CN102286616A - Method and kit for detecting mycobacterium tuberculosis isoniazide drug resistant gene mutation - Google Patents
Method and kit for detecting mycobacterium tuberculosis isoniazide drug resistant gene mutation Download PDFInfo
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Abstract
The invention belongs to the technical field of biomedical in vitro diagnostic reagents and particularly discloses a method and a kit for detecting mycobacterium tuberculosis isoniazide drug resistant gene mutation. The kit comprises a double-polymerase chain reaction process based on the fluorescent quantitation polymerase chain reaction (PCR) technology and comprises PCR forward and reverse amplification primers and fluorescent probe sequences, wherein the PCR forward and reverse amplification primers aim at main nucleotide segments of isoniazide drug resistant genes of mycobacterium tuberculosis, and the mutation conditions of katG gene 315 sites and inhA gene-15 sites can be simultaneously detected in one reaction tube under the proper PCR condition. The method of the invention can be used for simply, conveniently and fast detecting the mycobacterium tuberculosis isoniazide drug resistant gene mutation in clinical samples and can be used for molecular drug resistant diagnosis of mycobacterium tuberculosis.
Description
Technical field
The invention belongs to biological medicine external diagnosis reagent technical field, be specifically related to the method and the test kit of a kind of rapid detection mycobacterium tuberculosis vazadrine drug resistant gene (katG 315 with inhA-15) sudden change.
Background technology
In recent years, the resistant tuberculosis problem is on the rise, and has become one of difficult problem of tuberculosis control.Because the treatment lack of standardization of antitubercular agent, the intravital tubercule bacillus of patient be to multiple antitubercular agent generation resistance, thereby form anti-multiple medicines tuberculosis, this is one of main reason of rising again of tuberculosis epidemic situation.Therefore, the foundation of quick diagnosis lungy and optimized individual treatment plan is one of significant challenge in the tuberculosis control.
Because the mycobacterium tuberculosis poor growth, the resistance measuring method of widespread use nowadays all is to be based upon on the basis of bacterial strain, though used new liquid nutrient medium, but still consuming time longer, postponed the time that obtains the bacterial strain responsive type.In present stage, each hospital of China extensively adopts the phlegm cultivation to add medicine susceptibility and tests the resistance that detects tubercule bacillus, promptly turns out tubercule bacillus earlier, gives antitubercular agent again, and observes the medicine fungistatic effect.The detected result of this method is reliable, but maximum weak point is a culture condition requirement height, and growth of bacillus tubercle is slow, often needs 6-8 week; Phlegm cultivation recall rate is low simultaneously, only is 30%, so the recall rate of drug sensitive test also only has about 30%, can not satisfy the clinical detection needs.Therefore, develop a kind of anti-detection method lungy of wanting of quick and precisely diagnosing more, for the propagation of early discovery, blocking-up resistant tuberculosis, the standard direction of medication usage, the curative ratio that improves resistant tuberculosis has very important significance.Reported the molecular biology method of many detection tuberculosis medicament-resistant mutations in recent years, however the substratum that these methods often need great amount of manpower and grow fine.Clinical diagnosis needs a kind of method simple, quick, good reproducibility that a series of transgenations relevant with the tuberculosis resistance are detected.The Real-time round pcr combines the advantages such as specificity of the susceptibility of PCR and DNA hybridization, has simple to operately, and the result judges characteristics such as directly perceived and pollution-free, has been widely used in the detection of multiple microbial pathogen DNA in clinical diagnosis.Therefore, the present invention successfully is incorporated into Real-time PCR (Taqman/MGB hydrolysis probes) technology in the field of mycobacterium tuberculosis drug resistant gene detection.Polymerase chain reaction (polymerase chain reaction, PCR) technological invention is 20 years so far, obtained continuous development during this period, the real-time fluorescence quantitative PCR of Chu Xianing (Real-time quantitative PCR) technology has realized the leap of PCR from qualitative to quantitative in recent years.It with high specificity, highly sensitive, good reproducibility, quantitatively accurately, advantages such as fast, the totally-enclosed reaction of speed become important tool in the molecular biology research.The employed fluorescence chemical method of real-time PCR mainly contains 5 big classes at present, is respectively: dna binding dye, hydrolysis probes (Taqman and MGB probe), molecular beacon (molecular beacon), fluorescent dye primer, hybridization probe.They can be divided into special and non-special detection two classes of extension increasing sequence again.The