CN116769939A - Primer combination for detecting fluoroquinolone drug-resistant mutation of mycobacterium tuberculosis - Google Patents

Primer combination for detecting fluoroquinolone drug-resistant mutation of mycobacterium tuberculosis Download PDF

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CN116769939A
CN116769939A CN202310571194.XA CN202310571194A CN116769939A CN 116769939 A CN116769939 A CN 116769939A CN 202310571194 A CN202310571194 A CN 202310571194A CN 116769939 A CN116769939 A CN 116769939A
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resistant
mycobacterium tuberculosis
fluoroquinolone
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夏雪山
张敏
冯悦
刘高文
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Kunming University of Science and Technology
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Abstract

The invention discloses a primer combination for detecting drug-resistant mutation of fluoroquinolone of mycobacterium tuberculosis, which designs a primer and a probe according to a fluoroquinolone drug-resistant determining region of mycobacterium tuberculosis, constructs a single-tube multiplex real-time PCR system, detects the drug-resistant mutation of fluoroquinolone of mycobacterium tuberculosis on samples such as sputum, and can detect codons of 6 amino acid loci corresponding to gyrA gene and gyrB gene of fluoroquinolone drug-resistant determining region of mycobacterium tuberculosis, cover 13 drug-resistant mutation types, judge whether drug-resistant mutation and specific mutation types are generated based on detection results, can also be used for detecting heterogeneous drug resistance, monitor the proportion of wild strain to drug-resistant mutation strain in a patient before and after drug administration, and can provide more accurate and scientific auxiliary diagnosis and treatment basis for patients infected by mycobacterium tuberculosis.

Description

Primer combination for detecting fluoroquinolone drug-resistant mutation of mycobacterium tuberculosis
Technical Field
The invention belongs to the technical field of biology, and particularly relates to a primer and a method for detecting fluoroquinolone drug-resistant mutation of mycobacterium tuberculosis based on a double-labeled oligonucleotide probe dissolution curve method.
Background
Tuberculosis (TB) is a chronic infectious disease caused by mycobacterium tuberculosis infection and is widely prevalent worldwide. At present, tuberculosis is still a global important public health problem, and the number of new tuberculosis patients worldwide reaches 1060 ten thousand per year. Wherein, the success rate of the treatment of the sensitive tuberculosis is more than 90 percent, and the cure rate of the global drug-resistant tuberculosis is only about 50 percent. Treatment of drug-resistant patients has been a major concern at home and abroad. The drug-resistant tuberculosis currently referred to by us comprises rifampicin resistance (RR-TB, whether resistant to other antitubercular drugs or not), multi-drug-resistant tuberculosis (MDR-TB, resistant to at least two or more first-line antitubercular drugs including isoniazid and rifampicin), quasi-broadly-resistant tuberculosis (Pre-XDR-TB, according to the definition of MDR-TB/RR-TB, and resistant to any fluoroquinolone drug) and broadly-resistant tuberculosis (XDR-TB, according to the definition of MDR-TB/RR-TB), and resistant to at least one of fluoroquinolone drug, bedaquiline and linezolid. Compared with common tuberculosis, the drug-resistant tuberculosis has the advantages of complex diagnosis, difficult treatment, long treatment course, poor curative effect and high cost. Therefore, diagnosis of drug-resistant tuberculosis is a key ring of control of tuberculosis, and is a serious problem in realizing 'stopping tuberculosis'.
At present, MDR-TB and XDR-TB are very difficult to treat. Antitubercular Fluoroquinolones (FQ) are widely used for the treatment of multi-drug resistant tuberculosis. FQs resistance is typically associated with mutations in the gyrA and gyrB genes encoding DNA gyrase. Up to now, 21 SNP sites associated with gyrA mutation and 29 SNP sites associated with gyrB have been known to be used for susceptibility prediction analysis of genotype to FQ resistance. Wherein the drug-resistant sites definitely related to the fluoroquinolone-resistant drugs are mainly concentrated at codons corresponding to amino acids 88, 90, 91 and 94 in the gyrA gene, and codons corresponding to amino acids 499 and 501 in the gyrB gene. The fluoroquinolone drugs can be used for a long time, and can be accompanied with the generation of drug resistance, and early, sensitive and accurate detection technology is required to be developed for monitoring.
