CN115786333A

CN115786333A - Detection reagent for drug resistance gene mutation of mycobacterium tuberculosis fluoroquinolone drugs and kit and method thereof

Info

Publication number: CN115786333A
Application number: CN202211491843.7A
Authority: CN
Inventors: 刘亚宝; 周慧玲
Original assignee: Sichuan Yazhong Gene Technology Co ltd
Current assignee: Sichuan Yazhong Gene Technology Co ltd
Priority date: 2022-11-25
Filing date: 2022-11-25
Publication date: 2023-03-14

Abstract

The invention discloses a detection reagent for drug-resistant gene mutation of mycobacterium tuberculosis fluoroquinolone drugs, a kit and a method thereof, and relates to the field of biological detection, wherein a primer-probe combination provided by the invention comprises a primer pair with a sequence shown as SEQ ID No. 1-2 and probes shown as SEQ ID No.3, SEQ ID No.4 or SEQ ID No.5, and can be used for detecting drug resistance of the mycobacterium tuberculosis drugs, the coverage rate of the drug-resistant gene mutation of the mycobacterium fluoroquinolone drugs can reach more than 90%, and the probes are specially modified, so that four drug-resistant mutations of mycobacterium tuberculosis gyrA genes in sputum, alveolar lavage fluid or isolated culture samples can be better detected and distinguished; based on the detection result and the clinical experience, the drug resistance condition of specific mutation sites and types in the clinic can be judged, and more accurate and scientific auxiliary diagnosis and treatment basis is provided for patients infected by mycobacterium tuberculosis.

Description

Detection reagent for drug resistance gene mutation of mycobacterium tuberculosis fluoroquinolone drugs and kit and method thereof

Technical Field

The invention relates to the field of biological detection, in particular to a detection reagent for drug resistance gene mutation of mycobacterium tuberculosis fluoroquinolone drugs, a kit and a method thereof.

Background

Tuberculosis (TB) is a chronic infectious disease, mostly occurring in the lung, with Tuberculosis being the most common. When the common tuberculosis patients have poor treatment compliance, unreasonable treatment scheme, poor absorption of the digestive system and the like, the mycobacterium tuberculosis in the body becomes more serious and can not be killed by one or more antitubercular drugs (namely, the antitubercular drugs have resistance). If the patient is infected with mycobacterium tuberculosis and has drug resistance to one or more antituberculosis drugs, the drug-resistant tuberculosis is obtained. As estimated by WHO/IUATLD's latest drug resistance monitoring, 10.2% of new patients are resistant to at least one antitubercular drug, and 1.1% are multidrug-resistant tuberculosis (MDR-TB); of the re-treated patients, 18.4% of patients were resistant to at least one antituberculotic drug, with an MDR-TB resistance rate of 7.0%. Multiple drug resistant tuberculosis poses a great challenge for the prevention and treatment of tuberculosis.

The fluoroquinolone medicine is artificially synthesized medicine and is used in clinical treatment of drug resistant tuberculosis. Fluoroquinolones are currently used clinically to try to serve as first-line drugs, shorten the course of treatment for TB, and are used for a long period of time. Their use is accompanied by the development of resistance to drugs, which requires the development of early, sensitive, accurate detection techniques for monitoring.

The fluoroquinolone drug-resistant tuberculosis needs to be diagnosed by a traditional drug sensitivity test or a molecular biological rapid detection technology. The report cycle of the traditional drug sensitivity test is as long as 6-8 weeks, which is not beneficial to the rapid diagnosis and treatment of mycobacterium tuberculosis infection.

At present, tuberculosis drug-resistant molecular diagnostic reagents applied to the market have some problems; for example, the patent (CN 201110137832.4) of Mycobacterium tuberculosis drug-resistant mutation detection reagent of Xiamen ameliota invented the specific mutation site type that could not distinguish fluoroquinolone drug-resistance; 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.

In view of this, the invention is particularly proposed.

Disclosure of Invention

The invention aims to provide a detection reagent for drug resistance gene mutation of mycobacterium tuberculosis fluoroquinolone drugs, a kit and a method thereof.

The invention is realized in the following way:

in a first aspect, embodiments of the present invention provide a nucleic acid composition comprising: the nucleotide sequence is shown as a primer pair of SEQ ID No. 1-2.