basis of the non-specific detection method of extension increasing sequence is a DNA bonded fluorescence molecule, as fluorescence dyes such as SYBR Green I.After SYBR Green I fluorescence dye mixes the dna double chain specifically, but the emitting fluorescence signal.The advantage of fluorescence dye is to detect the amplification of any dsDNA sequence, does not need the design of probe, makes detection method become easy, has also reduced the cost that detects simultaneously.But just because of fluorescence dye can and any dsDNA combination, so it also can combine with non-specific dsDNA (for example primer dimer), makes to test to be easy to generate the false positive signal.The extension increasing sequence method for detecting specificity is to utilize the gene specific oligonucleotide probe of mark fluorescent group to detect amplified production in the PCR reaction.It can be divided into direct method and indirect method again.Indirect method is exactly to utilize the strategy of hydrolysis probes.The most widely used Taqman system uses this principle exactly in real-time PCR at present.When adding a pair of primer, add a specificity fluorescent probe during pcr amplification.This probe is one section oligonucleotide, and two ends are mark report fluorophor and a cancellation fluorophor respectively.When probe is complete, detect the fluorescence that sends less than this probe 5 ' end fluorophor.But in pcr amplification, after the template sex change in the solution during low-temperature annealing, primer and probe are simultaneously and template tuberculosis.Under the mediation of primer, extend to probe forward in conjunction with going out along template, 5 '-3 ' 5 prime excision enzyme activity of Taq enzyme cuts down probe 5 ' fluorophor from probe, be free in the system, thereby break away from the shielding of 3 ' end fluorescent quenching group and send fluorescent signal, and be DNA chain of every amplification, just there is the success of fluorescence molecule luminous, it is synchronous fully that the accumulation that has realized fluorescent signal and PCR product form.For obtaining higher resolving power and outstanding signal to noise ratio, can also select Taqman MGB probe for use.Its remarkable advantage of MGB probe is that length is short, and signal to noise ratio is good, is non-fluorescent quenching group, stable performance and fluorescence interference is little; The resolving power height can be distinguished a base.Base difference can cause the significantly reduction of Tm value as 20 degree, and mispairing both can be negative findings, was widely used in snp analysis.And conventional Taqman probe has certain fault-tolerant ability, and 1-3 base mispairing might not influence detection, and the Tm value is more or less the same, and the result is still positive.
The vazadrine is crucial a kind of in 5 kind of one line medicine in the tuberculosis clinical treatment, also is the chemical sproof antitubercular agent of the easiest appearance.Studies show that, the vazadrine resistance main with coding catalase-peroxidase the katG gene and the promoter gene of the inhA gene of coding nicotinamide adenine dinucleotide reduced dependency enoyl-carrier proteins reductase enzyme suddenly change relevant.What wherein mutation frequency was the highest is katG 315 sites and inhA-15 site.
Summary of the invention
The detection method and the test kit that the purpose of this invention is to provide a kind of vazadrine resistance mycobacterium tuberculosis.
The detection method of mycobacterium tuberculosis provided by the invention vazadrine drug-tolerant gene mutation adopts the method for fluorescently-labeled polymerase chain reaction (PCR), and concrete steps comprise:
Amplification tuberculosis molecule bacillus gene group sequence in an amplified reaction, wherein, the sudden change in the described genomic zone has caused the vazadrine resistance of mycobacterium tuberculosis;
The amplification system of described amplified reaction comprises the oligonucleotide probe of 2 marks, 2 pairs of amplimers and a thermostable DNA polymerases;
The oligonucleotide probe of described mark and the hybridization of the genome sequence of amplification produce decipherable amplification curve diagram;
The oligonucleotide probe of described mark and target nucleotide sequences hybridization region comprise an oxyr gene mutational site at least, and the oligonucleotide probe of described mark and the complementation of wildtype target Nucleotide;
Described oligonucleotide probe is the double-tagging probe, comprises a report mark fluorescent group and a cancellation labelling groups;
Described report mark is a fluorescein base group, and it can be marked at oligonucleotide probe 5 ' end, kind can be among FAM, TET, JOE, HEX, CY3, ROX, the CY5 any, also be not limited only to this; Described cancellation mark is fluorescence or non-fluorophor, and it can be marked at probe 3 ' end, kind can be among TAMRA, BHQ1, BHQ2, BHQ3, the MGB any, also be not limited only to this;
Among the present invention, described genome area is selected from katG gene, the inhA gene promoter area relevant with anti-vazadrine (INH);
Sudden change in the described genome area comprises: 315 sites of katG gene ,-15 sites of inhA gene.
