CN102312001A - Method for detecting drug resistance of Mycobacterium tuberculosis with multiple PCR-reverse dot blot technique - Google Patents
Method for detecting drug resistance of Mycobacterium tuberculosis with multiple PCR-reverse dot blot technique Download PDFInfo
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Abstract
The invention relates to a method for detecting the drug resistance of Mycobacterium tuberculosis with a multiple PCR-reverse dot blot technique, that is, a method for detecting the drug resistance of Mycobacterium tuberculosis based on the multiple PCR-reverse dot blot technique, comprising a design of primers, a design of a probe, hybridization test and result determination. According to the invention, the drug resistance of Mycobacterium tuberculosis to RIF, INH and EMB can be determined simultaneously with short determination time and reliable results.
Description
Technical field
The present invention has set up a kind of being based upon and on the multiplex PCR basis mycobacterium tuberculosis has been carried out the reverse serial probe hybridization method of the resistance rapid detection of anti-Rifampin (RIF), vazadrine (INH) and Tibutol (EMB) medicine, can be relevant with the INH resistance through detection and RIF, EMB
RpoB,
KatG,
InhAThe generegulation district with
EmbBThe gene base mutation is judged the drug susceptibility of bacterial strain, and can carry out strain identification to mycobacterium tuberculosis complex simultaneously.
Background technology
White plaque is seriously to jeopardize the healthy public health problem of global human; Since eighties of last century the fifties; Constantly find effective antitubercular agent such as Rimactazid, Tibutol, Streptomycin sulphate etc., make popular certain control that obtained lungy, but because many national to ignorance lungy; Reduced financial input, added Increase of population, the propagation of floating population's increase, HIV infection; The particularly appearance of resistance mycobacterium tuberculosis and propagation makes the popular decline of white plaque slow, also gos up to some extent even aggravate in the countries and regions that have.China's tuberculosis patient is one of maximum in the world country; Be only second to India; Occupy the second in the world, the difficult point of controlling tuberculosis is control and the particularly anti-multiple medicines white plaque of treatment resistance [World Health Organisation, Global tuberculosis control:surveillance; Planning and financing, 2007. WHO/HTM/TB/2007.376.].
The best approach of the blindness of minimizing treatment is based on the drug susceptibility of patient's mycobacterium tuberculosis and treats.Based on the gold method that the traditional susceptibility laboratory method on the microbial culture basis is the detection of Mycobacterium tuberculosis drug-resistant property, the result is the most reliable, because this method directly reflects the medicaments insensitive implementations of bacterium, can observe colonial morphology and growing state.But because tubercule bacillus belongs to the poor growth bacterium, traditional diagnosis of tuberculosis detects with susceptibility, needs for 6 to 8 all times from turning out bacterium colony at least to drug sensitive test, can not satisfy the needs of clinical treatment far away, causes the generation and the propagation of resistant organism easily.BACTEC960 liquid nutrient medium method also is based on the drug sensitive experiment method of cultivation, is an innovation of phenotype detection method, though shortened the time of test; But compare with traditional drug sensitive test; BACTEC960 liquid nutrient medium method can't be seen the colony growth situation, and needs dependence on import expensive instrument and reagent, can't be in the many developing country's large-scale promotion of case; So; Start with from molecular diagnosis, seek novel mycobacterium tuberculosis resistance detection technique, become the focus of tubercule bacillus research.Disclosed gradually along with tubercule bacillus produces more drug-fast molecular mechanisms, made that setting up the molecular diagnosis means becomes possibility.Research shows, about 95% mycobacterium tuberculosis to RFP resistance and RNA polymerase beta subunit encoding sox (
RpoB) the relevant [Afanas of sudden change in 81-bp mutantional hotspot district of corresponding intestinal bacteria 507 to 533 codons
,Ev MV, Ikryannikova LN, J Antim Chemo, 2007,59:1057-1064.].And the generation of anti-INH relates to more gene.Research show the INH resistance with
KatG,
InhARegulatory region, or
AhpCRelevant Deng transgenation.Some evidences show that EMB is through directly suppressing
EmbABCThe pectinose based transferase of coding stops arabogalactan synthetic, plays effect [Zhang, SL, the Qi H of bacteria growing inhibiting; Qiu DL, Li DX, Zhang J, Du CM; Wang GB, Yang ZR, Sun Q. Biochem Genet. 2007. 45:281-290.].