Optionally, the nucleic acid composition also comprises a probe, and the nucleotide sequence of the probe is shown as SEQ ID No.3.

Optionally, the 5' end of the probe has 1 to 5 base sequences that do not pair with the target.

In a second aspect, embodiments of the present invention provide a detection reagent comprising the nucleic acid composition of the preceding embodiments.

In a third aspect, the embodiments of the present invention provide a detection kit, which includes the nucleic acid composition described in the previous embodiments or the detection reagent described in the previous embodiments.

In a fourth aspect, the embodiments of the present invention provide an application of the nucleic acid composition described in the foregoing embodiments in preparing a detection reagent or a kit for drug resistance of mycobacterium tuberculosis fluoroquinolone drugs or mutations in drug resistance genes of mycobacterium tuberculosis fluoroquinolone drugs.

In a fifth aspect, an embodiment of the present invention provides a molecular detection method for drug-resistant gene mutation of mycobacterium tuberculosis fluoroquinolone drugs, including: the PCR detection of the sample using the nucleic acid composition or the detection reagent or the detection kit of the preceding examples is not directly aimed at the diagnosis or treatment of the disease.

The invention has the following beneficial effects:

the primer probe combination provided by the invention is used for detecting the drug resistance of the fluoroquinolone drugs, and the coverage rate of amplified fragments on the drug resistance gene mutation of the fluoroquinolone drugs can reach more than 90%; and a probe specially modified at the 5' end is adopted, so that four fluoroquinolone drug-resistant mutations, namely A90V, D G, D A and D94N of a gyrA gene of mycobacterium tuberculosis in sputum, alveolar lavage fluid or isolated culture samples can be detected and distinguished; according to clinical experience, the drug resistance (drug resistance gene type and concentration) of specific mutation sites and types in clinic is preliminarily judged, and more accurate and scientific auxiliary diagnosis and treatment basis is provided for patients infected by mycobacterium tuberculosis.

Drawings

In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings needed to be used in the embodiments will be briefly described below, it should be understood that the following drawings only illustrate some embodiments of the present invention and therefore should not be considered as limiting the scope, and for those skilled in the art, other related drawings can be obtained according to the drawings without inventive efforts.

FIG. 1 shows the results of detection of four drug-resistant mutant genes, namely, gyrA gene A90V, D G, D A and D94N;

FIG. 2 is a comparison of a probe having 2 tail bases at the 5' end with a conventional probe;

FIG. 3 is a comparison of a probe having 5 tail bases at the 5' end with a conventional probe;

FIG. 4 is a review of the detection ratio of gyrA A90V mutation sites;

FIG. 5 is a review of the detection ratio of gyrA D94G mutation sites;

FIG. 6 is a review of the detection ratio of gyrA D94A mutation sites;

FIG. 7 is a review of the detection ratio of gyrA D94N mutation sites;

FIG. 8 is a gyrA W wild (sensitive) lowest detection limit study;

FIG. 9 is a gyrA A90V minimum detection limit study;

FIG. 10 is a review of the gyrA D94G minimum detection limit;

FIG. 11 is a gyrA D94A minimum detection limit inspection;

fig. 12 is a gyrA D94N minimum detection limit test.

Detailed Description

In order to make the objects, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. The examples, in which specific conditions are not specified, were conducted under conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products available commercially.

First, an embodiment of the present invention provides a nucleic acid composition, including: the nucleotide sequence is shown as a primer pair of SEQ ID No. 1-2.

The primer pair provided by the invention covers 90 and 94 amino acid sites of a fluoroquinolone drug-resistant gene gyrA gene, and can be used for detecting at least one of the following mutation sites: the primer pairs of the invention have better detection specificity and sensitivity compared with other existing primer combinations, namely gyrA 90V, gyrA D94G, gyrA D94A and gyrA D94N.

In some embodiments, the nucleic acid composition further comprises a probe for detecting at least one mutation site in the gyrA gene a90V, D G, D a and D94N.

Optionally, the nucleotide sequence of the probe is shown as SEQ ID No.3.