Among the present invention, described archaeal dna polymerase is the archaeal dna polymerase with 5 ' exonuclease activity.
Among the present invention, described amplified reaction is a multi-PRC reaction, has two probes with different genes group area hybridization in the reaction at least; Each bar of described at least two probes can mate fully with wild sequence, also can all not exclusively mate, also can be wherein one with wild-type sequence coupling, an other incomplete coupling; Described two probes can be included in the same or similar fluorescent mark that detects in the single signal detection passage, also can be included in the different fluorescent marks that detect in the unlike signal sense channel.
The present invention uses combination 1: the primer katG FP in the sequence table (SEQ ID NO.1), katG RP (SEQ ID NO.2) and probe katG TW(SEQ ID NO.5) detect katG gene 315 sites, use and make up 2: the primer inhA FP(SEQ ID NO.3 in the sequence table), inhA RP (SEQ ID NO.4) and probe inhA TW(SEQ ID NO.6) detect inhA gene-1 5 sites.
The present invention also provides a kind of probe that detects mycobacterium tuberculosis vazadrine drug-tolerant gene mutation, and this probe is made up of 13 ~ 19 Nucleotide, and above-mentioned probe comprises the report mark and the cancellation mark that is marked at 3 ' end that are marked at 5 ' end.At PCR reaction annealing stage, this probe can with 315 zones of katG and inhA promotor-15 area hybridization of tuberculosis molecule bacillus, then in archaeal dna polymerase catalysis primer extension process with 5 '-3 ' 5 prime excision enzyme activity, shredded by archaeal dna polymerase, order report marked free comes out, and sends fluorescence.Described two probes are shown in SEQ ID NO.5～NO.6.
The present invention also provides a test kit that detects mycobacterium tuberculosis vazadrine drug-tolerant gene mutation from sample, described test kit comprises: the oligonucleotide probe of at least two pairs of amplimers and at least two marks, a kind of thermally-stabilised and have the archaeal dna polymerase of 5 ' exonuclease activity and needed other usual component of pcr amplification.Wherein said two pairs of amplimers are the SEQ ID NO.1 ~ SEQ ID NO.4 in the sequence table; Described two probes are SEQ ID NO.5 and the SEQ ID NO.6 in the sequence table.
Detect the primer and the probe oligonucleotides sequence table of mycobacterium tuberculosis vazadrine drug-tolerant gene mutation
| Sequence numbering | The sequence title | Oligonucleotide sequence (5'-3') |
| SEQ ID NO.1 | katG FP | cttgggctggaagagc |
| SEQ ID NO.2 | katG RP | cgtacaggatctcgagga |
| SEQ ID NO.3 | inhA FP | tacgctcgtggacataccgatt |
| SEQ ID NO.4 | inhA RP | accaggactgaacgggatacg |
| SEQ ID NO.5 | katG TW | tcaccagcg gcatcg |
| SEQ ID NO.6 | inhA TW | cggcgagac gataggt |
Core content of the present invention and principle are as follows:
1. design two pairs of Auele Specific Primers, near near the mycobacterium tuberculosis genomic dna in katG 315 sites of increasing respectively and inhA-15 site, and do not disturb each other.
2. design two oligonucleotide probes, respectively with mycobacterium tuberculosis genomic dna katG 315 sites of wild-type and inhA-15 site near sequence complementary fully.Probe length is between 13 ~ 19 bases.The fluorescence report group of probe 5 ' end mark can be any among FAM, TET, JOE, HEX, VIC, ROX, TAMRA, CY3, the CY5, also is not limited only to this.The quenching group of probe 3 ' end mark can be any among TAMRA, DABCYL, the MGB, also is not limited only to this.