According to mycobacterium tuberculosis resistance molecular mechanism, set up the multiple molecular detecting method that is used for the mycobacterium tuberculosis drug resistance related gene at present, like dna direct PCR sequencing PCR, single-strand conformation polymorphism analysis (SSCP) [Lee H, Cho SN; Bang HE, Lee JH, Bae GH, Kim SJ; Kim JD, Int J Tuberc Lung Dis. 1998,2 (7): 585-589.], lines probe hybridization DNA microarray [Suresh N, Singh UB; Arora J, Pande JN, Seth P, Samantaray JC; Diagnosti Micro Infec Dis, 2006,56:133-140.], real-time quantitative PCR [Torres MJ; Criado A, Palomares JC, Aznar J, J Clin Microbiol; 2000,38,3194-3199.], the technology such as allos polymorphism analysis of gene chips and temperature mediation.Dna sequencing, though technology results such as real-time quantitative PCR and gene chips accurately, reliably need comparatively expensive specialized equipment and reagent, can't be at the many developing country's large-scale promotion applications of case; Single-strand conformation polymorphism analysis does not need expensive instrument; Only the PCR product is carried out sex change and polyacrylamide gel electrophoresis; But be not sure of the position and the character of drug-tolerant gene mutation, can't distinguish missense mutation and same sense mutation, and the safety and the repeatability of test are not strong.The reverse serial probe hybridization of PCR-based and reverse dot blot hybridization technique is technological owing to easy, direct, cheap, quick, and multiple medicines drug resistance information can be provided in single test, in various molecular diagnosis methods, shows one's talent; Be the most promising mycobacterium tuberculosis resistance Molecular Detection means of it seems at present, be successfully applied to rapid detection [Zhang SL, the Shen JG of drug resistant gene at present; Xu PH, Sun Q, Yang ZQ; Et al; J Appl Microbiol, 2007,103 (4): 1262-1271.].This probe array can obtain several genes or site mutation information simultaneously in a hybrid experiment, greatly reduce the time and the cost of test.But some test kit prices of external exploitation are still expensive, and can only be used for
RpoBThe mutantional hotspot district of gene is detected, and can not detect sudden change situation [Hillemann D, the Weizenegger M of the drug resistant gene of anti-INH of mycobacterium tuberculosis and EMB simultaneously; Kubica T, Richter E, Niemann S; J Clin Microbiol. 2005,43:3699-3703.].
Find as yet at present to have and to detect mycobacterium tuberculosis simultaneously and the chemical sproof technology of RFP, INH and three kinds of medicines of EMB is used in reality or reported.
Summary of the invention
The object of the present invention is to provide a kind of method for quick based on molecular biological resistant tuberculosis; Particularly a kind of reverse dot blot hybridization technique based on multiplex PCR detects the chemical sproof method of mycobacterium tuberculosis; Set up a kind of being based upon and on the multiplex PCR basis mycobacterium tuberculosis is carried out the reverse serial probe hybridization method of the resistance rapid detection of anti-Rifampin (RIF), vazadrine (INH) and Tibutol (EMB) medicine, can be relevant with the INH resistance through detection and RIF, EMB
RpoB,
KatG,
InhAThe generegulation district with
EmbBThe gene base mutation is judged the drug susceptibility of bacterial strain, and can carry out bacterial classification (MTC) to mycobacterium tuberculosis complex simultaneously and identify, has wherein comprised pcr amplification primer sequence, hybridization probe sequence.
Solution of the present invention is based on round pcr and reverse probe hybridization technology.
Particular content of the present invention is following:
A kind of multiplex PCR-reverse dot blot hybridization technique detects the chemical sproof method of mycobacterium tuberculosis, promptly is that a kind of reverse dot blot hybridization technique based on multiplex PCR detects the chemical sproof method of mycobacterium tuberculosis, it is characterized in that:
Being reflected in the pipe of A, multiplex PCR accomplished, and at least four dna fragmentations to be detected of disposable pcr amplification;
Low density probe array described in B, the reverse dot blot hybridization comprise at least 19 can detect simultaneously with
RFP, EMB are relevant with the INH resistance
RpoB,
KatG,
InhAThe generegulation district with
EmbBThe base mutation of transgenation hot zone and the probe that the compound crowd of tubercule bacillus is carried out bacterial classification differential diagnosis;
C, describedly have carboxyl and be the Hybond membrane of negative electricity,, form bridge architecture with the oligonucleotide probe covalent attachment of amino labeled;
Primer in D, the multiplex PCR adopts Streptavidin-SEAP bonded vitamin H to carry out mark, forms coloured material through catalytic substrate BCIP/NBT reaction and judges the positive and negative of hybridization, and reflection PCR product and probe have or not sudden change.