The diagnosis technology of drug-resistant tuberculosis is mainly divided into two main categories: the first is the phenotypic method (Roche Medium Scale, H710/11 scale, MGIT 960); the second type is a genotype method (molecular beacon, reverse hybridization and dissolution curve), and compared with the genotype method, the phenotype method is a gold standard for drug resistance diagnosis, has more detection drugs, and has the defects of long detection period, high biological safety requirement and the like, and is not beneficial to rapid diagnosis of drug resistance tuberculosis. The genotype method has the advantages of short detection period, good repeatability of detection results and low biosafety, and has been widely used. Currently, tuberculosis drug-resistant molecular diagnostic reagents applied to the market have some problems; for example CN201110137832.4 does not distinguish between specific mutation site types for fluoroquinolone resistance; CN202211491843.7 can only detect gyrA gene, and cannot cover the existing major drug-resistant mutation site. In addition, the existing tuberculosis drug-resistant molecular diagnostic reagent also has the problems of long detection time, high cost, low detection sensitivity, low specificity and the like.
Disclosure of Invention
The invention provides a primer combination for detecting drug-resistant mutation of mycobacterium tuberculosis fluoroquinolone, which is based on a double-labeled oligonucleotide probe dissolution curve analysis technology and can rapidly, sensitively and specifically detect the drug-resistant mutation of mycobacterium tuberculosis fluoroquinolone.
The aim of the invention is achieved by the following technical scheme:
(1) Extracting mycobacterium tuberculosis sample DNA;
(2) Designing primers and probes according to a fluoroquinolone resistance determining region (QRDR) of the mycobacterium tuberculosis by using Primer design software Primer 5;
the specific method for designing the primer probe is as follows:
A. the primer is designed at two ends of a mutation site covered by the gyrA gene and the gyrB gene, and the mutation site is covered by the probe;
B. the number of the probes is 3, and Probe A: covering codons corresponding to amino acids 88, 90 and 91 of the gyrA gene, marking FAM at the 5 'end and BHQ1 at the 3' end; probe B: covering the codon of the gyrA gene corresponding to the 94 th amino acid, marking ROX at the 5 'end and BHQ2 at the 3' end; probe C: covering the 499 th and 501 th amino terminal codons of gyrB gene, and marking HEX at 5 'end and BHQ1 at 3' end. In addition, to better distinguish wild-type strains from mutant strains, LNA was used to modify the major mutation sites.
The specific sequences of the primer and the probe are as follows:
gyrA-F：5’-TGCCGAGACCATGGGCAAC-3’
gyrA-R：5’-GAAGTTGCCCTGGCCGTC-3’
gyrB-F：5’-CAATGTGGAGAAAGCGCGC-3’
gyrB-R：5’-CTTGCCGATATCGAACTCGTC-3’
Probe A：5’-FAM-ACGGCGACGCGTCGA-BHQ1-3’
Probe B：5’-ROX-TACGACAGCCTGGTGCGCAT-BHQ2-3’
Probe C：5’-HEX-AAAGAACACCGAAGTTCAGGCG-BHQ1-3’；
(3) The most common mutation site gyrAAP 94Asn (GAC > AAC) of fluoroquinolone is selected as a research object, a single monochromatic real-time PCR system is established, PCR detection of drug-resistant mutation of fluoroquinolone of mycobacterium tuberculosis is carried out, the difference of melting points (Tm values) of dissolution curves between a sample to be detected and a wild type positive standard is compared, and whether the sample is mutated or not and mutation types are judged.
