Determination Determination of Mycoplasma genitalium drug resistance mutation results: After the PCR reaction is completed, the effectiveness of the test is first determined by internal standard quality control, and then the melting curve is analyzed. Based on the difference in melting point and shape of the melting peak, wild type and mutant type are distinguished, thus completing the detection of Mycoplasma genitalium fluoroquinolone drug resistance mutation.

[0119] The CY5 fluorescence channel was used to detect macrolide resistance mutations, and the ROX fluorescence channel was used to detect fluoroquinolone resistance mutations. Wild-type and mutant types of both drugs were interpreted separately. The specific methods for distinguishing between wild-type and mutant types are as follows: If the wild-type positive control Tm value -2℃ < the sample Tm value < the wild-type positive control Tm value +2℃, it is determined to be a wild-type sample; if the sample Tm value is 55℃ < the wild-type positive control Tm value ≤ the wild-type positive control Tm value -2℃, it is determined to be a mutant sample.

[0120] 5. Reference values ​​for drug resistance gene mutation detection The Tm value range of the wild type in the above reaction system and detection channel is as follows: The Tm value of the wild type positive control peak in the CY5 detection channel is 75.1℃ ( Figure 18 The Tm value of the wild-type positive control peak in the ROX detection channel was 67.8℃. Figure 2 ).

[0121] The Tm values ​​are common values ​​obtained on a specific Macrostone SLAN-96S instrument and are used as a reference. When using other instruments, the Tm values ​​may vary slightly. The Tm values ​​obtained from the wild-type positive control in this test shall prevail. The Tm value should be obtained automatically by the instrument. When the instrument gives more than one Tm value, please refer to the peak shape of the wild-type positive control to select the effective Tm value. When the instrument cannot automatically give the Tm value, obtain the Tm value by adjusting the baseline or by direct manual interpretation.

[0122] The results are as follows Figures 17-21 As shown, the results indicate that the kit of this invention can simultaneously complete the identification of Mg pathogens, macrolide resistance (23S rRNA), and fluoroquinolone resistance (parC) dual-drug resistance mutation typing in a single tube reaction. Combined with human internal standard quality control, the three sets of primers and probes exhibit no cross-interference or amplification competition, and all detections are completed synchronously in a single tube with multiple channels. Whether it is a macrolide 23S rRNA hotspot mutation or a fluoroquinolone parC hotspot mutation, wild-type / mutant coexisting samples can still be clearly distinguished by melting curves in a multiplex system. There are no missed detections at 30% low mutation abundance, and the typing accuracy is comparable to that of a single-target independent detection system.

[0123] Comparative Example 1: Kits containing different probe mixtures The only difference between Comparative Example 1 and Example 2 is the parC detection probe. The primer-probe mixture was prepared according to the composition in Table 8.

[0124] Table 8 Comparative Example 1 Primer-Probe Mixture

[0125] Note: In the table, "P" represents a probe; "+" represents locked nucleic acid modification.

[0126] Comparative Example 2: Kits containing different probe mixtures The only difference between Comparative Example 2 and Example 2 is the composition of the primer-probe mixture. The primer-probe mixture was prepared according to the composition in Table 9.

[0127] Table 9 Comparative Example 2 Primer-Probe Mixture

[0128] Note: In the table, "P" represents a probe; "+" represents locked nucleic acid modification.

[0129] Experimental Example 1 Using the primer-probe mixture in Table 8 of Comparative Example 1, the kit was prepared according to the method described in Example 2 to test the samples. The samples used for testing were G248T (S83I) and G259T (D87Y) plasmid samples with a 70% mutation frequency.

[0130] The test results of Comparative Example 1 are as follows Figure 22 As shown, in this experiment, both wild-type and mutant peaks were detected in the G259T (D87Y) sample with a 70% mutation frequency, but only the wild-type peak was detected in the G248T (S83I) sample with a 70% mutation frequency. In Comparative Example 1, the probe design and locked nucleic acid modification only covered the D87 amino acid position and could not simultaneously detect mutations at the S83 and D87 amino acid positions.

