Primer probe combination for detecting drug-resistant gene of mycoplasma genitalium as well as application and product of primer probe combination

Abstract

The invention provides a primer probe combination for detecting drug-resistant genes of mycoplasma genitalium as well as application and a product of the primer probe combination, and belongs to the technical field of biological detection. The invention provides a primer probe combination. The primer probe combination comprises a primer and a probe of MG 23S rRNA (ribosomal Ribonucleic Acid), the primer has a nucleotide sequence as shown in SEQ ID NO. 1-2; the primer has a nucleotide sequence as shown in SEQ ID NO. The probe disclosed by the invention has a nucleotide sequence disclosed as SEQ ID NO. 3. The kit prepared by the primer probe combination provided by the invention can be used for accurately distinguishing mutant samples from wild samples. The method can be used for detecting gene mutation of bacterium 23SrRNA, and a novel method for detecting macrolide drug resistance of mycoplasma genitalium is established.

Description

Technical Field

[0001] The present invention belongs to the technical field of biological detection, and in particular relates to a primer-probe combination for detecting drug-resistant genes of Mycoplasma genitalium, and applications and products thereof. Background Art

[0002] Macrolide antibiotics have strong antibacterial activity against Gram-positive bacteria and some Gram-negative bacteria, and also have good effects on microorganisms such as Chlamydia and Mycoplasma, and are widely used in respiratory and reproductive tract infections. In recent years, due to the irrational use of a large number of antibiotics in clinical practice, the drug resistance of bacteria related to clinical diseases has continued to increase, causing the imbalance of normal flora and the emergence of a large number of drug-resistant strains.

[0003] The drug resistance of Mycoplasma genitalium (MG) is developing rapidly. How to control its development and find new drugs for treatment has become a global problem that needs to be solved urgently. Mycoplasma genitalium infection is the main pathogen of non-chlamydial and non-gonococcal urethritis in men, and is associated with cervicitis and pelvic inflammatory disease (PID) in women. Persistent infection with Mycoplasma genitalium and its complications are serious, and uncured Mycoplasma genitalium has the risk of drug resistance spreading. There is currently no effective vaccine against Mycoplasma genitalium, and effective antibacterial therapy remains the main means of treating and controlling Mycoplasma genitalium infection.

[0004] Azithromycin, a macrolide antibiotic, is the first-line drug for Mycoplasma genitalium infection. However, due to the wide application of azithromycin and the high dosage, the drug resistance rate in many Mycoplasma genitalium infections has reached as high as 50%. Given the high resistance to macrolides, azithromycin is not recommended without resistance testing. Due to the widespread existence of macrolide resistance, all Mycoplasma genitalium tests that are positive must be tested for macrolide resistance-mediating mutations (MRMMs). After MRMMs, treatment is carried out according to the results of resistance testing, and the cure rate can be increased to more than 90%.

[0005] Studies have shown that the resistance of Mycoplasma genitalium to macrolides is mainly related to the gene mutation of bacterial 23SrRNA. The base mutation in the V region of this gene can change the ribosome structure and prevent the drug from binding to the ribosome. The main mutation sites of the 23SrRNA gene are A2058G, A2059G and A2058T. In addition, mutations can also occur at the A2058C, A2059C and A2059T sites. WujianKe et al., in a study titled Three Commercial PCR Assays for the Detection of Macrolide Resistance in Mycoplasma genitalium, showed that among 101 samples with macrolide resistance mutations, A2059G accounted for 58.42%, A2058G accounted for 20.79%, A2058T accounted for 19.80%, and A2059C accounted for 0.99%.

[0006] Currently, there is no product for detecting macrolide resistance of Mycoplasma genitalium. Provided are a primer-probe combination for detecting the resistance gene of Mycoplasma genitalium, its application and product. Summary of the invention

[0007] In view of the above shortcomings, the present invention provides a primer-probe combination for detecting drug-resistant genes of Mycoplasma genitalium, and its application and product. The present invention provides a primer-probe combination, including primers and probes of MG 23S rRNA; the primers have nucleotide sequences as shown in SEQ ID NO.1-2; the probes have nucleotide sequences as shown in SEQ ID NO.3. The kit prepared by the primer-probe combination provided by the present invention can be used to accurately distinguish mutant samples from wild-type samples. It can be used to detect gene mutations of bacterial 23SrRNA, and a new method for detecting macrolide resistance of Mycoplasma genitalium has been established.