The single-tube monochromatic real-time PCR system comprises: the 25. Mu.L PCR reaction system contained 1 XPCR buffer (10 mM Tris-HCl, pH8.6, 50mM KCl,5% glycerol), 2.0mM MgCl 2 、dNTP Mix 0.2mM、2U Titanium Taq DNAPolymerase、0.06μM gyrA-F、0.6μM gyrA-R、0.4μM probe-B；
The PCR reaction program is as follows: pre-denaturation at 95℃for 3min; denaturation at 95℃for 10s, annealing at 58℃for 15s, extension at 63℃for 25s,55 cycles, and fluorescence signal collection at 58℃for 15 s; denaturation at 95℃for 2min; preserving heat at 45 ℃ for 2min; carrying out dissolution curve analysis at 45-90 ℃ at a heating rate of every 0.1 ℃ from 45-90 ℃, and collecting a fluorescent signal of a ROX channel in a dissolution curve program;
(4) Based on a single-color real-time PCR system, a single-tube multiple fluoroquinolone drug-resistant mutation detection system is established, FAM channels detect codons corresponding to amino acids 88, 90 and 91 of a gyrA gene, ROX channels detect codons corresponding to amino acid 94 of the gyrA gene, and HEX channels detect codons corresponding to amino acid 499 and 501 of the gyrB gene;
the single-tube multiplex real-time PCR system is as follows: the 25. Mu.L PCR reaction system contained 1 XPCR buffer (10 mM Tris-HCl, pH8.6, 50mM KCl,5% glycerol), 2.0mM MgCl 2 、dNTP Mix 0.2mM、2U Titanium Taq DNAPolymerase、0.06μM gyrA-F、0.6μM gyrA-R、0.06μM gyrB-F、1.2μM gyrB-R、0.4μM probe-A、0.4μM probe-B、0.4μM probe-C；
The PCR reaction program is as follows: pre-denaturation at 95℃for 3min; denaturation at 95℃for 10s, annealing at 58℃for 15s, extension at 63℃for 25s,55 cycles, and fluorescence signal collection at 58℃for 15 s; denaturation at 95℃for 2min; preserving heat at 45 ℃ for 2min; and (3) carrying out dissolution curve analysis at 45-90 ℃ at a heating rate of every 0.1 ℃ from 45-90 ℃, and collecting fluorescence signals of three channels of FAM, ROX and HEX in a dissolution curve program.
Compared with the existing method for detecting the fluoroquinolone resistance of the mycobacterium tuberculosis, the invention has the following advantages:
(1) The primer probe combination provided by the invention is used for detecting the drug resistance of fluoroquinolone drugs, and the amplified fragment covers gyrA and gyrB genes related to the drug resistance of fluoroquinolone drugs and comprises main drug resistance gene mutation sites of codons of 88 th, 90 th, 91 th and 94 th amino acids corresponding to the gyrA genes and codons of 499 th and 501 th amino acids corresponding to the gyrB genes; compared with the existing detection method, the method has the advantages that more drug-resistant sites are covered;
(2) By adopting the double-labeled oligonucleotide probe, the probe can select multiple types of fluorescent groups and quenching groups, and LNA modification can be performed, so that the wild strain and the mutant strain can be better distinguished;
the invention greatly improves the sensitivity and specificity of the detection of the drug-resistant mutation of the fluoroquinolone of the mycobacterium tuberculosis, can assist a clinician in primarily judging the drug-resistant condition (drug-resistant gene type and concentration) of specific mutation sites and types in clinic, and provides more accurate and scientific auxiliary diagnosis and treatment basis for patients infected by the mycobacterium tuberculosis.
Drawings
FIG. 1 shows the detection result of a single monochromatic real-time PCR reaction system;
FIG. 2 shows the results of the codon mutation detection of the gyrA gene for amino acids 88, 90 and 91;
FIG. 3 shows the result of detecting the mutation of the codon corresponding to amino acid 94 in the gyrA gene;
FIG. 4 shows the results of codon mutations corresponding to amino acids 499 and 501 in the gyrB gene;
FIG. 5 shows the results of a ratio study of the detected mutation sites of gyrA Asp94Asn (GAC > AAC);
FIG. 6 shows the results of the lowest limit of detection of the mutation site of gyrA Asp94Asn (GAC > AAC).
