A composition for detecting mycobacterium tuberculosis complex and use thereof

Abstract

The application belongs to the technical field of molecular biology detection, and particularly relates to a composition for detecting mycobacterium tuberculosis complex and application thereof. The application provides a primer pair and a probe for detecting mycobacterium tuberculosis complex, and simultaneously uses IS6110 and IS1081 genes specific to the mycobacterium tuberculosis complex as detection targets, and introduces a RNase P endogenous internal standard gene fragment, and the primer and the probe are respectively designed according to the conservative sequences of the IS6110 and IS1081 genes of the mycobacterium tuberculosis complex, and the primer and the probe have good accuracy, specificity and sensitivity for detecting the mycobacterium tuberculosis complex. The results of the examples show that the detection accuracy of the primer pair and the probe provided by the application for the mycobacterium tuberculosis complex can reach 100%, and no fluorescence signal is found for detecting other non-mycobacterium tuberculosis species.

Description

Technical Field

[0001] This invention belongs to the field of molecular biology detection technology, specifically relating to a composition for detecting Mycobacterium tuberculosis complex. Background Technology

[0002] Tuberculosis is a chronic infectious disease caused by Mycobacterium tuberculosis, Mycobacterium bovis, and Mycobacterium africanum, among others. It can affect all organs of the body, with pulmonary tuberculosis being the most common. The primary pathogen of tuberculosis is Mycobacterium tuberculosis, followed by Mycobacterium bovis.

[0003] In the bacteriological examination of tuberculosis, mycobacteria are usually classified into the Mycobacterium tuberculosis complex. (Mycobacterium tuberculosis complex MTBC) and nontuberculous mycobacteria ( Non-tuberculosis mycobacteria The tuberculosis complex includes Mycobacterium tuberculosis (NTM). M. tuberculosis Mycobacterium bovis, Mycobacterium africanum ( M. africanum Mycobacterium tuberculosis complex includes *Mycobacterium tuberculosis* and *Mycobacterium microti*. Among the various species within the Mycobacterium tuberculosis complex, only *Mycobacterium microti* is non-pathogenic to humans; the other three are pathogenic, producing largely the same clinical manifestations. Therefore, clinically, only the Mycobacterium tuberculosis complex is identified, not at the subspecies level. Nucleic acid detection reagents used for auxiliary diagnosis of tuberculosis often use nucleic acid sequences common to the Mycobacterium tuberculosis complex as targets for detection.

[0004] Currently, the main methods for detecting Mycobacterium tuberculosis include smear methods and culture methods, as well as PCR-based diagnostic techniques (such as Xpert MTB / RIF, LAMP, fluorescent PCR, and fluorescent PCR melting curve analysis). While direct smear methods are simple and easy to perform, they have low sensitivity. Culture methods are the gold standard for tuberculosis detection; although highly sensitive, the time to obtain results is too long—4–6 weeks for the modified Roche solid culture method and 1–3 weeks for the rapid liquid culture method. Furthermore, the complex operation and high laboratory requirements limit the clinical guidance significance of the results. In recent years, with the development and popularization of molecular biology techniques, more and more molecular biology methods have been used for rapid tuberculosis detection. For example, Xpert MTB / RIF and loop-mediated isothermal amplification (LAMP) are WHO-recommended rapid diagnostic methods for tuberculosis. However, the former relies on expensive instruments and consumables, and the latter's result interpretation mainly depends on the color change of the reaction system, which can lead to difficult-to-interpret results in samples with low bacterial loads. Therefore, these factors limit the widespread adoption and application of these technologies.

[0005] Nucleic acid testing is an important reference for the etiological diagnosis of pulmonary tuberculosis. Compared with other laboratory testing methods, the value of nucleic acid testing lies in: (1) it can quickly distinguish between Mycobacterium tuberculosis complex and non-tuberculous mycobacteria, improving the diagnostic specificity of smear-positive pulmonary tuberculosis; (2) compared with smears, it has higher sensitivity and can improve the detection rate of smear-negative pulmonary tuberculosis; (3) compared with culture, it is faster to operate and can provide earlier and correct medical treatment.