In some embodiments, 1-5 base sequences unpaired with a target sequence are introduced into the 5 'end of the probe, so that the 5' tail tilting probe cannot be hydrolyzed by DNA polymerase, and the purposes of saving the probe dosage and improving the fluorescent signal are achieved.

Preferably, the nucleotide sequence of the probe is shown as SEQ ID No.4 or SEQ ID No.5.

The probe provided by the invention can detect and distinguish four drug-resistant mutations of a gyrA gene A90V, D94G, D A, D N, quickly identify whether a fluoroquinolone drug-resistant condition exists in a mycobacterium tuberculosis infected patient within 3 hours, judge the specific mutation type according to the detection result, and preliminarily determine the drug-resistant type, the drug-resistant concentration and the like of the infected patient to the fluoroquinolone drug.

In some embodiments, the probe has a fluorescent reporter attached to the 5 'end and a fluorescent quencher attached to the 3' end.

In some embodiments, the fluorescent reporter group is selected from: any one of 5-FAM, 6-FAM, HEX, TET, VIC, JOE, cy3, cy3.5, NED, TAMRA, ROX, texas Red, cy5, cy5.5, and Quasar 670.

In some embodiments, the fluorescence quencher group is selected from the group consisting of: any one of TAMRA, BHQ1, BHQ2, BHQ3 and MGB.

In some embodiments, the molar ratio of the forward primer to the reverse primer is 1: (1-10). Specifically, the molar ratio may be 1: 1. 1: 2. 1: 3. 1: 4. 1: 5. 1: 6. 1: 7. 1: 8. 1:9 and 1:10 or a range between any two.

In another aspect, embodiments of the invention provide a detection reagent comprising a nucleic acid composition as described in any of the preceding embodiments.

In some embodiments, the detection reagent is used for detecting drug resistance of mycobacterium tuberculosis fluoroquinolone drugs or drug resistance gene mutation of mycobacterium tuberculosis fluoroquinolone drugs.

In another aspect, the present invention provides a detection kit, which includes the nucleic acid composition described in any of the preceding embodiments or the detection reagent described in any of the preceding embodiments.

In some embodiments, the kit further comprises: and (4) PCR detection reagents.

In some embodiments, the PCR detection reagents comprise: at least one of PCR reaction buffer, taq enzyme, magnesium ion, dNTP and water.

In another aspect, the embodiment of the present invention provides an application of the nucleic acid composition described in any of the foregoing embodiments in preparing a detection reagent or a kit for a mycobacterium tuberculosis fluoroquinolone drug resistance gene mutation.

In addition, the embodiment of the invention also provides a detection method of mycobacterium tuberculosis fluoroquinolone drug resistance gene mutation, which comprises the following steps: performing PCR detection on a sample by using the nucleic acid composition described in any of the preceding embodiments or the detection reagent described in any of the preceding embodiments or the detection kit described in any of the preceding embodiments.

In some embodiments, the detection method is not directed toward the diagnosis or treatment of a disease.

In some embodiments, the detection method judges the detection result by a fluorescence PCR dissolution curve method: and judging whether corresponding mutation occurs according to the Tm value and the dissolution peak detected by each channel.

The invention analyzes and designs a primer probe combination capable of detecting the drug-resistant gene mutation by the gyrA gene sequence of the fluoroquinolone drug-resistant poly-determining region, and simultaneously realizes the detection of a PCR reaction system containing four drug-resistant mutant genes, namely A90V, D G, D A and D94N, in one reaction system by adopting an asymmetric PCR amplification method and combining a fluorescence PCR-dissolution curve method. The method has the advantages of short detection time, low cost, high detection sensitivity, strong specificity and the like, and the result judgment is convenient and intuitive, so that the effective detection of the drug resistance and the drug-resistant gene mutation can be realized.

In some embodiments, the upstream primer and the downstream primer are present in a molar ratio of 1: (1-10). The molar ratio may be specifically as described in any of the foregoing examples.

In some embodiments, the concentration of the upstream primer and the downstream primer is 0.01 to 1 μ M independently, and specifically may be any one or a range between any two of 0.01 μ M, 0.05 μ M, 0.1 μ M, 0.2 μ M, 0.4 μ M, 0.6 μ M, 0.8 μ M and 1 μ M, and preferably 0.04 to 0.4 μ M.