Detailed Description
The present invention will be described in further detail below with reference to the drawings and examples, which should not be construed as limiting the invention, wherein the methods of the present invention, unless otherwise specified, are all conducted in conventional manner, and wherein the reagents are employed, either as conventional reagents or as conventionally configured reagents, unless otherwise specified;
example 1: design of fluoroquinolone drug mutant gene detection primer probe
Using FMCA technology principle, when TaqMan probe is free in solution and in random coiled state, the fluorescent group marked on the probe is relatively close to the quenching group, and the fluorescent signal is weaker due to fluorescence energy resonance transfer (fluorescence resonance energy transfer, FRET). When the target sequence exists in the reaction system, the probe and the target sequence are complementarily paired to form an extended state, and the fluorescent group and the quenching group are relatively far away and emit stronger fluorescent signals. When the amplification is finished, the product quantity reaches the highest peak, the fluorescence signal is also strongest, at the moment, the amplification product is heated up again, then the probe hybridized on the product is gradually dissociated from the target sequence to form a random coil state, the fluorescence signal is gradually weakened, and a change curve graph of the temperature and the fluorescence signal is drawn, so that the melting curve analysis can be performed. In addition, in order to allow multiple different mutation sites to be detected in the same fluorescent channel, LNA is used in probe design to modify the main mutation site so that the ΔTm value between the wild-type Tm and the more common mutant Tm is as large as possible.
The NCBI database is used for searching related gene sequences, primer probes are designed according to the gene gyrA and gyrB of the fluoroquinolone drug resistance determining region of the mycobacterium tuberculosis, and the software Oligo 7 is used for designing the primers and the probes, so that the problem of nonspecific amplification or low amplification efficiency caused by mutual pairing between a secondary structure and a nucleic acid sequence is avoided. The primer is arranged at two ends of the probe, the probe covers codons corresponding to 88 th, 90 th, 91 th and 94 th amino acids in the gyrA gene and codons corresponding to 499 th and 501 th amino acids in the gyrB gene, and the probe is modified by adopting a fluorescence reporter group, a fluorescence quenching group and LNA.
The sequence of the fluoroquinolone drug mutant gene detection reagent primer and probe is as follows:
name of the name Sequence(s)
gyrA-F 5’-TGCCGAGACCATGGGCAAC-3’
gyrA-R 5’-GAAGTTGCCCTGGCCGTC-3’
gyrB-F 5’-CAATGTGGAGAAAGCGCGC-3’
gyrB-R 5’-CTTGCCGATATCGAACTCGTC-3’
Probe A: 5’-FAM-ACGGCGACGCGTCGA-BHQ1-3’
Probe B 5’-ROX-TACGACAGCCTGGTGCGCAT-BHQ2-3’
Probe C： 5’-HEX-AAAGAACACCGAAGTTCAGGCG-BHQ1-3’
Remarks: the underlined sections are modified with locked nucleic acids (Lock Nucleotid Acid, LNA).
Example 2: artificially synthesized target sequence dissolution profile analysis
To examine the ability of the designed probes to distinguish wild type from mutant, we designed 14 target sequences, including 2 wild type target sequences (shown as SEQ ID NO:1, SEQ ID NO: 11) and 12 mutant target sequences (shown as SEQ ID NO:1-10, SEQ ID NO: 12-14) for codons corresponding to amino acids 88, 90, 91 and 94 in the gyrA gene and codons corresponding to amino acids 499 and 501 in the gyrB gene, and analyzed the dissolution profile;
examination of the dissolution Curve analysis System of the Probe and the artificially synthesized target sequence, 25. Mu.L of the PCR reaction System contained 1 XPCR buffer (10 mM Tris-HCl, pH8.6, 50mM KCl,5% glycerol), 2.0mM MgCl 2 0.25 mu M probe, 0.4 mu M fluorescent probe.