3.PCR in the reaction system, need to add a kind of archaeal dna polymerase that has 5 ' exonuclease activity (Taq polysaccharase) of thermostability.
4. in the annealing steps of PCR reaction, under suitable annealing temperature condition, the double-tagging fluorescent probe that mates fully with the wild-type template firmly at first is attached on the target sequence.In extending step, archaeal dna polymerase moves the extension of catalysis primer along template strand from 5 ' to 3 '.When archaeal dna polymerase runs into the fluorescent probe that is attached on the template, can bring into play 5 ' exonuclease activity, cut away fluorescent probe gradually, make the fluorophor of fluorescent probe 5 ' separate with 3 ' quenching group, dissociate out, thereby break away from the cancellation of quenching group, send detectable fluorescence fluorophor.And the quantity of luminous fluorophor (fluorescence intensity) is directly proportional with amplified production quantity in the system, thereby realizes detecting in real time the PCR reaction.Under identical annealing temperature, mutant template (mainly being the sudden change of 315 of katG and inhA-15 position) can not be mated fully with fluorescent probe, therefore can not mortise, therefore can not cut by archaeal dna polymerase, its luminophore and quenching group keep distance closely always, so fluorescent signal keeps the cancellation state always.Thereby realize differentiation to wild-type template and mutant template.
Advantage of the present invention: the present invention can in same reaction tubes, detect simultaneously two with the relevant gene mutation site in the anti-vazadrine of tuberculosis, but big time saver, reduce cost, and prevent to pollute, lowering false positive results occurs, improve the accuracy that detects, be more suitable for the demand of Clinical Laboratory.
Description of drawings
Fig. 1: 315 wild-types of mycobacterium tuberculosis katG, inhA-15 a mutant amplification curve diagram.
Fig. 2: 315 mutants of mycobacterium tuberculosis katG, inhA-15 a wild-type amplification graphic representation.
Fig. 3: 315 mutants of mycobacterium tuberculosis katG, inhA-15 a mutant amplification curve diagram.
Fig. 4: 315 wild-types of mycobacterium tuberculosis katG, inhA-15 a wild-type amplification graphic representation.
Embodiment
Embodiment 1:The design of primer and probe is with synthetic
According to literature search, occurrence frequency is the highest in the gene locus relevant with mycobacterium tuberculosis vazadrine resistance is 315 of katG and inhA-15, therefore is chosen to be target detect of the present invention site.Download katG gene order and inhA gene promoter sequence from the NCBI website, use Primer express 3.0 softwares to carry out the design of primer and probe then, draw two pairs of Auele Specific Primers (SEQ ID NO.1 ~ SEQ ID NO.4) and two specific probes (SEQ ID NO.5 ~ SEQ ID NO.6) according to suitable parameters.
Entrust specialized company's synthetic primer and probe, wherein primer is the PAGE purifying, and probe is HPLC or PAGE purifying.Article two, probe one 5 ' end flag F AM fluorophor wherein, other one 5 ' end mark HEX fluorophor; But two 3 ' of probe hold mark TAMRA probe or MGB probe.Article two, probe can be placed in the same PCR reaction, thereby reaches the purpose of two transgenations of a PCR reaction detection.
Embodiment 2:The extraction of sample DNA (1)
Learn from else's experience and be accredited as mycobacterium tuberculosis male clinical sample strain isolated after cultivating, 80 degree deactivations 2 hours, carry out the extraction of sample DNA again with commercial genome DNA extracting reagent kit, the genomic dna that obtains is directly used in tuberculosis Drug Resistance Detection or-20 degree storages, standby.
Embodiment 3:The extraction of sample DNA (2)
Get sputum 2ml, add 2.5 times of volume 4%NaOH, 37 degree temperature are bathed 30min.With the centrifugal supernatant that goes of sputum after the liquefaction, precipitation is used for the extraction of sample DNA.Press embodiment 2 then and continue operation.