[0008] The technical solution of the present invention is as follows: In a first aspect, the present invention provides a primer-probe combination, wherein the primer-probe combination comprises a primer and a probe of MG 23SrRNA.

[0009] Specifically, the primers include a forward primer and a reverse primer, and have a nucleotide sequence as shown in SEQ ID NO.1-2.

[0010] Preferably, the forward primer has a nucleotide sequence as shown in SEQ ID NO.1; and the reverse primer has a nucleotide sequence as shown in SEQ ID NO.2.

[0011] Specifically, the probe has a nucleotide sequence as shown in SEQ ID NO.3.

[0012] Preferably, the probe may be labeled with a fluorescent group or a quencher group at the 5' end or the 3' end.

[0013] Preferably, the fluorescent group is selected from one or more of FAM, VIC, TET, CAL Gold 540, JOE, HEX, TAMRA, ROX, CY3, and CY5, and the quenching group is selected from one or more of DABCYL, BHQ1, BHQ2, BHQ3, and ECLIPE.

[0014] Preferably, the probe is labeled with a FAM fluorescent group at the 5' end and a BHQ1 quenching group at the 3' end.

[0015] Specifically, the final concentration ratio of the forward primer, reverse primer and probe is 0.2:1:1.

[0016] Preferably, the final concentration of the forward primer is 0.08 μM, the final concentration of the reverse primer is 0.4 μM, and the final concentration of the probe is 0.4 μM.

[0017] In a second aspect, the present invention provides the use of the above primer-probe combination in the preparation of a Mycoplasma genitalium drug resistance gene detection kit.

[0018] In a third aspect, the present invention provides a kit for detecting drug-resistant genes of Mycoplasma genitalium, the kit comprising the above-mentioned primer-probe combination.

[0019] Specifically, the kit also includes a PCR reaction solution, a positive control substance, and a negative control substance.

[0020] Preferably, the PCR reaction solution comprises the above primer-probe combination, PCR buffer, DNA polymerase, dNTP enzyme, dUTP enzyme, MgCl2 and nuclease-free water.

[0021] Preferably, the positive control is a wild-type target gene plasmid.

[0022] Preferably, the negative control substance is physiological saline.

[0023] Preferably, the kit is used for detecting macrolide resistance of Mycoplasma genitalium.

[0024] In a fourth aspect, the present invention provides a method for detecting a Mycoplasma genitalium resistance gene, wherein the method comprises using the above-mentioned kit, comprising the following steps: S1. Use a nucleic acid extraction kit to extract nucleic acid from the sample to be tested; S2, preparing the primer-probe combination into a PCR reaction solution; S3, performing PCR amplification detection; S4. Interpretation of results.

[0025] Preferably, the conditions for PCR amplification in step S3 are: Step 1: 37°C, 2 min; Step 2: 95°C, 2 min; Step 3: 95°C, 10s; 58°C, 20s; 72°C, 20s; 45 cycles; collect fluorescence signals at 58°C, 20s, and the fluorescence channel is FAM; Step 4: 95°C, 1 min; 45°C, 5 min; Step 5: 45-90℃, collect fluorescence signals every 0.2℃, keep constant temperature for 8s, and use FAM as the fluorescence channel.

[0026] Preferably, the standard for interpreting the results in step S4 is: if the positive control Tm value -2°C < sample Tm value < positive control Tm value + 2°C, it is determined to be a wild-type sample; if 55°C < sample Tm value ≤ positive control Tm value -2°C, it is determined to be a mutant sample.