The dissolution profile analysis procedure was: pre-denaturation at 95 ℃ for 2min at 45 ℃ and incubation for 2min at 45 ℃ followed by dissolution profile analysis from 45 ℃ to 90 ℃ at a rate of rise per 0.1 ℃, fluorescence signals of three channels of FAM, ROX and HEX were collected in the dissolution profile procedure, dissolution profile analysis was performed on a SLAN-96S real-time fluorescence PCR instrument (macrostone, shanghai), and the ability of 3 probes to discriminate between wild-type and mutant templates (Δtm values) was examined. The Tm values of hybridization of the probe with the corresponding synthetic target sequence are shown in the table below; as can be seen from the table, since Probe A, probe B and Probe C were perfectly matched with the wild-type template, a dissolution peak with a higher Tm value (77.4 ℃ C., 71.8 ℃ C., 74.2 ℃ C., respectively) was generated, and a dissolution peak with a lower Tm value was generated with the corresponding mutant template, and the range of ΔTm for each Probe in the table was between 3 and 9 ℃ C. In summary, the probes designed by the invention can meet the requirement that the delta Tm value between the wild type target sequence and the mutant type target sequence is more than 2 ℃. Therefore, the delta Tm value of the sample to be tested and the wild-type control can be compared to determine whether mutation occurs.
Example 3: establishment of single monochromatic real-time PCR system
Establishing a single monochromatic real-time PCR system by using a most common mutation site gyrAAP 94Asn (GAC > AAC) of fluoroquinolone, performing PCR detection of drug-resistant mutation of mycobacterium tuberculosis fluoroquinolone, comparing the difference of melting points (Tm values) of dissolution curves between a sample to be detected and a wild type positive standard, and judging whether the sample is mutated or not and the mutation type;
the single-tube monochromatic real-time PCR system is as follows: the 25. Mu.L PCR reaction system contained 1 XPCR buffer (10 mM Tris-HCl, pH8.6, 50mM KCl,5% glycerol), 2.0mM MgCl 2 、dNTP Mix 0.2mM、2U Titanium Taq DNAPolymerase、0.06μM gyrA-F、0.6μM gyrA-R、0.4μM probe-B；
mu.L of a mixture containing gyraap 94Asn (GAC)>AAC) mutated DNA as template at a concentration of 10 4 Wild type positive plasmid of copies/. Mu.L was used as positive control, ddH 2 O was used as a negative control, and the wild type positive plasmid was derived from artificial synthesis.
The PCR reaction procedure was:
the first step: pre-denaturation at 95℃for 3min;
and a second step of: denaturation at 95℃for 10s, annealing at 58℃for 15s, extension at 63℃for 25s,55 cycles, and fluorescence signal collection at 58℃for 15 s;
and a third step of: denaturation at 95℃for 2min;
fourth step: preserving heat at 45 ℃ for 2min;
fifth step: carrying out dissolution curve analysis from 45 ℃ to 90 ℃ at a temperature rising rate of every 0.1 ℃ from 45 ℃ to 90 ℃, collecting a fluorescent signal of a ROX channel in a dissolution curve program, wherein a detection result is shown in a graph shown in figure 1, and the graph shows that a gyrA wild type template is completely matched with a probe, and a dissolution peak with a higher Tm value (71.8 ℃) is generated through the dissolution curve analysis; whereas the mutant template of gyraap 94Asn (GAC > AAC) had a mutation of G > a, which did not match the probe perfectly, resulting in a dissolution peak with a lower Tm value (67.4 ℃). The delta Tm value between the wild template and the mutant template is more than 2 ℃, so that whether the detection sample is mutated or not can be accurately judged, and further the method established by the invention can be used for detecting the fluoroquinolone drug-resistant mutation of the mycobacterium tuberculosis.
Example 4: establishment and analysis performance investigation of multiple real-time PCR system
Based on a single-color real-time PCR system, a single-tube multiple fluoroquinolone drug-resistant mutation detection system is established, FAM channels detect codons corresponding to amino acids 88, 90 and 91 of a gyrA gene, ROX channels detect codons corresponding to amino acid 94 of the gyrA gene, and HEX channels detect codons corresponding to amino acid 499 and 501 of the gyrB gene;
the single-tube multiplex real-time PCR system is as follows: the 25. Mu.L PCR reaction system contained 1 XPCR buffer (10 mM Tris-HCl, pH8.6, 50mM KCl,5% glycerol), 2.0mM MgCl 2 、dNTP Mix 0.2mM、2U Titanium TaqDNAPolymerase、0.06μM gyrA-F、0.6μM gyrA-R、0.06μM gyrB-F、1.2μM gyrB-R、0.4μMprobe-A、0.4μM probe-B、0.4μM probe-C。
The 12 pieces of mutant sequence DNA artificially synthesized in example 2 were taken as 5. Mu.L of a template, and the concentration was taken to be 10 4 Wild type positive plasmid of copies/. Mu.L was used as positive control, ddH 2 O was used as a negative control.