[0006] Current molecular biology methods primarily target repetitive sequences in Mycobacterium tuberculosis. IS6110, with a copy number of 10-20 in the Mycobacterium tuberculosis complex, has been an excellent target for diagnosis of the Mycobacterium tuberculosis complex since the 1990s. For example, it has up to 22 copies in Mycobacterium tuberculosis strain AP018035.1, 8 copies in Mycobacterium vulnificus strain CP010333.1, 3 copies in Mycobacterium capsulatum strain CP016401.1, and 1 copy in Mycobacterium africanum strain CP014517.1, Mycobacterium bovis strain CP015773.2, and Mycobacterium bovis strain BCG. However, reports indicate that IS6110 is not very effective in some regions (such as Southeast Asia and Vietnam), mainly because some strains contain only one copy of IS6110 in their genome.

[0007] Therefore, there is an urgent need to develop a composition that has good accuracy, specificity and sensitivity for the detection of Mycobacterium tuberculosis complex. Summary of the Invention

[0008] The purpose of this invention is to provide a composition for detecting Mycobacterium tuberculosis complex. The composition provided by this invention simultaneously detects the IS6110 and IS1081 genes, which can improve the sensitivity and specificity of Mycobacterium tuberculosis complex detection and avoid false negatives.

[0009] To achieve the above objectives, the present invention provides the following technical solution: This invention provides a composition for detecting Mycobacterium tuberculosis complex, the composition comprising: The first primer pair consists of the upstream primer shown in SEQ ID NO.1 and the downstream primer shown in SEQ ID NO.2; the first probe consists of the sequence shown in SEQ ID NO.3; The second primer pair consists of the upstream primer shown in SEQ ID NO.4 and the downstream primer shown in SEQ ID NO.5; the second probe has the sequence shown in SEQ ID NO.6; The third primer pair consists of the upstream primer shown in SEQ ID NO.7 and the downstream primer shown in SEQ ID NO.8; the third probe has the sequence shown in SEQ ID NO.9.

[0010] Preferably, the first probe, the second probe, and the third probe are all fluorescently labeled probes.

[0011] More preferably, the 5' end of the first probe and the second probe are labeled with a FAM fluorescent reporter group, and the 3' end is labeled with a first quencher group; the 5' end of the third probe is labeled with a ROX fluorescent reporter group, and the 3' end is labeled with a second quencher group; the first quencher group is DBQ1, and the second quencher group is BHQ2.

[0012] The present invention also provides a detection reagent for binding mycobacteria, the detection reagent comprising the above-described composition.

[0013] The present invention also provides a kit for detecting Mycobacterium tuberculosis, the kit comprising the above-described composition, lysis buffer, magnetic beads, washing buffer, and elution buffer.

[0014] This invention also provides a method for detecting Mycobacterium tuberculosis, comprising the following steps: S1. Extract DNA from the sample to be tested; S2. Using the DNA of the sample to be tested as a template, perform PCR amplification using the above primer pairs and the probe of the Mycobacterium tuberculosis gene; S3. The fluorescence quantitative PCR instrument detects the reaction products. The appearance of a fluorescence signal indicates that the sample contains Mycobacterium tuberculosis, and vice versa.

[0015] Preferably, the PCR amplification reaction system is 25 μL, wherein the concentrations of each primer and probe are as follows: the primer concentrations shown in SEQ ID NO. 1 and 2 are both 0.4 μM, the primer concentrations shown in SEQ ID NO. 4 and 5 are both 0.4 μM, the primer concentrations shown in SEQ ID NO. 7 and 8 are both 0.1333 μM, the probe concentrations shown in SEQ ID NO. 3 and 6 are both 0.2 μM, and the probe concentration shown in SEQ ID NO. 9 is 0.0667 μM.

[0016] Preferably, the PCR amplification reaction conditions are: 50℃, 2 min; 95℃, 30 s; 95℃, 10 s, 55℃, 30 s, for a total of 45 cycles.