Embodiment 4:The selection of positive reference substance
Mycobacterium tuberculosis type strain H37Ra spent the night shake bacterium, high-temperature inactivation.Embodiment 2 is installed then carries out DNA extraction.With the DNA that obtains quantitatively after, be diluted to 10 ng/ul with TE buffer or ultrapure water, as the positive reference substance of wild-type mycobacterium tuberculosis.
Embodiment 5:The PCR reaction system is formed
According to the form below preparation PCR reaction solution also adds the mycobacterium tuberculosis dna profiling, and each experiment must comprise blank and wild-type positive control.Vortex concussion mixing after preparation is finished, machine is gone up in centrifugal back.
Table 1:PCR system each become to be grouped into:
| Each component title | Final concentration |
| PCR Buffer | 1 Χ |
| MgCl 2 | 2.5mM |
| dNTPs | 0.2mM |
| KatG-FP (katG forward primer) | 0.3uM |
| KatG-RP (katG reverse primer) | 0.3uM |
| KatG-TW (katG probe) | 0.15uM |
| InhA-FP (inhA forward primer) | 0.3uM |
| InhA-RP (inhA reverse primer) | 0.3uM |
| InhA-TW (inhA probe) | 0.15uM |
| The Taq enzyme | 1 U |
| Aseptic ultrapure water | In right amount |
| Dna profiling | 1 ul |
| |
20 ul |
Embodiment 6:Response procedures is set
The sense channel of collecting FAM and VIC (HEX) fluorescent signal is set respectively, the PCR reaction tubes is put into quantitative real time PCR Instrument, begin amplification, set response procedures following (is example with ABI 7500 fast system)
Table 2:Real-time PCR program
* collect fluorescent signal in step 3.
Embodiment 7:The result judges
Behind the quantitative real time PCR Instrument end of run, carry out the analysis of amplification curve with its supporting analysis software.The wild-type template of positive control and corresponding gene has typically " S " type amplification curve, and the amplification curve of the mutant template of blank and corresponding gene is the straight line of a level substantially.
Embodiment 8:Quality control standard
Table 3:Real-time PCR quality control standard
Embodiment 9:Use this test kit to detect vazadrine drug-tolerant gene mutation in the clinical isolating mycobacterium tuberculosis bacterial strain
For detecting accuracy and the validity that test kit of the present invention detects vazadrine drug-tolerant gene mutation in the mycobacterium tuberculosis bacterial strain, 23 routine clinical samples are carried out pcr amplification and fluoroscopic examination, whether undergo mutation to judge 315 of vazadrine drug resistant gene katG and inhA-15 position.23 routine clinical isolates strains prepare dna profiling by method described in the embodiment 2, and then detect (the part detected result is seen the patent specification accompanying drawing) with the decision method as a result among detection of the PCR response procedures among the PCR reaction system described in the embodiment 5, the embodiment 6 and the embodiment 7.The result of Real-time PCR result and sequence measurement compares, with the validity (the results are shown in Table 4) of identifying detection method clinical diagnosis of the present invention.
Table 4: mycobacterium tuberculosis vazadrine drug-tolerant gene mutation quantitative fluorescent PCR detects comparative result with order-checking
Table 4 is the result show, fluorescence quantifying PCR method of the present invention compares 23 routine clinical samples measurement results and sequencing (gold standard), and accuracy rate is 100%.
Mycobacterium tuberculosis of the present invention vazadrine drug-tolerant gene mutation PCR kit for fluorescence quantitative, testing process simple, fast, can detect topmost two gene mutation sites relevant easily with the vazadrine, more favourable than existing other molecular biology methods.Can improve more extensive case research.
Below clear interpretation explanation: test kit of the present invention be a kind of can be easy, accurately detect the instrument of mycobacterium tuberculosis vazadrine drug resistant gene, for the early diagnosis and the monitoring of resistant tuberculosis effect is arranged very.