The Tm value of many primers in the multiplex PCR according to the invention differs≤and 3 ℃, the amplified production clip size differs the 50-77 base pair.
Probe according to the invention is 8 oligonucleotide probes and 1 MTC group specificity probe, and said probe length is 20 ± 3 bases; Mismatched bases site (mutational site) is arranged on the probe middle part as far as possible; The Tm value of each probe is close as far as possible, differs to answer≤3 ℃.
The constructive method of multiple oligonucleotide probe array of the present invention is following: use 0.5mol/L Na
2CO
3/ NaHCO
3Damping fluid (pH 8.4) dissolving probe, weaker concn is 20 μ M, fully behind the mixing, refrigerator is preserved subsequent use.Be cut into a certain size to Biodyne C nylon membrane, and use the pencil defined area.Application 1 6% ethyl dimethylamino-propyl carbodiimide (EDAC) the immersion treatment nylon membrane 10min of elder generation cleans with zero(ppm) water with activation nylon membrane (this solution can be reused) then, dries.Use micro sample adding appliance and put 2 μ l probe solutions on Biodyne C nylon membrane corresponding position, react 15min, make the carboxyl covalent attachment of the probe and the nylon membrane of amino labeled; Handle 10min with 0.1mol/L NaOH again, zero(ppm) water cleans, and is subsequent use after drying.Fig. 2 is seen in the arrangement of each probe on the diaphragm.The left side generic point wild-type probe of nylon membrane, the mutant probe in corresponding site is put on the right.
Multiplex PCR amplification of the present invention is meant in 1 amplification system increases
RpoB,
KatG,
InhAThe generegulation district with
EmbB4 kinds of fragments of gene make a PCR reaction produce four drug resistant gene fragments.Each reaction system is got bacterial genomes DNA 1 μ l (about 20ng), (contains 0.2mmol/L dNTPs, dna profiling 100 ng in the 50 μ L reaction response systems in 50 μ l reaction systems; Primer 0.2mmol/L; Taq enzyme 1.5U, 10 * PCR damping fluid of 1/10 volume, 1.5mmol/L MgCl
2).Amplification program: 95 ℃, 5 min; 95 ℃, 1 min, 56 ℃, 1 min and 72 ℃, 1 min, totally 40 circulations; At last 72 ℃ of 10 min extensions down.
The method of reverse dot blot hybridization according to the invention is following: get biotin labeled pcr amplification product 40 μ L and add 1 * SSPE, 200 μ L in the EP of 0.5mL pipe; After 95 ℃ of 15 min carries out sex change; Put into ice rapidly and prevent PCR product renaturation; Learn from else's experience the then PCR product of sex change adds and places in advance and preheating nylon membrane and 3mL hybridization solution (5 * SSPE; 0.5% SDS) centrifuge tube, 57 ℃ of hybridization 30 min.Take out the film bar, move to washing lotion (1 * SSPE is housed; 0.5% SDS) and through the centrifuge tube of 57 ℃ of preheatings, 57 ℃ of each 15min washed twice.All the other operating process are at room temperature carried out: 3mL connects in the liquid (including Streptavidin-SEAP) hatches 15min, uses the washing lotion room temperature washing then 2 times, each 10 min.Use substrate buffer solution (PBS) washing more once, add 3mL substrate reactions liquid BCIP/NBT then, jog 30 min use water washing with diaphragm under the room temperature, drying, and promptly visible blue spot manifests.
Being reflected in the pipe of multiplex PCR of the present invention accomplished, and disposable pcr amplification is four dna fragmentations to be detected.
Low density probe array described in the reverse dot blot hybridization of the present invention comprises that 19 detections simultaneously and RIF, EMB are relevant with the INH resistance
RpoB,
KatG,
InhAThe generegulation district with
EmbBThe base mutation of transgenation hot zone and the probe that the compound crowd of tubercule bacillus is carried out bacterial classification differential diagnosis.