The PCR reaction program is as follows:
the first step: pre-denaturation at 95℃for 3min;
and a second step of: denaturation at 95℃for 10s, annealing at 58℃for 15s, extension at 63℃for 25s,55 cycles, and fluorescence signal collection at 58℃for 15 s;
and a third step of: denaturation at 95℃for 2min;
fourth step: preserving heat at 45 ℃ for 2min;
fifth step: carrying out dissolution curve analysis from 45 ℃ to 90 ℃ at a temperature rise rate of every 0.1 ℃ at 45 ℃ to 90 ℃, collecting fluorescence signals of three channels of FAM, ROX and HEX in a dissolution curve program, and detecting results shown in figures 2, 3 and 4; from the figure, it can be seen that the FAM channel produced 5 lytic peaks, comprising 1 gyrA wild-type lytic peak and 4 mutant lytic peaks of Gly88 Cys, gly88Ala, ala90Val, ser 91Pro; the ROX channel produces 6 lytic peaks, comprising 1 gyrA wild-type lytic peak and 5 mutant lytic peaks of Asp94Asn, asp94Tyr, asp94 His, asp94Gly, asp94 Ala; the HEX channel produced 4 lytic peaks, comprising one gyrB wild-type lytic peak and Asn 499Ala, asn 499Asp, gln 501Asp3 mutant lytic peaks. In summary, the single tube multiplex fluoroquinolone drug resistance mutation detection system established by the invention can detect 12 drug resistance mutation types related to fluoroquinolone drug resistance.
To examine the ability of heterogeneous drug resistance detection, we mixed wild-type genomic DNA with gyrA Asp94Asn (GAC > AAC) mutant DNA in equal proportions to prepare a mixed template containing 0%, 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% and 100% mutant DNA and examined the results are shown in fig. 5, and the results show that the detection system of the present invention can detect mutation ratios as low as 5%.
System sensitivity investigation: will contain gyrA Asp94Asn (GAC)>AAC) mutated genome was subjected to 10-fold gradient dilution to prepare DNA containing genome of 5X 10, respectively 5 /mL、5×10 4 /mL、5×10 3 /mL、5×10 2 /mL、5×10 1 template/mL and 5 copies/mL. Negative controls and no template controls were set simultaneously. The detection result is shown in FIG. 6, and the result shows that the lowest detection limit of the detection system is 5 multiplied by 10 1 The copies/mL has better detection sensitivity.
Example 5: clinical application for detecting fluoroquinolone drug-resistant mutation of mycobacterium tuberculosis based on double-labeled oligonucleotide probe dissolution curve method
1. 50 sputum cultures of patients with Mycobacterium tuberculosis infection were collected in Yunnan province infectious disease hospital;
2. mycobacterium tuberculosis DNA extraction
The extraction procedure can be performed using a commercial nucleic acid DNA extraction kit (gram positive g+ genomic DNA extraction kit, beijing Zhuang Meng) according to commercial kit instructions, and the purified nucleic acid is used for detection as follows:
A. collecting 1-loop of Mycobacterium tuberculosis grown on solid medium with 22SWG standard inoculating loop, suspending in 250 μl TBDNA extracting solution (kit self-contained), and extracting DNA with the kit;
B. mycobacterium tuberculosis grown in liquid medium 1mL was centrifuged at 10000rpm for 15min, the supernatant was discarded and the bacteria were resuspended in 250 μLTB DNA extract (kit self-contained); extracting DNA from the resuspended thalli by using a kit;
C. preparing a 25 mu L PCR reaction system, and performing single-tube multiplex real-time PCR in the same manner as in example 4;
D. taking 5 mu L of the DNA extracted in the step B as a template, and taking the concentration as 10 4 Wild type positive plasmid of copies/. Mu.L was used as positive control, ddH 2 O is used as a negative control;
3. amplification and dissolution curve analysis were both performed on a SLAN-96S real-time fluorescent PCR instrument (macrostone, shanghai) with the following reaction procedure:
the first step: pre-denaturation at 95℃for 3min;
and a second step of: denaturation at 95℃for 10s, annealing at 58℃for 15s, extension at 63℃for 25s,55 cycles, and fluorescence signal collection at 58℃for 15 s;
and a third step of: denaturation at 95℃for 2min;
fourth step: preserving heat at 45 ℃ for 2min;
fifth step: and (3) carrying out dissolution curve analysis at 45-90 ℃ at a temperature rising rate of every 0.1 ℃ from 45-90 ℃, and collecting fluorescence signals of three channels of FAM, ROX and HEX in a dissolution curve program.