In some embodiments, the concentration of the probe is 0.1-0.5. Mu.M, and specifically can be any one or a range between any two of 0.1. Mu.M, 0.2. Mu.M, 0.3. Mu.M, 0.4. Mu.M, and 0.5. Mu.M.

In some embodiments, the reaction sequence for the PCR is as follows: 94-96 ℃ for 4.8-5.2 min; 10-20s at 94-96 ℃, 58-62 ℃, collecting fluorescence signals for 15-25s, 15-25s at 70-74 ℃ and 45-50 cycles; 94-96 ℃ for 0.8-1.2min, 33-37 ℃ for 2-3 min; the fluorescence signal is continuously collected at the temperature of 35-90 ℃ every 0.04 ℃/s.

In some embodiments, the dNTP is used at a concentration of 0.1 to 5mM, specifically, 0.1mM, 0.5mM, 1mM, 2mM, 3mM, 4mM, and 5mM, or a range between any two of them, in PCR assay.

In some embodiments, the concentration of Taq enzyme applied when performing the PCR assay is any one or a range between any two of 0.01U, 0.02U, 0.04U, 0.06U, 0.08U, 0.1U, 0.2U, 0.4U, 0.6U, 0.8U, and 1.0U.

In some embodiments, the sample can be a nucleic acid sample, an environmental sample containing a nucleic acid sample, or a manually configured sample (such as a negative control or a positive control).

The features and properties of the present invention are described in further detail below with reference to examples.

Example 1

A design method of fluoroquinolone drug mutant gene detection reagent primer probes.

The technical principle is as follows: the probe is in a flexible state in a free transition state, the fluorescent group is closer to the quenching group, the fluorescence of the fluorescent group is quenched, and the fluorescence cannot be detected at the moment. When the target sequence exists, the probe can be complementary with the target sequence, the luminescent group and the quenching group are far away due to extension, the fluorescence of the fluorescent group can not be quenched by the quenching group, and then the fluorescence signal can be detected. In the detection process of the melting analysis, as the temperature is increased, the probe and the target sequence show a process from complete binding to dissociation, so that the matching degree of the probe and the target sequence can be identified through the difference of melting points. In addition, when the same channel detects different mutation sites, the Tm values of different detection probes need to be designed to be 5-10 degrees different.

The NCBI database is used for searching related gene sequences, primer probes are designed according to gyrA genes in a fluoroquinolone drug resistance decision zone of mycobacterium tuberculosis, and the primers and the probes are designed by using software Oligo 7, so that the problem of non-specific amplification or low amplification efficiency caused by mutual pairing of a secondary structure and a nucleic acid sequence is avoided. The primers are at both ends of the probe, and the probe covers gyrA A90V, D94G, D A, D N and the site. The probe adopts a special design of 5 'tail warping, namely 1 to 5 base sequences which are not matched with a target sequence are introduced into the 5' end of the probe, so that the hydrolysis reaction of the probe is avoided.

The probe is modified by a fluorescence reporter group and a fluorescence quenching group.

The sequences of the fluoroquinolone drug mutant gene detection reagent primers and probes are as follows.

TABLE 1 primer Probe sequences

Remarking: primer is a Primer, F is an upstream Primer, R is a downstream Primer, and Probe is a Probe; the TA base at the 5' end (bold) of Probe-P1 is 2 waning tail bases which are not matched with the target;

the TATAT base at the 5' end (bold) of Probe-P2 is the introduced 5 tail bases.

Example 2

A detection reagent for drug resistance or drug resistance gene mutation of mycobacterium tuberculosis fluoroquinolone drugs specifically comprises the following components.

1. The combinations of primers and probes are shown below.

2. The PCR reaction solution contains: PCR reaction buffer solution, 0.1-0.5 mmol/L deoxyribonucleoside triphosphate, upstream primer and downstream primer of target gene 0.1-1 mu mol/L, positive and negative primer ratio is 1:10, 0.1-1 mu mol/L of a probe of a target gene, and 0.005-0.05U/mu L of Taq DNA polymerase. The specific reagents were configured as follows.

The combination can detect four drug-resistant mutant genes of gyrA 90V, D94G, D A, D N and different mutation combinations thereof by a single tube. The detection results are shown in FIG. 1.