[0017] The beneficial effects of this invention are: The conserved sequences of the IS6110 and IS1081 genes described in this invention exhibit high homology within the Mycobacterium tuberculosis complex and show no homology with other microorganisms. Primers and probes were designed based on the conserved sequences of the IS6110 and IS1081 genes of this Mycobacterium tuberculosis complex. These primers and probes demonstrate good accuracy, specificity, and sensitivity in the detection of the Mycobacterium tuberculosis complex. Experiments have confirmed that the detection accuracy of the primer pairs and probes provided in this invention for the Mycobacterium tuberculosis complex can reach 100%, and no fluorescent signal was observed when detecting other non-tuberculous mycobacterial species. Detailed Implementation

[0018] This invention provides a composition for detecting Mycobacterium tuberculosis complex and its application. This invention uses the Mycobacterium tuberculosis complex-specific genes IS6110 and IS1081 simultaneously as detection targets. IS1081 is present in all Mycobacterium tuberculosis complexes, and its copy number in the Mycobacterium tuberculosis genome is 5-7. Simultaneous detection of both IS6110 and IS1081 genes can improve the sensitivity and specificity of Mycobacterium tuberculosis complex detection and avoid false negatives.

[0019] NCBI-Blast analysis revealed that the conserved sequences of the IS6110 and IS1081 genes showed high homology within the Mycobacterium tuberculosis complex and no homology with other microorganisms. Primers and probes were designed based on the conserved sequences of the IS6110 and IS1081 genes of this Mycobacterium tuberculosis complex. These primers and probes demonstrated good accuracy, specificity, and sensitivity for the detection of the Mycobacterium tuberculosis complex. The results of the examples show that the detection accuracy of the primer pairs and probes provided by this invention can reach 100% for the Mycobacterium tuberculosis complex, and no fluorescent signal was observed when detecting other non-tuberculous mycobacterial species.

[0020] This invention provides two pairs of primers for the detection of Mycobacterium tuberculosis complexes by quantitative real-time PCR, and a set of primers for human RNase P, including an upstream primer and a downstream primer, the sequences of which are as follows: Upstream primer IS-F1: 5'-GACCACCAGCACCTAACCG-3' (SEQ ID NO.1); Downstream primer IS-R1: 5'-GTGACAAAGGCCACGTAG-3' (SEQ ID NO.2); Upstream primer IS-F2: 5'-TACCGCCACCGTGATTTC-3' (SEQ ID NO.4); Downstream primer IS-R2: 5'-TCCGGGAAATAGCTGCC-3' (SEQ ID NO.5); Upstream primer F3: 5'-AGATTTGGACCTGCGAGCG-3' (SEQ ID NO.7); Downstream primer R3: 5'-GAGCGGCTGTCTCCACAAGT-3' (SEQ ID NO.8).

[0021] The Mycobacterium tuberculosis complex fluorescence quantitative PCR amplification detection reagent containing the above primers includes the following components: Upstream primers IS-F1, IS-F2, F3; downstream primers IS-R1, IS-R2, R3; and probes P1-FAM, P2-FAM, and P3-ROX. The sequences and structures of the probes P1-FAM, P2-FAM, and P3-ROX are shown below: Probe P1-FAM: 5'-F-ACCTATGTGTCGACCTGGGCAG-Q1-3' (SEQ ID NO.3); Probe P2-FAM: 5'-F-CCGTGCCGCAACCATCGAC-Q1-3' (SEQ ID NO.6); Probe P3-ROX: 5'-R-TTCTGACCTGAAGGCTCTGCGCGC-Q2-3' (SEQ ID NO.9); Where F = FAM, R = ROX, Q1 = DBQ1 (double quenching), Q2 = BHQ2.

[0022] To further illustrate the present invention, the technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0023] Unless otherwise specified, the production processes, experimental methods, or testing methods involved in the embodiments of this invention are all conventional methods in the prior art, and their names and / or abbreviations are all conventional names in the field, which are very clear and distinct in the relevant application areas. Those skilled in the art can understand the conventional process steps based on the names and apply the corresponding equipment, and implement them according to conventional conditions or the conditions recommended by the manufacturer.

[0024] The various instruments, equipment, raw materials or reagents used in the embodiments of this invention are not subject to any special restrictions on their source. They are all conventional products that can be purchased through regular commercial channels and can be prepared according to conventional methods known to those skilled in the art.