[0131] Experimental Example 2 Using the primer-probe mixture from Table 9 of Comparative Example 2, the kit was prepared according to the method described in Example 2 to test the samples. The VIC and ROX fluorescence channels were detected simultaneously during the test. The samples used for testing were G248T (S83I) samples with a 20% mutation frequency from Example 5.

[0132] The test results of Comparative Example 2 are as follows Figure 23 As shown, primer probe combination 2 showed no obvious melting peak, while primer probe combination 3 could only detect one melting peak. The primer probe combination of the present invention can detect mutant and wild-type peaks in samples with a mutation frequency of 20%.

[0133] Finally, it should be noted that the above content is only used to illustrate the technical solution of the present invention, and is not intended to limit the scope of protection of the present invention. Simple modifications or equivalent substitutions made by those skilled in the art to the technical solution of the present invention do not depart from the essence and scope of the technical solution of the present invention.

Claims

1. A primer-probe combination for detecting drug resistance genes in Mycoplasma genitalium, characterized in that, The forward primer has the nucleotide sequence shown in SEQ ID NO.1; the reverse primer has the nucleotide sequence shown in SEQ ID NO.2; and the probe has the nucleotide sequence shown in SEQ ID NO.

3.

2. The primer-probe combination according to claim 1, characterized in that, The probe can be labeled with a fluorescent group or a quencher group at its 5' or 3' end.

3. The primer-probe combination according to claim 2, characterized in that, The fluorescent group is selected from one or more of ROX, VIC, TET, CAL Gold 540, JOE, HEX, TAMRA, ROX, CY3, and CY5; the quenching group is selected from one or more of DABCYL, BHQ1, BHQ2, BHQ3, and ECLIPE.

4. The primer-probe combination according to claim 1, characterized in that, The final concentration ratio of the forward primer, reverse primer and probe is 1:(2-10):(1.2-5).

5. The use of the primer-probe combination according to any one of claims 1-4 in the preparation of a kit for detecting mycoplasma genitalium drug resistance genes.

6. A kit for detecting drug resistance genes in Mycoplasma genitalium for non-diagnostic and non-therapeutic purposes, characterized in that, The kit comprises the primer-probe combination as described in any one of claims 1-4.

7. The reagent kit according to claim 6, characterized in that, The kit also includes PCR reaction solution, wild-type positive control, and negative control.

8. The reagent kit according to claim 6, characterized in that, The kit described is used for the detection of fluoroquinolone and macrolide resistance in Mycoplasma genitalium.

9. The reagent kit according to claim 8, characterized in that, The kit is a multiplex assay kit, which also includes primers and probes for detecting macrolide resistance genes and internal standard primers and probes.

10. The reagent kit according to claim 6, characterized in that, The detection method of the aforementioned kit is as follows: S1. Extract nucleic acid from the sample to be tested; S2. Prepare the primer and probe combination into a PCR reaction solution; S3. Perform PCR amplification detection; S4. Result Interpretation.

11. The reagent kit according to claim 10, characterized in that, The PCR amplification conditions described in step S3 are as follows: Step 1: 37℃, 2 minutes; Step 2: 95℃, 2 minutes; Step 3: 95℃, 10s; 58℃, 20s; 72℃, 20s; 45 cycles; collect fluorescence signal at 58℃, 20s, using ROX fluorescence channel; Step 4: 95℃, 1 min; 45℃, 5 min; Step 5: Continuously melt at a heating rate of 0.05℃ / s from 45-90℃, and collect the fluorescence signal. The fluorescence channel is ROX.

12. The reagent kit according to claim 10, characterized in that, The criteria for interpreting the results in step S4 are as follows: if the wild-type positive control Tm value -2℃ < sample Tm value < wild-type positive control Tm value +2℃, then it is determined to be a wild-type sample; if 55℃ < sample Tm value ≤ wild-type positive control Tm value -2℃, then it is determined to be a mutant sample.

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