The present invention is multiplex PCR and reverse hybridized combining; Four dna fragmentations to be detected of disposable pcr amplification; Through designing a novel low density probe array, be applied to the quick Drug Resistance Detection of tubercule bacillus, detect the sudden change situation of a plurality of drug resistant genes in like this can single test.All Hybond membranes of the present invention are Biodyne C type nylon membrane; Produced by U.S. Pall BioSupport company, this nylon membrane is negative electricity because of having carboxyl, can with the oligonucleotide probe covalent attachment of amino labeled; Form bridge architecture, can improve hybrid rate greatly.And primer carries out mark with vitamin H, can use Streptavidin-SEAP to combine, and forms coloured material through catalytic substrate BCIP/NBT reaction and judges the positive and negative of hybridization, and then reflect that PCR product and probe have or not sudden change.
The present invention is based on the reverse serial probe hybridization technology of PCR and reverse dot blot hybridization technique; Have easy, direct, cheap, characteristics fast, multiple medicines drug resistance information can be provided in single test, particularly can detect the resistance of mycobacterium tuberculosis simultaneously RIF, INH and three kinds of medicines of EMB; The time and the cost of test have been reduced; The result is accurate, reliable, and need not expensive specialized equipment and reagent, helps large-scale promotion application.
Materials and methods according to the invention comprises following content:
1. the design of primer and oligonucleotide probe and preparation
From GenBank search tubercule bacillus
RpoB,
KatG,
InhAThe generegulation district with
EmbBGene; And confirm above-mentioned drug-tolerant gene mutation hot zone; According to the conservative region difference Using P rimer Premier 5.0 and four pairs of Auele Specific Primers of Oligo 6.0 software designs on its next door, in order to carry out multiplex PCR, the Tm value of four primers is close; The amplified production clip size differs the 50-77 base pair and is beneficial to electrophoretic separation (table 1), and reverse primer is all used biotin labeling.
According to the drug-tolerant gene mutation focus, Using P rimer Premier 5.0 biosoftwares such as grade design 18 oligonucleotide probes altogether.In addition, according to
RpoB1 MTC group specificity of sequences Design probe.The probe design principle is: probe length is about 20 bases; Mismatched bases site (mutational site) is arranged on the probe middle part as far as possible; The Tm value of each probe is close as far as possible, differs about 3 ℃.Shown in each probe specifying information table 2.All probe 5 ' ends carry out amido modified.Primer and probe are synthetic by Invitrogen Shanghai company.
Table 1 pcr amplification drug resistant gene
RpoB,
KatG,
InhAThe generegulation district with
EmbBThe primer of gene
2. multiple oligonucleotide probe array
Use 0.5mol/L Na
2CO
3/ NaHCO
3Damping fluid (pH 8.4) dissolving probe, weaker concn is 20 μ M, fully behind the mixing, refrigerator is preserved subsequent use.Be cut into a certain size to Biodyne C nylon membrane, and use the pencil defined area.Application 1 6% ethyl dimethylamino-propyl carbodiimide (EDAC) the immersion treatment nylon membrane 10min of elder generation cleans with zero(ppm) water with activation nylon membrane (this solution can be reused) then, dries.Use micro sample adding appliance and put 2 μ L probe solutions on Biodyne C nylon membrane corresponding position, react 15min, make the carboxyl covalent attachment of the probe and the nylon membrane of amino labeled; Handle 10min with 0.1mol/L NaOH again, zero(ppm) water cleans, and is subsequent use after drying.Fig. 2 is seen in the arrangement of each probe on the diaphragm.The left side generic point wild-type probe of nylon membrane, the mutant probe in corresponding site is put on the right.Nylon membrane with this method is handled can be preserved half a year down at 4 ℃.
Table 2 drug resistant gene
RpoB,
KatG,
InhAThe generegulation district with
EmbBThe gene oligonucleotide probe
aPR:?
rpoB；
b?K:?
katG；
c?E?
embB；
3. PCR amplification
Amplification
RpoB,
KatG,
InhAThe generegulation district with
EmbB4 kinds of fragments of gene increase at 1 amplification system, make a PCR reaction produce four drug resistant gene fragments.Each reaction system is got bacterial genomes DNA1 μ L (about 20ng), in 50 μ l reaction systems (contain 0.2mmol/L dNTPs in the 50 μ L reaction response systems, dna profiling 100 ng, primer 0.2 mmol/L,
TaqEnzyme 1.5U, 10 * PCR damping fluid of 1/10 volume, 1.5mmol/L MgCl
2).Amplification program: 95 ℃, 5 min; 95 ℃, 1 min, 56 ℃, 1 min and 72 ℃, 1 min, totally 40 circulations; At last 72 ℃ of 10 min extensions down.