[0027] The beneficial effects of the present invention are: The present invention provides a primer-probe combination, including primers and probes of MG 23S rRNA; the primers have nucleotide sequences as shown in SEQ ID NO.1-2; the probes have nucleotide sequences as shown in SEQ ID NO.3. The kit prepared by the primer-probe combination provided by the present invention can be used to accurately distinguish mutant samples from wild-type samples. It can be used to detect gene mutations of bacterial 23SrRNA, and a new method for detecting macrolide resistance of Mycoplasma genitalium is established. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 is the melting peak of the wild-type sample (100% wild-type).

[0029] Figure 2 Schematic diagram of the results for A2059G (100% mutant).

[0030] Figure 3 Schematic diagram of the results for A2058G (100% mutant).

[0031] Figure 4 Schematic diagram of the results for A2058T (100% mutant).

[0032] Figure 5 Schematic diagram of the results for A2059C (100% mutant).

[0033] Figure 6 Schematic diagram of the results for A2058C (100% mutant).

[0034] Figure 7 It is the melting peak of 80% mutant + 20% wild-type samples.

[0035] Figure 8 It is the melting peak of 70% mutant + 30% wild-type samples.

[0036] Fig. 9 It is the melting peak of 30% mutant + 70% wild-type samples.

[0037] Fig.10 It is the melting peak of 20% mutant + 80% wild-type samples.

[0038] Fig.11 This is the amplification curve of the gradient dilution sample (20% mutant + 80% wild-type sample melting peak).

[0039] Fig.12 This is a schematic diagram of the melting peak results of the 50fg sample.

[0040] Fig.13 This is a schematic diagram of the melting peak results of the 5fg sample.

[0041] Fig.14 This is a schematic diagram of the melting peak results of the 0.5fg sample.

[0042] Fig.15 It is the melting peak of the sample in Comparative Example 1.

[0043] Fig.16 It is the melting peak of the sample of Comparative Example 2.

[0044] Fig.17 It is the melting peak of the sample of comparative example 3.

[0045] Fig.18 It is the melting peak of the sample of Comparative Example 4.

[0046] Fig.19 It is the melting peak of the sample in comparative example 5. DETAILED DESCRIPTION

[0047] The technical solutions in the embodiments of the present invention will be clearly and completely described below in combination with the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, rather than all the embodiments; based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of the present invention.

[0048] Example 1 Primer-probe composition for detecting drug-resistant genes of Mycoplasma genitalium Primers and probes were designed using the primer design software Primer Premier 5 according to the 23S ribosomal RNA sequence NR_077054.1 in the genomic nucleic acid sequence of Mycoplasma genitalium G-37 in the GenBank database; the specific method of primer and probe design is as follows: (1) Primers are designed at both ends of the mutation to be detected, and the probe covers the mutation site; (2) The fluorescent group of the MG 23S rRNA probe is the universal FAM probe labeling dye, and the quenching group is the universal BHQ1 probe labeling quencher. The sequence information of the primers and probes is shown in Table 1.

[0049] Table 1 Primer and probe sequence information

[0050] Note: In the table, “F” stands for forward primer; “R” stands for reverse primer; “P” stands for probe; “+” stands for locked nucleic acid (LNA) modification.

[0051] Example 2 Kit for detecting drug resistance genes of Mycoplasma genitalium and method of use 1. Reagent preparation (liquid preparation area) (1) First, take all reagents out of the refrigerator and equilibrate them to room temperature.

[0052] (2) Preparation of primer-probe mixture: Prepare primer-probe mixture according to the composition in Table 2.

[0053] Table 2 Composition of primer-probe mixture

[0054] Note: In the table, "F" stands for forward primer; "R" stands for reverse primer; "P" stands for probe.

[0055] (3) Prepare PCR reaction solution according to the composition in Table 3, add to a 1.5 mL centrifuge tube, shake to mix for a few seconds, and centrifuge at 3000 rpm for a few seconds. The prepared PCR reaction solution can be stored at 4°C for 1 hour or stored at -20°C for 4 hours.

[0056] Table 3 Composition of PCR reaction solution

[0057] (4) Aliquot the PCR reaction solution into PCR thin-walled reaction tubes at 20 μL per tube.

[0058] (5) Transfer the prepared PCR reaction tube to the extraction room and store it at 4°C or -20°C until the sample extraction is completed.