Example 3

A method for detecting drug resistance or drug resistance gene mutation of a mycobacterium tuberculosis fluoroquinolone drug comprises the following steps.

1. Collecting samples: a mycobacterium tuberculosis culture.

2. Sample pretreatment:

2.1 sputum treatment mode: the sample and the liquefied liquid 1:1 are mixed and liquefied until the sample reaches the desired fluidity. 200 μ L of the liquefied solution was used for subsequent nucleic acid extraction.

2.2 Mycobacterium tuberculosis isolated culture treatment mode:

mycobacterium tuberculosis grown on solid medium, bacterial 1-loop was harvested with 22SWG standard inoculation loop and resuspended in 200. Mu.L of TB DNA extract for subsequent nucleic acid extraction.

The Mycobacterium tuberculosis grown in the liquid culture medium is taken to be 1mL, centrifuged at 12000rpm for 15min, the supernatant is discarded, and the precipitate is resuspended in 200 mu L of TB DNA extracting solution for subsequent nucleic acid extraction.

2.3 alveolar lavage fluid treatment: 1-5 ml of alveolar lavage fluid is taken, centrifuged at 10000rpm for 1 minute, the supernatant is discarded, and the precipitate is resuspended in 200. Mu.L of TB DNA extract for subsequent nucleic acid extraction.

3. Extracting nucleic acid of a sample:

commercial nucleic acid DNA extraction kits (e.g., qiagen DNA Mini Kit, etc.) can be used, the extraction process can be performed according to the instructions of the commercial Kit, and the purified nucleic acid can be used for subsequent PCR amplification.

In this example, a Qiagen DNA Mini Kit was used, and the specific procedures were as follows:

3.1 to a 1.5ml EP tube containing 200. Mu.L of the sample treatment solution, 180. Mu.l of buffer ATL and 20. Mu.L of proteinase K were added, and incubated for 10min after vortexing and shaking for 10 seconds.

3.2 mu.L of buffer AL was added to the aforementioned EP tube. Mix well and vortex for 15s.

3.3 Incubate at 70 ℃ for 10min. Add 200. Mu.L of alcohol (95% -100%) and vortex for 15s. After brief centrifugation, the supernatant was transferred to a QIAamp mini-column, and the mini-column was placed in a 2ml collection tube, which was capped.

3.4 centrifuge the collection tube 6000 Xg for 1min. The old collection tube was discarded along with the filtrate.

3.5 QIAamp mini-columns were placed in new 2ml collection tubes. To QIAamp mini column was added 500. Mu.l of buffer AW1. The lid was closed and centrifuged at 6000 Xg for 1min. Discarding the old collection tube together with the filtrate;

3.6 QIAamp mini columns were placed in new 2ml collection tubes. To QIAamp mini column was added 500. Mu.l of buffer AW2. Cover the lid and centrifuge at 20000 Xg for 3min.

3.7 put QIAamp mini column into a new 2ml collection tube, and the old tube together with the filtrate discarded. Centrifuge at 6000 Xg for 1min.

3.8 QIAamp mini-columns were placed in clean 1.5ml EP tubes. The old collection tube containing the filtrate was discarded. Carefully open the lid and add 200. Mu.l buffer AE. The lid was closed and then allowed to stand at room temperature for 1 minute.

3.9 centrifugation at 8000rpm for 1 minute. The QIAamp mini column was discarded, and the EP tube containing the eluate was retained and subsequently available for PCR amplification.

3. Adding a reagent:

3.1 adding 20. Mu.L of PCR reagent into the PCR tube according to the detection reagent prepared in example 2;

3.2 adding 5 μ L of the extracted nucleic acid into the PCR amplification system, mixing, covering the cap of the PCR tube, and waiting for loading.

Setting and operating a PCR amplification program:

4.1 PCR amplification is carried out on fluorescent quantitative PCR instruments such as Roche LightCycler 480, SLAN-96P, etc.

4.2 open the instrument and place the PCR tube to be tested.

4.3 set up the amplification program. The corresponding fluorescence channel FAM channel (Reporter: FAM, quencher: none) was selected. And carrying out sample editing.

4.4 run the amplification program.