[0025] Example 1 Two sets of primers for quantitative real-time PCR detection of Mycobacterium tuberculosis complexes, and one set of primers for human RNase P, including upstream and downstream primers, are provided below: Upstream primer IS-F1: 5'-GACCACCAGCACCTAACCG-3' (SEQ ID NO.1); Downstream primer IS-R1: 5'-GTGACAAAGGCCACGTAG-3' (SEQ ID NO.2); Upstream primer IS-F2: 5'-TACCGCCACCGTGATTTC-3' (SEQ ID NO.4); Downstream primer IS-R2: 5'-TCCGGGAAATAGCTGCC-3' (SEQ ID NO.5); Upstream primer F3: 5'-AGATTTGGACCTGCGAGCG-3' (SEQ ID NO.7); Downstream primer R3: 5'-GAGCGGCTGTCTCCACAAGT-3' (SEQ ID NO.8); The Mycobacterium tuberculosis complex fluorescence quantitative PCR amplification detection reagent containing the above primers includes the following components: Upstream primers IS-F1, IS-F2, F3; downstream primers IS-R1, IS-R2, R3; and probes P1-FAM, P2-FAM, and P3-ROX. The sequences and structures of the probes P1-FAM, P2-FAM, and P3-ROX are shown below: Probe P1-FAM: 5'-F-ACCTATGTGTCGACCTGGGCAG-Q1-3' (SEQ ID NO.3); Probe P2-FAM: 5'-F-CCGTGCCGCAACCATCGAC-Q1-3' (SEQ ID NO.6); Probe P3-ROX: 5'-R-TTCTGACCTGAAGGCTCTGCGCGC-Q2-3' (SEQ ID NO.9); Where F = FAM, R = ROX, Q1 = DBQ1 (double quenching), Q2 = BHQ2.

[0026] The 25 μL detection reagent contains the following primer and probe concentrations: 0.4 μM upstream primer IS-F1, 0.4 μM upstream primer IS-F2, 0.1333 μM upstream primer F3, 0.4 μM downstream primer IS-R1, 0.4 μM downstream primer IS-R2, 0.1333 μM downstream primer R3, 0.2 μM probe P1-FAM, 0.2 μM probe P2-FAM, and 0.0667 μM probe P3-ROX.

[0027] Example 2: Method for detecting nontuberculous mycobacteria and other pathogens using quantitative real-time PCR with detection reagents. 2.1 DNA Extraction 1. Sputum liquefaction: Add the sputum sample to a test tube with a screw cap. Depending on the sample's characteristics, add 1-2 times the volume of 4% NaOH digestion solution to the tube. Tighten the screw cap and vortex for 1 minute to fully homogenize the sputum. Place the tube in a biosafety cabinet at room temperature for approximately 15-20 minutes, shaking several times during this period to ensure complete liquefaction. Centrifuge the liquefied sputum at 13000 rpm for 5 minutes, collect the precipitate, add 1 mL of physiological saline to the precipitate, wash, centrifuge again, and resuspend the precipitate in 0.5 mL of physiological saline and mix well for later use.

[0028] 2. The specific procedures for DNA extraction are as follows: S1. Take 200 μL of the above resuspension, add 400 μL of lysis buffer, mix, heat at 80℃ for 15 minutes, and mix again 2 to 3 times during the process. S2. Add 15 μL of magnetic bead suspension (mix the magnetic beads thoroughly before use), shake to mix for 1 min, then place on a rotary mixer or mix manually for 10 min. S3. Place the centrifuge tube on the magnetic rack for 1 minute to allow the liquid to become completely clear and the magnetic beads to be fully adsorbed. Then, use a pipette to remove the liquid from the tube and try to dry it as much as possible. S4. Remove the centrifuge tube, add 500 μL of washing solution, shake to mix for 1 min, place the centrifuge tube on a magnetic rack for 1 min to adsorb, and wait until the liquid is completely clear and the magnetic beads in the tube are fully adsorbed. Then, use a pipette to remove the liquid in the tube and try to dry it as much as possible. S5. Repeat step S4 twice; S6. Remove the centrifuge tube, add 100 μL of elution buffer, and incubate at 65°C with shaking for 3 min. If a centrifuge tube is not available, remove the tube and shake 3-4 times during the process to ensure complete elution of nucleic acids, which will then be the DNA template for PCR amplification.

[0029] 3. Detection Method The PCR amplification system consisted of 25 μL of 2×PCR amplification buffer, IS-F1, IS-F2, F3, IS-R1, IS-R2, R3, P1-FAM, P2-FAM, and P3, with purified water added to a final volume of 20 μL, and 5.0 μL of sample DNA. This system was placed in a real-time PCR instrument, and the PCR program was set as follows: 50℃, 2 min; 95℃, 30 s; (95℃, 10 s, 55℃, 30 s) for 45 cycles. Positive and negative controls were included in the experiment.