[0059] 2. Sample extraction (extraction area) (1) DNA extraction from Mycoplasma genitalium samples Nucleic acid extraction or purification reagents are used to perform nucleic acid extraction, enrichment, purification and other steps. In the embodiment of the present invention, MagaBio plus viral DNA / RNA purification kit III (purchased from Biori Technology, item number BSC86S1B) is used, and the nucleic acid enrichment method is the magnetic bead capture method.

[0060] (2) Use a micropipette to add 5 μL of the corresponding DNA extraction sample, negative control or positive control into each PCR thin-walled reaction tube and immediately cover the tube tightly; (3) Transfer the PCR thin-walled reaction tube with template added to the PCR amplification area.

[0061] 3. PCR amplification (PCR amplification area) (1) The PCR amplification program was set as follows: Step 1: 37°C, 2 min; Step 2: 95°C, 2 min; Step 3: 95°C, 10s; 58°C, 20s; 72°C, 20s; 45 cycles; collect fluorescence signals at 58°C, 20s, and the fluorescence channel is FAM; Step 4: 95°C, 1 min; 45°C, 5 min; Step 5: 45-90℃, collect fluorescence signals every 0.2℃, keep constant temperature for 8s, and use FAM as the fluorescence channel.

[0062] (2) After the program is completed, take out the PCR thin-walled reaction tube and place it in a concave-convex bag, seal it tightly, and handle it as a contamination source.

[0063] 4. Result determination Determination of the results of the macrolide resistance mutation of Mycoplasma genitalium: After the PCR reaction is completed, the product is analyzed by melting curve, and the wild type and mutant type are distinguished based on the difference in melting point and shape of the melting peak, thus completing the detection of the macrolide resistance mutation of Mycoplasma genitalium.

[0064] The specific method for distinguishing wild type and mutant is as follows: If the positive control Tm value is -2℃<sample Tm value<positive control Tm value+2℃, it is judged as a wild-type sample; if 55℃<sample Tm value≤positive control Tm value-2℃, it is judged as a mutant sample.

[0065] 5. Reference values ​​for drug-resistant gene mutation detection The Tm value range of the positive control, that is, the wild type in each system and each channel is as follows: The Tm value of the wild type control peak in the FAM channel of reaction system A is 74.8℃ ( Figure 1 ).

[0066] Each Tm value is a common value obtained on a specific Macrostone SLAN-96S instrument and is used as a reference. When other instruments are used, the Tm value may vary slightly and shall be based on the Tm value obtained for the positive control of the test. The Tm value shall be based on the automatic interpretation of the instrument. When the instrument gives more than one Tm value, please refer to the peak shape of the positive control and negative control to select a valid Tm value. When the instrument cannot automatically give a Tm value, obtain the Tm value by adjusting the baseline or directly interpreting it manually.

[0067] Example 3 Use of a kit for detecting different drug-resistant mutation types of Mycoplasma genitalium The mutant samples are MG 23S rRNA mutant plasmids, synthesized by Shanghai Jierui Biotechnology Co., Ltd., including A2059G, A2058G, A2058T, A2059C and A2058C, a total of five mutant plasmids. Each mutant plasmid was independently synthesized and quantified.

[0068] Each plasmid was diluted to an appropriate concentration using TE buffer, and the sample was assayed using the kit described in Example 2, referring to the method described in Example 2. Figure 2 As shown, the A2058G result diagram is as follows Figure 3 As shown, the A2058T result diagram is as follows Figure 4 As shown, the A2059C result diagram is as follows Figure 5 As shown, the A2058C result diagram is as follows Figure 6 As shown. The results show that the Mycoplasma genitalium drug resistance gene detection kit of the present invention can accurately distinguish mutant samples from wild-type samples. It can be used to detect gene mutations of bacterial 23SrRNA and for the detection of macrolide resistance of Mycoplasma genitalium.

[0069] Example 4 Use of a kit for detecting the ratio of different drug-resistant mutations of Mycoplasma genitalium The sample was assayed using the kit described in Example 2 and the method described in Example 2.