＜110〉Fudan University
＜120〉a kind of method and test kit that detects mycobacterium tuberculosis vazadrine drug-tolerant gene mutation
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Claims (7)
1. the detection method of a mycobacterium tuberculosis vazadrine drug-tolerant gene mutation is characterized in that concrete steps comprise:
By amplified reaction, amplification tuberculosis molecule bacillus gene group sequence, wherein, the sudden change in the described genomic zone has caused the vazadrine resistance of mycobacterium tuberculosis;
The amplification system of described amplified reaction comprises the oligonucleotide probe of 2 marks, 2 pairs of amplimers and a thermostable DNA polymerases;
The oligonucleotide probe of described mark and the hybridization of the genome sequence of amplification produce decipherable amplification curve diagram;
The oligonucleotide probe of described mark and target nucleotide sequences hybridization region comprise an oxyr gene mutational site at least, and the oligonucleotide probe of described mark and the complementation of wildtype target Nucleotide;
Described oligonucleotide probe is the double-tagging probe, comprises a report mark fluorescent group and a cancellation labelling groups.
2. the detection method of mycobacterium tuberculosis according to claim 1 vazadrine drug-tolerant gene mutation is characterized in that described genome area is selected from katG gene, the inhA gene promoter area relevant with anti-vazadrine;
Sudden change in the described genome area comprises: 315 sites of katG gene ,-15 sites of inhA gene.
3. the detection method of mycobacterium tuberculosis according to claim 1 and 2 vazadrine drug-tolerant gene mutation, it is characterized in that described 2 oligonucleotide probes are shown in SEQ ID NO.5 and the SEQ ID NO.6, described 2 pairs of amplimers are SEQ ID NO.1 and SEQ ID NO.2, shown in SEQ ID NO.3 and the SEQ ID NO.4.
4. according to the detection method of claim 1 or 2 or 3 described mycobacterium tuberculosis vazadrine drug-tolerant gene mutations, it is characterized in that described archaeal dna polymerase is the archaeal dna polymerase with 5 ' exonuclease activity.
5. according to the detection method of the described mycobacterium tuberculosis of one of claim 1-4 vazadrine drug-tolerant gene mutation, it is characterized in that described report mark is a fluorophor, it is marked at oligonucleotide probe 5 ' end, kind be among FAM, TET, JOE, HEX, CY3, ROX, the CY5 any; Described cancellation mark is fluorescence or non-fluorophor, and it is marked at probe 3 ' end, kind be among TAMRA, BHQ1, BHQ2, BHQ3, the MGB any.
6. probe that detects mycobacterium tuberculosis vazadrine drug-tolerant gene mutation is characterized in that this probe is made up of 13 ~ 19 Nucleotide, shown in SEQ ID NO.5 and SEQ ID NO.6; Above-mentioned probe comprises the report mark fluorescent group and the cancellation labelling groups that is marked at 3 ' end that are marked at 5 ' end; At PCR reaction annealing stage, 315 zones of the katG of this probe and tuberculosis molecule bacillus and inhA promotor-15 area hybridization, then in archaeal dna polymerase catalysis primer extension process with 5 '-3 ' 5 prime excision enzyme activity, cut off by archaeal dna polymerase, order report marked free comes out, and sends fluorescence.
7. test kit that from sample, detects mycobacterium tuberculosis vazadrine drug-tolerant gene mutation, it is characterized in that comprising: the oligonucleotide probe of at least two pairs of amplimers and at least two marks, a kind of thermally-stabilised and have the archaeal dna polymerase of 5 ' exonuclease activity and needed other usual component of pcr amplification; Wherein said two pairs of amplimers are SEQ ID NO.1 and SEQ ID NO.2, shown in SEQ ID NO.3 and the SEQ ID NO.4; Described two probes are shown in SEQ ID NO.5 and the SEQ ID NO.6.
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| CN108950025B (en) * | 2018-01-29 | 2021-12-17 | 中国人民解放军第四军医大学 | Primer group and kit for rapid detection of mycobacterium tuberculosis katG gene mutation and application |
| CN108611358A (en) * | 2018-04-10 | 2018-10-02 | 佛山科学技术学院 | A method of isoniazid nicotinamide adenine dinucleotide is prepared by synthetic biology |
| CN110672576A (en) * | 2019-11-14 | 2020-01-10 | 中南民族大学 | Method for measuring isoniazid by quantum dot fluorescence quenching method |
| CN110672576B (en) * | 2019-11-14 | 2021-11-12 | 中南民族大学 | Method for measuring isoniazid by quantum dot fluorescence quenching method |
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