Interpretation of the results: and judging whether the sample is mutated or not by comparing the difference of melting point Tm values of melting curves between the detected sample and the wild type template, wherein the melting point of the sample is consistent with that of the wild type template, namely, the sample is judged to be wild type when the error is not more than 1 ℃, and if the sample is inconsistent with the melting point Tm values, judging which mutation type the sample to be detected is by referring to the Tm values in the table of the embodiment 2.
50 sputum cultures were tested by the test method established by the invention, and the results: the fluoroquinolone drug resistance samples are 11, and the rest 39 samples show that the fluoroquinolone drug resistance is negative. Meanwhile, 50 samples are detected by adopting a culture method drug sensitivity experiment, and the result shows that the fluoroquinolone drug resistance samples are 12, the other drug resistance samples are 19, and the number of the complete wild type samples is 19. A first-generation sequencing verification is carried out on one sample with inconsistent drug sensitivity test and detection result of the invention, and the result shows that the sample is gyrB Asp494Ala (GAC > GCC) and is not contained in the detection site of the invention, so that the detection cannot be carried out.
The statistics of the clinical sample detection results are as follows:
in conclusion, compared with a drug-sensitive method, the fluoroquinolone drug-resistant mutation detection method disclosed by the invention is simple to operate, short in time consumption and good in accuracy, and can meet the requirement of clinical auxiliary diagnosis of the fluoroquinolone drug mutation gene detection of mycobacterium tuberculosis.
The above description is only of the preferred embodiments of the present invention and is not intended to limit the present invention, but various modifications and variations can be made to the present invention by those skilled in the art. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims (1)

1. Application of a group of primers in preparation of reagent for detecting fluoroquinolone drug-resistant mutation of mycobacterium tuberculosis, wherein the primers are as follows: gyrA-F:5'-TGCCGAGACCATGGGCAAC-3'
gyrA-R：5’-GAAGTTGCCCTGGCCGTC-3’
gyrB-F：5’-CAATGTGGAGAAAGCGCGC-3’
gyrB-R：5’-CTTGCCGATATCGAACTCGTC-3’
Probe A：5’-FAM-ACGGCGACGCGTCGA-BHQ1-3’
Probe B：5’-ROX-TACGACAGCCTGGTGCGCAT-BHQ2-3’
Probe C：5’-HEX-AAAGAACACCGAAGTTCAGGCG-BHQ1-3’；
The detection was performed using a double-labeled oligonucleotide probe dissolution profile method.
CN202310571194.XA 2023-05-20 2023-05-20 Primer combination for detecting fluoroquinolone drug-resistant mutation of mycobacterium tuberculosis Pending CN116769939A (en)

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN117230222A (en) * 2023-10-18 2023-12-15 鲲鹏基因（北京）科技有限责任公司 Composition, kit and method for detecting quinolone resistance of mycobacterium tuberculosis

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN117230222A (en) * 2023-10-18 2023-12-15 鲲鹏基因（北京）科技有限责任公司 Composition, kit and method for detecting quinolone resistance of mycobacterium tuberculosis

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