[0070] The wild-type sample is the MG 23S rRNA wild-type plasmid, synthesized by Shanghai Jierui Biotechnology Co., Ltd. The mutant sample is the MG 23S rRNA mutant plasmid, synthesized by Shanghai Jierui Biotechnology Co., Ltd. Each mutant plasmid was synthesized and quantified independently.

[0071] In this embodiment, taking the A2059G mutation type as an example, TE buffer was used to dilute each plasmid to the same concentration, and samples with different drug-resistant mutation ratios were prepared. The method is shown in Table 4 below: Table 4 Sample preparation with different mutation ratios

[0072] The sample was assayed using the kit described in Example 2 and the method described in Example 2.

[0073] The results of 80% mutant + 20% wild-type samples are shown in the figure below. Figure 7 As shown; the result diagram of 70% mutant + 30% wild type samples is shown Figure 8 As shown; the results of 30% mutant + 70% wild-type samples are shown in Fig. 9 As shown; the result diagram of 20% mutant + 80% wild type samples is shown Fig.10 As shown. The results show that the Mycoplasma genitalium drug resistance gene detection kit of the present invention can accurately distinguish mutant samples from wild-type samples. It can be used to detect gene mutations of bacterial 23SrRNA and for the detection of macrolide resistance of Mycoplasma genitalium.

[0074] Example 5 Sensitivity of the Mycoplasma genitalium resistance gene detection kit The wild-type sample is the MG 23S rRNA wild-type plasmid, synthesized by Shanghai Jierui Biotechnology Co., Ltd. The mutant sample is the MG 23S rRNA mutant plasmid, synthesized by Shanghai Jierui Biotechnology Co., Ltd. Each plasmid was synthesized and quantified independently.

[0075] The wild-type and mutant plasmids were diluted 10-fold using TE buffer to 10fg / μL, 1fg / μL, and 0.1fg / μL, respectively. Subsequently, the wild-type and mutant plasmids of the same concentration were mixed to a 20% mutation ratio. The sample mixing method is shown in Table 5 below: Table 5 Sensitivity test sample preparation (20% mutation ratio)

[0076] Each concentration gradient sample was prepared according to Table 5. The samples were measured using the kit described in Example 2 and the method described in Example 2. The sample addition volume was 5 μL, so the total amount of plasmid sample added was 50 fg, 5 fg and 0.5 fg, respectively.

[0077] The schematic diagram of the amplification curve results of samples with different concentration gradients is as follows Fig.11 As shown, the schematic diagram of the melting peak diagram of the 50fg sample is as follows Fig.12 As shown, the schematic diagram of the melting peak diagram of the 5fg sample is as follows Fig.13 As shown, the schematic diagram of the melting peak diagram of the 0.5fg sample is as follows Fig.14 As shown. The results show that the Mycoplasma genitalium drug resistance gene detection kit of the present invention can accurately distinguish mutant samples from wild-type samples. It can be used to detect gene mutations of bacterial 23SrRNA and for the detection of macrolide resistance of Mycoplasma genitalium.

[0078] Comparative Example 1 Kits containing different primer-probe mixtures The difference between Comparative Example 1 and Example 2 is that the composition of the primer-probe mixed solution is different. The primer-probe mixed solution is prepared according to the composition in Table 6.

[0079] Table 6 Primer-probe mixture of Comparative Example 1

[0080] Note: In the table, "F" stands for forward primer; "R" stands for reverse primer; "P" stands for probe.

[0081] Comparative Example 2 Kits containing different primer-probe mixtures The difference between Comparative Example 2 and Example 2 is that the composition of the primer-probe mixed solution is different. The primer-probe mixed solution is prepared according to the composition in Table 7.

[0082] Table 7 Primer-probe mixture of Comparative Example 2

[0083] Note: In the table, “F” stands for forward primer; “R” stands for reverse primer; “P” stands for probe; “+” stands for locked nucleic acid (LNA) modification.

[0084] Comparative Example 3 Kits containing different primer-probe mixtures The difference between Comparative Example 3 and Example 2 is that the composition of the primer-probe mixed solution is different. The primer-probe mixed solution is prepared according to the composition in Table 8.