[0030] 2.2 Examples of Cross-Reactivity Detection for Nontuberculous Mycobacteria (NTM) Experiments have confirmed that this invention produces negative results for the following pathogens: Mycobacterium kansasii, Mycobacterium sugaensis, Mycobacterium marineum, Mycobacterium guildrums, Mycobacterium terrestris, Mycobacterium occulta, Mycobacterium minorum, Mycobacterium smegmatis, Mycobacterium ulcerativeum, Mycobacterium abscessus, Mycobacterium Gordonum, Mycobacterium gastritis, Mycobacterium bufotae, Mycobacterium intracellulare, Mycobacterium avium, Mycobacterium spp., and Mycobacterium scrofula, with no cross-reactivity. This kit also shows no cross-reactivity with the following pathogens: Streptococcus pneumoniae, Staphylococcus aureus, Saccharobacter influenzae, Nocardia, Escherichia coli, Pseudomonas aeruginosa, Staphylococcus epidermidis, Candida albicans, and Cryptococcus.

[0031] Comparative Example 1 The extraction kit described in this invention is compared with commercially available alcohol extraction kits.

[0032] 1.1 Component Comparison Commercially available alcohol extraction kits include pretreatment solution, lysis buffer, magnetic beads, washing solution I, washing solution II, and elution buffer, wherein washing solution I and washing solution II contain alcohol. The extraction reagent of this invention includes lysis buffer, magnetic beads, washing solution, and elution buffer, wherein the elution buffer does not contain alcohol, eliminating the need for a drying step during washing, making it safer and simpler.

[0033] 1.2 Comparison of Extraction Steps 1.2.1 Extraction steps of the present invention: S1. Take 200 μL of the pretreated sputum suspension, add 400 μL of lysis buffer, mix, heat at 80℃ for 15 minutes, and mix again 2 to 3 times during the process. S2. Add 15 μL of magnetic bead suspension (mix the magnetic beads thoroughly before use), shake to mix for 1 min, then place on a rotary mixer or mix manually for 10 min. S3. Place the centrifuge tube on the magnetic rack for 1 minute to allow the liquid to become completely clear and the magnetic beads to be fully adsorbed. Then, use a pipette to remove the liquid from the tube and try to dry it as much as possible. S4. Remove the centrifuge tube, add 500 μL of washing solution, shake to mix for 1 min, place the centrifuge tube on a magnetic rack for 1 min to adsorb, and wait until the liquid is completely clear and the magnetic beads in the tube are fully adsorbed. Then, use a pipette to remove the liquid in the tube and try to dry it as much as possible. S5. Repeat step S4 twice; S6. Remove the centrifuge tube, add 100 μL of elution buffer, and incubate at 65°C with shaking for 3 min. If a centrifuge tube is not available, remove the tube and shake 3-4 times during the process to ensure complete elution of nucleic acids, which will then be the DNA template for PCR amplification.

[0034] 1.2.2 Extraction steps using a commercially available alcohol extraction kit: S1. Take 200 μL of the pretreated sputum suspension, add 400 μL of lysis buffer, mix, heat at 70℃ for 10 minutes, and mix again 2 to 3 times during the process. S2. Take 400 μL of magnetic bead suspension (mix the magnetic beads thoroughly before use), place it on a magnetic rack for 1 min to adsorb, discard the supernatant, add the lysed sample mixture, place it on a rotary mixer or mix manually for 10 min. S3. Place the centrifuge tube on the magnetic rack for 1 minute to allow the liquid to become completely clear and the magnetic beads to be fully adsorbed. Then, use a pipette to remove the liquid from the tube and try to dry it as much as possible. S4. Remove the centrifuge tube, add 500 μL of washing solution I, shake to mix for 1 min, place the centrifuge tube on a magnetic rack for 1 min to adsorb, and wait until the liquid is completely clear and the magnetic beads in the tube are fully adsorbed. Then, use a pipette to remove the liquid in the tube and try to dry it as much as possible. S5. Remove the centrifuge tube, add 500 μL of washing solution II, and repeat the washing step S4 above. S6. Repeat step S5 and wash once with washing solution II. S7. Open the cap and leave it open for 3 minutes to allow the reagents inside to dry. Then add 100 μL of elution buffer and incubate at 65°C with shaking for 3 minutes. If you do not have an incubator, you may need to remove the tube and shake it 3-4 times during this period to ensure that the nucleic acid is fully eluted. This will give you the DNA template for PCR amplification.