4.5 specific amplification procedures were as follows:

the first step is as follows: 5min at 95 ℃;

the second step is that: 1, collecting fluorescence signals at the positions of 95 ℃ and 15s,55 ℃ and 30s,72 ℃ and 30s, and performing 50 cycles at 55 ℃ and 30 s;

the fifth step: continuously collecting fluorescence signals at 40-90 ℃, wherein FAM is selected as a fluorescence channel.

5. And (4) analyzing results:

5.1 after the reaction is finished, the result is automatically stored by the instrument, and the automatic analysis can be carried out by utilizing the software of the instrument.

5.2 judging whether the sample is mutated or not by comparing the difference of melting curve melting points (Tm) between the detected sample and a wild control; judging the wild type when the difference delta Tm of the melting point of the detection sample and the melting point of the wild control is less than or equal to +/-1 ℃; and when the difference delta Tm between the melting point of the detection sample in any one of the 4 channels and the melting point of the wild control is more than or equal to +/-2 ℃, determining the drug-resistant mutation type, and then determining the specific drug-resistant mutation site information according to the difference between the melting points of the corresponding channel and the wild control.

6. And (4) interpretation of results:

the specific interpretation rule of the detection result can refer to the following table:

type of sample	FAM	Interpretation of results
			Wild control	74.20±1℃	Sensitive strain
Negative control	-	-
			gyrA A90V	-8.42	Drug resistance of fluoroquinolones
gyrA D94G	+2.91	Drug resistance of fluoroquinolones
			gyrA D94A	+4.67	Drug resistance of fluoroquinolones
gyrA D94N	-3.52	Drug resistance of fluoroquinolones

Note: 1"-" denotes no signal; 2 "\\" means that the item has no referential meaning;

example 4

This example provides a comparison of the effectiveness of the use of a 5 'tail-tipped design probe and a 5' tail-less design probe.

1. Designing a common probe, a tail tilting probe 1 and a tail tilting probe 2, wherein the nucleic acid sequences of the probes are SEQ ID NO.3, SEQ ID NO.4 and SEQ ID NO.5 respectively.

2. Wild type and gyrA A90V, D94G, D A, D N mutation sites were detected using the method of example 3.

3. The detection result shows that the detection effect of the tail raising probe 1 and the tail raising probe 2 is superior to that of a common probe. The detection results are shown in fig. 2 and fig. 3.

Example 5

In this example, the plasmid was used to simulate clinical samples, and the detection ratios of different mutation sites under the mixed template condition and the minimum detection limits of different mutation sites were examined.

1. Wild type plasmids gyrA W and gyrA A90V, D94G, D A, D N mutant plasmids are diluted to working concentration of 1 × 10 ⁵ CP/. Mu.L, different volumes of wild-type plasmid and mutant plasmid were mixed, and different mixed samples were prepared at mutation ratios of 0%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, and 100%.

2. Wild type of gyrA gene, A90V, D94G, D A, D N mutant plasmid was diluted to working concentration of 1X10 ⁷ CP/μL、1×10 ⁶ CP/μL、1×10 ⁵ CP/μL、1×10 ⁴ CP/μL、1×10 ³ CP/. Mu.L samples of different concentrations.

3. The different samples were amplified and subjected to dissolution curve analysis as in example 3, and the lowest detection ratio and detection limit of the different mutant samples were examined.

4. The results showed that the gyrA gene had a minimum detection rate of A90V of 5%, a minimum detection rate of D94G of 30%, a minimum detection rate of D94A of 5%, and a minimum detection rate of D94N of 20%. The results of the tests are shown in FIGS. 4 to 7.

5. The result shows that the minimum detection limit of four drug-resistant mutant genes of the gyrA gene A90V, D94G, D A, D N is 1X10 ⁴ CP/mL. The results of the tests are shown in FIGS. 8 to 12.

Example 6

A clinical application example of a fluoroquinolone drug-resistant gene mutation nucleic acid detection method by using a mycobacterium tuberculosis culture.

1. The sputum culture of the mycobacterium tuberculosis infection patient is collected in 26 cases in the urban public health clinical medical center, and simultaneously compared with the result of the drug sensitivity test of the culture method, other types of drug-resistant strains are excluded from the sample.