4. reverse film hybridization
Get biotin labeled pcr amplification product 40 μ L and add 1 * SSPE, 200 μ L in the EP of 0.5mL pipe; After 95 ℃ of 15 min carries out sex change; Put into ice rapidly and prevent PCR product renaturation; Learn from else's experience the then PCR product of sex change adds and places in advance and preheating nylon membrane and 3ml hybridization solution (5 * SSPE; 0.5% SDS) centrifuge tube, 57 ℃ of hybridization 30 min.Take out the film bar, move to washing lotion (1 * SSPE is housed; 0.5% SDS) and through the centrifuge tube of 57 ℃ of preheatings, 57 ℃ of each 15min washed twice.All the other operating process are at room temperature carried out: 3ml connects in the liquid (including Streptavidin-SEAP) hatches 15min, uses the washing lotion room temperature washing then 2 times, each 10 min.Use substrate buffer solution (PBS) washing more once, add 3ml substrate reactions liquid BCIP/NBT then, jog 30 min use water washing with diaphragm under the room temperature, drying, and promptly visible blue spot manifests.
5. the result judges
The method that the result judges: the judgement detected result that puts in order of probe on the control film bar, blue hybridization spot appears, be judged to be this probe hybridization positive, otherwise then hybridization is negative.
Verification method:
1, bacterium source: 99 strains supply examination mycobacterium tuberculosis strain isolated from the tuberculosis prevention & care inst, Chengdu, and the clinical separation strain and 20 strains that have different resistance patterns comprising 79 strains are to Rimactazid and the equal responsive clinical separation strain of Tibutol.All bacterial classifications are collected, are identified and preserve the rules of all formulating with WHO and carry out; Carry out mycobacterium cultivation, strain identification and drug sensitive test [Chinese anti-tuberculosis association with reference to diagnosis of tuberculosis bacteriological analysis rules; Middle national defence consumptive disease magazine, 1996,18:28-31.].
2, medicine and reagent:
Taq DNAPolysaccharase: available from the precious biotech firm in Dalian; DNTPs: available from Chengdu Bo Ruike biotech firm; The PCR product reclaims test kit: U.S. Qiagen Company products; Dna molecular amount standard DL-2000: precious biotechnology (Dalian) ltd product; Nylon membrane: Biodyne C type film, U.S. Pall BioSupport company; EDAC (ethyl dimethylamino-propyl carbodiimide): available from available from Shanghai Sangon Biological Engineering Technology And Service Co., Ltd; 20 * SSPE:3.6M NaCl, 0.2M Na
2PO
4, 0.02M EDTA (pH7.5); Hybridization solution: 5 * SSPE, 0.5% SDS; Washing lotion: 1 * SSPE, 0.5% SDS; Streptavidin-SEAP: available from Shanghai Sangon Biological Engineering Technology And Service Co., Ltd; Phosphoric acid buffer (PBS): 50mM sodium phosphate salt, 200mM NaCl (pH7.4); 5-bromo-4-chloro-3-indolylphosphate salt (5-bromo-4-chloro-3-indoylphosphate, BCIP): German Boehringer Mannheim Company products; Chlorination nitro tetrazole (nitroblue tetrazolium, NBT): German Boehringer Mannheim Company products.Other agents useful for same is homemade AR; Water is redistilled water.Modified Russell medium is all adopted in the cultivation of Mycobacterium tuberculosis and drug sensitive test.
3, pcr amplification and detection: to 99 strain mycobacterium tuberculosis clinical separation strains
RpoBGene, and the part bacterial strain
KatG,
InhARegulatory region with
EmbBGoing to increase in interior interval, gets PCR product 5 μ L respectively, carries out electrophoresis at 3% (w/v) sepharose, strength of electric field 5V/cm, and time 60min is soaked in the 0.5 μ g/mL ethidium bromide after electrophoresis finishes and dyes.Observe gel under the uv lamp and have or not the purpose amplified band, and compare, if purpose band correct position can be judged to the amplification positive with dna molecular amount standard.