[0085] Table 8 Primer-probe mixture of Comparative Example 3

[0086] Note: In the table, “F” stands for forward primer; “R” stands for reverse primer; “P” stands for probe; “+” stands for locked nucleic acid (LNA) modification.

[0087] Comparative Example 4 Kits containing different primer-probe mixtures The difference between Comparative Example 4 and Example 2 is that the composition of the primer-probe mixed solution is different. The primer-probe mixed solution is prepared according to the composition in Table 9.

[0088] Table 9 Primer-probe mixture of Comparative Example 4

[0089] Note: In the table, “F” stands for forward primer; “R” stands for reverse primer; “P” stands for probe; “+” stands for locked nucleic acid (LNA) modification.

[0090] Comparative Example 5 Kits containing different primer-probe mixtures The difference between Comparative Example 5 and Example 2 is that the composition of the primer-probe mixed solution is different. The primer-probe mixed solution is prepared according to the composition in Table 10.

[0091] Table 10 Primer-probe mixture of Comparative Example 5

[0092] Experimental Example 1 The primer-probe mixtures of Comparative Examples 1-5 were used to prepare a kit for testing samples according to the method described in Example 2. The samples used in the test were the "30% mutant + 70% wild type" samples in Example 4.

[0093] The test results of Comparative Example 1 are as follows Fig.15 As shown, the melting peak baseline is not flat; the detection results of comparative example 2 are as follows Fig.16 As shown, the melting peak baseline is not flat and the positions of the wild-type peak and the mutant peak cannot be determined; the detection results of comparative example 3 are as follows Fig.17 As shown in FIG. 4 , there is only a single melting peak and the Tm value is low. Fig.18 As shown, there is no obvious melting peak. The detection result diagram of comparative example 5 is as follows Fig.19 As shown in the figure, the wild-type melting peak position is accurate, but the mutant melting peak height is reduced. When the software uses the unified interpretation rules, the mutant melting peak cannot be accurately identified, which may easily cause some mutant samples to be missed.

[0094] The above detailed description is a specific description of one of the feasible embodiments of the present invention, and the embodiment is not intended to limit the scope of the present invention. It should be pointed out that any equivalent implementation or change that does not deviate from the present invention should be included in the scope of the technical solution of the present invention. Therefore, the protection scope of the patent of the present invention shall be based on the attached requirements.

Claims

1. A primer-probe combination, characterized in that: The primer-probe combination includes primers and probes of MG 23S rRNA; The primers include a forward primer and a reverse primer, and have a nucleotide sequence as shown in SEQ ID NO.1-2; the probe has a nucleotide sequence as shown in SEQ ID NO.3; The final concentration ratio of the forward primer, reverse primer and probe is 0.2:1:

1.

2. The primer-probe combination according to claim 1, characterized in that: 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.

3. The primer-probe combination according to claim 1, characterized in that: The probe may be labeled with a fluorescent group or a quenching group at the 5' end or the 3' end.

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

5. Use of the primer-probe combination according to any one of claims 1 to 4 in the preparation of a kit for detecting drug-resistant genes of Mycoplasma genitalium.

6. A kit for detecting drug-resistant genes of Mycoplasma genitalium, characterized in that: The kit comprises the primer-probe combination according to any one of claims 1 to 4.

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

8. The kit according to claim 7, characterized in that The negative control substance is physiological saline; the positive control substance is a wild-type target gene plasmid.

9. A method for detecting Mycoplasma genitalium resistance genes, characterized in that: The method comprises using the kit according to any one of claims 6 to 7, comprising the following steps: S1. Use a nucleic acid extraction kit to extract nucleic acid from the sample to be tested; S2, preparing the primer-probe combination into a PCR reaction solution; S3, performing PCR amplification detection; S4. Interpretation of results.

10. The method according to claim 9, characterized in that The standard for interpreting the results in step S4 is: if the positive control Tm value -2°C < sample Tm value < positive control Tm value +2°C, it is determined to be a wild-type sample; if 55°C < sample Tm value ≤ positive control Tm value -2°C, it is determined to be a mutant sample.

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