2. The fluoroquinolone drug gene nucleic acid detection is carried out according to the method in example 3. The detection results are as follows:

the sputum samples collected in this example totaled 26, wherein the fluoroquinolone drug-resistant samples were 7, the fluoroquinolone drug-resistant undetected samples were 1, and the sensitive samples were 18. The difference result sample is verified by sequencing, and the result shows that the sample is a fluoroquinolone drug-resistant rare mutation site and is not included in the detection range.

The statistics of the clinical sample detection results are as follows:

in conclusion, compared with a drug sensitive method, the method provided by the invention is simple to operate, short in time consumption and good in accuracy, and can meet the requirement of mycobacterium tuberculosis fluoroquinolone drug mutant gene detection on clinical auxiliary diagnosis.

The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention, and various modifications and changes may be made by those skilled in the art. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. A nucleic acid composition, comprising: the nucleotide sequence is shown as a primer pair of SEQ ID No. 1-2.

2. The nucleic acid composition of claim 1, further comprising a probe for detecting at least one mutation site in the gyrA gene A90V, D G, D A and D94N;

preferably, the nucleotide sequence of the probe is shown as SEQ ID No. 3;

preferably, the 5' end of the probe has 1 to 5 base sequences which are not paired with the target;

preferably, the nucleotide sequence of the probe is shown as SEQ ID No.4 or 5;

preferably, the 5 'end of the probe is connected with a fluorescence reporter group, and the 3' end of the probe is connected with a fluorescence quenching group;

preferably, the fluorescent reporter group is selected from: any one of 5-FAM, 6-FAM, HEX, TET, VIC, JOE, cy3, cy3.5, NED, TAMRA, ROX, texas Red, cy5, cy5.5, and Quasar 670;

preferably, the fluorescence quenching group is selected from: any one of TAMRA, BHQ1, BHQ2, BHQ3 and MGB.

3. The nucleic acid composition of claim 1 or 2, wherein the molar ratio of the upstream primer to the downstream primer in the primer pair is 1: (1-10).

4. A detection reagent comprising the nucleic acid composition according to any one of claims 1 to 3.

5. The detection reagent as claimed in claim 4, wherein the detection reagent is used for detecting drug resistance of Mycobacterium tuberculosis fluoroquinolone drugs or drug resistance gene mutation of Mycobacterium tuberculosis fluoroquinolone drugs.

6. A detection kit comprising the nucleic acid composition according to any one of claims 1 to 3 or the detection reagent according to claim 4 or 5.

7. The test kit of claim 6, wherein the kit further comprises: PCR detection reagent;

preferably, the PCR detection reagent comprises: at least one of PCR reaction buffer, taq enzyme, magnesium ion, dNTP and water.

8. Use of the nucleic acid composition according to any one of claims 1 to 3 for preparing a detection reagent or a kit for drug resistance of mycobacterium tuberculosis fluoroquinolone drugs or mutation of drug resistance genes of mycobacterium tuberculosis fluoroquinolone drugs.

9. A method for detecting drug resistance of mycobacterium tuberculosis fluoroquinolone drugs or drug resistance gene mutation of mycobacterium tuberculosis fluoroquinolone drugs is characterized by comprising the following steps: performing PCR detection on a sample by using the nucleic acid composition according to any one of claims 1 to 3 or the detection reagent according to claim 4 or 5 or the detection kit according to claim 6 or 7; the methods are not directed towards the diagnosis or treatment of disease.

10. The method according to claim 9, wherein the molar ratio of the upstream primer to the downstream primer in the primer pair is 1: (1-10);

preferably, when PCR detection is carried out, the action concentration of the upstream primer and the downstream primer is 0.01-1 μ M independently, and preferably 0.04-0.4 μ M independently;

preferably, when PCR detection is carried out, the action concentration of the probe is 0.1-0.5 mu M;

preferably, the reaction procedure of the PCR is as follows: 4-6 min at 94-96 ℃; 94-98 ℃ for 10-30s, 55-65 ℃ for 20-40s, 70-74 ℃ for 20-30s, and 30-50 cycles; collecting fluorescence signals at the temperature of 55-56 ℃ for 20-40 s; 94-96 ℃ for 1-3min, 33-37 ℃ for 1-3 min; and continuously collecting fluorescence signals at 40-90 ℃.