4, the PCR product carries out dna sequencing: send the PCR product the big gene of China to check order.
The checking result:
The sequencing result that 79 examples have different resistance type strain probe array detected results and PCR product contrasts, and is as shown in table 3, and nearly all probe in detecting result can both obtain the support of PCR product D NA The sequencing results.And for 20 routine medicaments insensitive strains, sequencing result also shows all not sudden change generations of their corresponding amplification region, and is all positive with wild-type probe hybridization, supports the detected result of probe array.
Table 3 probe array and dna sequence analysis detect the contrast of substance of medicines-resistant branched tubercle bacillus results of mutation
The application probe array detection 61 routine bacterial strains of anti-the Rifampin
RpoBThe mutantional hotspot district, 53 strains (86.9%) detect sudden change.49 strains are shown as the negative and correctly corresponding together mutant probe hybridization of wild-type probe hybridization; 4 strains demonstration wild-type probe is lost; Also hybridize feminine gender at the mutant probe of having listed, therefore can judge that this 4 strain bacterial strain is a Resistant strain, but its emergent properties is not sure of; Because do not place the probe of corresponding mutation type, so there is not mutant probe to hybridize with it.Modal mutation type is S531L (TCG → TTG), in the 61 routine bacterial strains of anti-RIF, detect 34 examples (55.8%) altogether.In addition, common mutation type (CTG → CCG), 516 (GAC → GGC) and the 526 (site such as GAC → TAC) that also comprises 511.Compare with the phenotype detected result of routine, it is 86.9 % that this probe array detects the Sichuan drug-fast susceptibility of area crowd mycobacterium tuberculosis RIF.
The result shows that in the 52 routine bacterial strains of anti-the vazadrine, 30 examples (57.7 %) exist
KatGCodon 315 sites detect sudden change, and S315T takes place, and (AGC → ACC), only 2 examples present wild-type probe KW and all hybridize negative pattern with mutant probe KM, the mutant probe of design correspondence on this probe array.Prompting
KatG315 of codons are undergone mutation, but the particular type that can not clearly suddenly change, their emergent properties need be confirmed through further dna sequencing.Simultaneously, detecting 4 examples (7.7%, 4/52) Resistant strain exists
InhA(C → T) sudden change, wherein 2 examples only take place in-15 sites generation of regulatory region
InhASudden change is not followed
KatG315 codon mutations; And 2 examples in addition
InhASudden change is followed simultaneously
KatG315 codon mutations.Generally, in the 52 routine bacterial strains of anti-the vazadrine, 34 examples (65.4%) exist
KatG315 bit codons or
InhAAt least detecting a kind of sudden change on-15 sites takes place.Phenotype Drug Resistance Detection method with routine is compared, and it is 65.4% that this probe array detects the drug-fast susceptibility of INH.
In the 47 strain bacterial strains of anti-the Tibutol, reverse hybridized demonstration 23 strains exist
EmbBSudden change is found in gene 306 sites, and ATG → GTG, ATG → ATA, ATG → ATC type sudden change are respectively 14,4,3 strains, and ratio is respectively 29.8%, 8.5%, 6.4%.Susceptibility is 48.9%.It is all negative that two strains are wild probe, mutant probe, and meaning is other mutation type.
The result shows that the present invention can be to mycobacterium tuberculosis r
PoBGene medicament-resistant mutation hot zone,
KatG315 sites,
InhAThe generegulation district with
EmbBThe most of mutational site of gene; Shortened the susceptibility detection time of mycobacterium tuberculosis greatly, cheap, simple to operate; Therefore might develop into being applied to the novel method that clinical labororatory carries out quick Drug Resistance Detection, for the correct chemotherapy regimen of clinical formulation and control measures provide foundation.For the control lungy of anti-multiple medicines with prevent that the bacterial strain of anti-the multiple medicines from propagating and play an important role.
Description of drawings
Fig. 1 is the PCR product electrophorogram of each drug resistant gene.Wherein, M:Marker; 1:
KatGGene; 2:
EmbBGene; 3:
RpoBGene; 4:
InhAGene
Shown application
RpoB,
KatG,
InhAThe generegulation district with
EmbB4 pairs of primer amplified tubercule bacillus of gene strain isolated, the result produces the dna fragmentation of expection, and does not all have the non-specific amplification band.Each primer carries out the result of pcr amplification separately.Produced the dna fragmentation in the mutantional hotspot district that comprises each drug resistant gene respectively.