[0035] 1.3 Comparison of Reagent Performance Four simulated sputum samples were collected, with bacterial concentrations of 10... 0 10 2 10 3 10 4Nucleic acid was extracted using the kit described in this invention and a commercially available alcohol-based extraction kit (comparative reagent 1) at CFU / mL. The corresponding nucleic acids were collected, and the two groups of nucleic acids were detected using the amplification method described in this invention. The results are shown in Table 1. Comparison revealed that the Ct value of the simulated sputum sample extracted using the kit of this embodiment was 0.2-0.8 Ct lower than that extracted using the kit of comparative reagent 1. There was a linear relationship between the Ct value and the logarithm of the starting template; a 10-fold difference in template amount corresponded to a Ct value difference of approximately 3.3 cycle numbers. The Ct value accurately characterizes the quantity and quality of the target nucleic acid. Corresponding to this invention, the recovery rate of the simulated sputum sample extraction kit for the target nucleic acid is approximately 1.1-1.7 times higher than that of the commercially available alcohol-based extraction kit (comparative reagent 1).

[0036] Table 1. Comparison of Reagent Performance

[0037] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, and not all embodiments. People can obtain other embodiments based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A composition for detecting Mycobacterium tuberculosis complex, characterized in that, The composition comprises: The first primer pair consists of the upstream primer shown in SEQ ID NO.1 and the downstream primer shown in SEQ ID NO.2; the first probe consists of the sequence shown in SEQ ID NO.3; The second primer pair consists of the upstream primer shown in SEQ ID NO.4 and the downstream primer shown in SEQ ID NO.5; the second probe has the sequence shown in SEQ ID NO.6; The third primer pair consists of the upstream primer shown in SEQ ID NO.7 and the downstream primer shown in SEQ ID NO.8; the third probe has the sequence shown in SEQ ID NO.

9.

2. The composition according to claim 1, characterized in that, The first probe, the second probe, and the third probe are all fluorescently labeled probes.

3. The composition according to claim 2, characterized in that, The first and second probes are labeled with a FAM fluorescent reporter group at their 5' end and a first quencher group at their 3' end; the third probe is labeled with a ROX fluorescent reporter group at its 5' end and a second quencher group at its 3' end; the first quencher group is DBQ1 and the second quencher group is BHQ2.

4. A detection reagent for binding mycobacteria, characterized in that, The detection reagent includes the composition of claim 1.

5. A kit for detecting Mycobacterium tuberculosis, characterized in that, The kit comprises the composition of claim 1, lysis buffer, magnetic beads, washing solution, and elution solution.

6. A method for detecting Mycobacterium tuberculosis, characterized in that, Includes the following steps: S1. Extract DNA from the sample to be tested; S2. Using the DNA of the sample to be tested as a template, perform PCR amplification reaction using the primer pair described in claim 1 and the probe of the Mycobacterium tuberculosis gene; S3. The fluorescence quantitative PCR instrument detects the reaction products. The appearance of a fluorescence signal indicates that the sample contains Mycobacterium tuberculosis, and vice versa.

7. The detection method according to claim 6, characterized in that, The PCR amplification reaction system was 25 μL, wherein the concentrations of each primer and probe were as follows: the primers shown in SEQ ID NO.1 and 2 were both 0.4 μM, the primers shown in SEQ ID NO.4 and 5 were both 0.4 μM, the primers shown in SEQ ID NO.7 and 8 were both 0.1333 μM, the probes shown in SEQ ID NO.3 and 6 were both 0.2 μM, and the probes shown in SEQ ID NO.9 were 0.0667 μM.

8. The detection method according to claim 6, characterized in that, The PCR amplification reaction conditions were: 50℃, 2 min; 95℃, 30 s; 95℃, 10 s, 55℃, 30 s, for a total of 45 cycles.

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