Fig. 2 is the figure as a result of multiplex PCR.Four kinds of primers produce four specific bands simultaneously in same PCR reaction system, be respectively
RpoB,
KatG,
InhAThe generegulation district with
EmbBThe mutantional hotspot district.
Fig. 3 is that oligonucleotide probe array mode chart and the hybridization of part tubercule bacillus strain isolated detect synoptic diagram.Wherein, (A) positive control (B) negative control; (C) wild type; (D)
RpoBL533P, CTG → CCG;
KatG315
,AGC → ACC; (E)
RpoBS531L, TCG → TTG,
EmbB306:ATG → GTG:
KatG315 AGC → ACC; (F)
RpoB511:CTG → CCG, 516:GAC → GGC,
EmbB306:ATG → GTG
Claims (9)
1. multiplex PCR-reverse dot blot hybridization technique detects the chemical sproof method of mycobacterium tuberculosis, it is characterized in that:
Being reflected in the pipe of A, multiplex PCR accomplished, and at least four dna fragmentations to be detected of disposable pcr amplification;
Low density probe array described in B, the reverse dot blot hybridization
At leastComprise that 19 detections simultaneously and RIF, EMB are relevant with the INH resistance
RpoB,
KatG,
InhAThe generegulation district with
EmbBThe base mutation of transgenation hot zone and the probe that the compound crowd of tubercule bacillus is carried out bacterial classification differential diagnosis;
C, describedly have carboxyl and be the Hybond membrane of negative electricity,, form bridge architecture with the oligonucleotide probe covalent attachment of amino labeled;
Primer in D, the multiplex PCR adopts Streptavidin-SEAP bonded vitamin H to carry out mark, forms coloured material through catalytic substrate BCIP/NBT reaction and judges the positive and negative of hybridization, and reflection PCR product and probe have or not sudden change.
2. a kind of multiplex PCR according to claim 1-reverse dot blot hybridization technique detects the chemical sproof method of mycobacterium tuberculosis; It is characterized in that: the Tm value of many primers in the said multiplex PCR differs≤and 3 ℃, the amplified production clip size differs the 50-77 base pair.
3. a kind of multiplex PCR according to claim 1-reverse dot blot hybridization technique detects the chemical sproof method of mycobacterium tuberculosis, and it is characterized in that: said probe is 18 oligonucleotide probes and 1 MTC group specificity probe.
4. a kind of reverse dot blot hybridization technique based on multiplex PCR according to claim 1 detects the chemical sproof method of mycobacterium tuberculosis, and it is characterized in that: said probe length is 20 ± 3 bases; The mismatched bases site is that the mutational site is arranged on the probe middle part as far as possible; The Tm value of each probe is close as far as possible, differs to answer≤3 ℃.
5. detect the chemical sproof method of mycobacterium tuberculosis according to claim 1 or 3 described a kind of multiplex PCRs-reverse dot blot hybridization technique, it is characterized in that: the constructive method of described multiple oligonucleotide probe array is following:
Use 0.5mol/L Na
2CO
3/ NaHCO
3Damping fluid dissolving probe, weaker concn is 20 μ M, and fully behind the mixing, refrigerator is preserved subsequent use, and the pH of damping fluid is 8.4;
Be cut into a certain size to Biodyne C nylon membrane, and use the pencil defined area;
The application 1 6% ethyl dimethylamino-propyl carbodiimide immersion treatment nylon membrane 10min of elder generation cleans with zero(ppm) water with the activation nylon membrane then, dries;
Use micro sample adding appliance and put 2 μ L probe solutions on Biodyne C nylon membrane corresponding position, react 15min, make the carboxyl covalent attachment of the probe and the nylon membrane of amino labeled;
Handle 10min with 0.1mol/L NaoH again, zero(ppm) water cleans, and is subsequent use after drying;
The left side generic point wild-type probe of nylon membrane, the mutant probe in corresponding site is put on the right.
6. a kind of multiplex PCR according to claim 1 and 2-reverse dot blot hybridization technique detects the chemical sproof method of mycobacterium tuberculosis, it is characterized in that:
Each reaction system is got bacterial genomes DNA 1 μ L in 50 μ L reaction systems; Contain 0.2mmol/L dNTPs in the 50 μ L reaction response systems, dna profiling 100 ng, primer 0.2 mmol/L,
TaqEnzyme 1.5U, 10 * PCR damping fluid of 1/10 volume, 1.5mmol/L MgCl
2
Amplification program: 95 ℃, 5 min; 95 ℃, 1 min, 56 ℃, 1 min and 72 ℃, 1 min, totally 40 circulations; At last 72 ℃ of 10 min extensions down.
7. a kind of multiplex PCR according to claim 1-reverse dot blot hybridization technique detects the chemical sproof method of mycobacterium tuberculosis, and it is characterized in that: the method for said reverse dot blot hybridization is following:
Get biotin labeled pcr amplification product 40 μ L and add 1 * SSPE, 200 μ L in the EP of 0.5mL pipe; After 95 ℃ of 15 min carries out sex change; Put into ice rapidly and prevent PCR product renaturation; Learn from else's experience the then PCR product of sex change adds and places in advance and preheating nylon membrane and 3ml hybridization solution centrifuge tube, hybridizes 30 min for 57 ℃;
Take out the film bar, move to the centrifuge tube that washing lotion and 57 ℃ of preheatings of warp are housed, 57 ℃ of each 15 min washed twice;
All the other operating process are at room temperature carried out: 3mL connects in the liquid hatches 15min, uses the washing lotion room temperature washing then 2 times, each 10 min;
With the substrate buffer solution washing once, add 3mL substrate reactions liquid BCIP/NBT then again, jog 30 min use water washing with diaphragm under the room temperature, drying, and promptly visible blue spot manifests.
8. a kind of multiplex PCR according to claim 1-reverse dot blot hybridization technique detects the chemical sproof method of mycobacterium tuberculosis; It is characterized in that: being reflected in the pipe of described multiplex PCR accomplished, and disposable pcr amplification is four dna fragmentations to be detected.
9. a kind of multiplex PCR according to claim 1-reverse dot blot hybridization technique detects the chemical sproof method of mycobacterium tuberculosis, it is characterized in that: the low density probe array described in the described reverse dot blot hybridization comprises that 19 detections simultaneously and RIF, EMB are relevant with the INH resistance
RpoB,
KatG,
InhAThe generegulation district with
EmbBThe base mutation of transgenation hot zone and the probe that the compound crowd of tubercule bacillus is carried out bacterial classification differential diagnosis.
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| CN104830848A (en) * | 2015-05-05 | 2015-08-12 | 中检国研(北京)科技有限公司 | Reverse dot blot hybridization kit for detection of mycobacterium tuberculosis and usage method thereof |
| CN105349620A (en) * | 2014-08-20 | 2016-02-24 | 北京百诺奇生物科技有限公司 | Kit for detection of K-ras gene mutation and application thereof |
| CN105349619A (en) * | 2014-08-20 | 2016-02-24 | 北京百诺奇生物科技有限公司 | Method for rapidly detecting target nucleic acid in sample and application thereof |
| CN105368920A (en) * | 2014-08-20 | 2016-03-02 | 北京百诺奇生物科技有限公司 | Kit for detecting mycobacterium tuberculosis and mutation of drug resistant gene thereof, and application thereof |
| CN105648039A (en) * | 2014-08-20 | 2016-06-08 | 北京百诺奇生物科技有限公司 | High-sensitivity reverse dot blot hybridization method and application |
| CN107022607A (en) * | 2017-03-16 | 2017-08-08 | 北京博奥晶典生物技术有限公司 | Pass through amplified allele unbalanced method during base mispairing solution multiplex PCR |
| CN108179182A (en) * | 2018-01-30 | 2018-06-19 | 昆明金域医学检验所有限公司 | The film preparation method of poor point mutation a kind ofly |
| CN116121347A (en) * | 2023-02-10 | 2023-05-16 | 合肥达徽基因科技有限公司 | Primer probe combination for detecting ATP7B gene and application thereof |
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| CN104830848A (en) * | 2015-05-05 | 2015-08-12 | 中检国研(北京)科技有限公司 | Reverse dot blot hybridization kit for detection of mycobacterium tuberculosis and usage method thereof |
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