CN102312001B

CN102312001B - Multiplex PCR-reverse dot blot hybridization technique detects the method for Drug Resistance of Mycobacterium Tuberculosis

Info

Publication number: CN102312001B
Application number: CN201110271351.2A
Authority: CN
Inventors: 孙群; 张舒林; 郭建华; 罗涛; 赵于丁; 汤科; 杨志荣
Original assignee: Sichuan University
Current assignee: Sichuan University
Priority date: 2011-09-14
Filing date: 2011-09-14
Publication date: 2016-04-13
Anticipated expiration: 2031-09-14
Also published as: CN102312001A

Abstract

The present invention relates to the method that a kind of multiplex PCR-reverse dot blot hybridization technique detects Drug Resistance of Mycobacterium Tuberculosis, namely be a kind of method that reverse dot blot hybridization technique based on multiplex PCR detects Drug Resistance of Mycobacterium Tuberculosis, include the design of primer, the design of probe, cross experiment, result judgement, the resistance of mycobacterium tuberculosis to RIF, INH and EMB tri-kinds of medicines can be detected simultaneously, substantially reduce the time of detection, reliable results.

Description

Multiplex PCR-reverse dot blot hybridization technique detects the method for Drug Resistance of Mycobacterium Tuberculosis

Technical field

The present invention establishes a kind of reverse serial probing procedure of resistance rapid detection mycobacterium tuberculosis being carried out to resistance to Rifampin (RIF), vazadrine (INH) and Tibutol (EMB) medicine be based upon on multiplex PCR basis, the drug susceptibility of bacterial strain can be judged by detection rpoB, katG, inhA generegulation district relevant to RIF, EMB and INH resistance and embB gene base mutation, and strain identification can be carried out to mycobacterium tuberculosis complex simultaneously.

Background technology

Tuberculosis seriously jeopardizes the public health problem of global human health, since eighties of last century the fifties, the effective antitubercular agent of continuous discovery is as Rifampin, vazadrine, Tibutol, Streptomycin sulphate etc., make lungyly popularly to obtain certain control, but because many countries are to ignorance lungy, decrease financial input, add the growth of population, the increase of floating population, the propagation of HIV (human immunodeficiency virus) infection, the particularly appearance of Drug-Resistant Mycobacterium tuberculosis and propagation, TB endemic is made to decline slowly, also go up to some extent and even aggravate in some countries and regions.China's tuberculosis patient is one of maximum in the world country, be only second to India, occupy the second in the world, control difficult point lungy to be to control and treatment resistance particularly multi-drug resistance tuberculosis [WorldHealthOrganisation, Globaltuberculosiscontrol:surveillance, planningandfinancing, 2007.WHO/HTM/TB/2007.376.].

The best approach of blindness reducing treatment is drug susceptibility based on patient's mycobacterium tuberculosis and treats.Based on the gold method that the traditional susceptibility laboratory method on microbial culture basis is Mycobacterium tuberculosis drug-resistant detection, result is the most reliable, because the method directly reflects the medicaments insensitive implementations of bacterium, can observe colonial morphology and growing state.But because tubercule bacillus belongs to poor growth bacterium, traditional diagnosis of tuberculosis and susceptibility detect, at least to need the time in 6 to 8 weeks to drug sensitive test from turning out bacterium colony, the needs of clinical treatment can not be met far away, easily cause generation and the propagation of resistant organism.BACTEC960 liquid nutrient medium method is also the drug sensitive experiment method based on cultivating, it is an innovation of phenotypic assays, although shorten the time of test, but compare with traditional drug sensitive test, BACTEC960 liquid nutrient medium method cannot see colony growth situation, and need instrument and the reagent of dependence on import costliness, cannot in the many developing country's large-scale promotion of case, so, start with from molecular diagnosis, find novel Drug Resistance of Mycobacterium Tuberculosis detection technique, become the focus of tubercule bacillus research.Some molecular mechanisms producing resistance along with tubercule bacillus are revealed gradually, and making to set up molecular diagnostics becomes possibility.Research shows, the mycobacterium tuberculosis of about 95% is to the sudden change relevant [Afanas of RFP resistance with the 81-bp mutantional hotspot district of corresponding intestinal bacteria 507 to 533 codon of RNA polymerase beta subunit encoding gene (rpoB) ^,evMV, IkryannikovaLN, JAntimChemo, 2007,59:1057-1064.].And the generation of resistance to INH relates to more gene.Research shows INH resistance and katG, inhA regulatory region, or the transgenation such as ahpC is relevant.Some evidences display EMB stops arabogalactan synthesis by directly suppressing the arabinofuranosyltransferase of embABC coding, play the effect [Zhang of bacteria growing inhibiting, SL, QiH, QiuDL, LiDX, ZhangJ, DuCM, WangGB, YangZR, SunQ.BiochemGenet.2007.45:281-290.].

According to drug resistance of Mycobacterium tuberculosis molecular mechanism, establish the multiple molecular detecting method for drug resistance of Mycobacterium tuberculosis genes involved at present, as DNA direct sequencing, single-strand conformation polymorphism analysis (SSCP) [LeeH, ChoSN, BangHE, LeeJH, BaeGH, KimSJ, KimJD, IntJTubercLungDis.1998, 2 (7): 585-589.], lines probe hybridization DNA microarray [SureshN, SinghUB, AroraJ, PandeJN, SethP, SamantarayJC, DiagnostiMicroInfecDis, 2006, 56:133-140.], real-time quantitative PCR [TorresMJ, CriadoA, PalomaresJC, AznarJ, JClinMicrobiol, 2000, 38, 3194-3199.], the technology such as the allos polymorphism analysis of gene chips and temperature mediation.DNA sequencing, although the technology such as real-time quantitative PCR and gene chips result accurately, reliable, all need specialized equipment costly and reagent, cannot at the many developing country's large-scale promotion application of case; Single-strand conformation polymorphism analysis does not need expensive instrument, only sex change and polyacrylamide gel electrophoresis are carried out to PCR primer, but can not determine position and the character of drug-tolerant gene mutation, missense mutation and same sense mutation cannot be distinguished, and the reproducibility and reliability of test is not strong.The reverse serial probe hybridization technology of PCR-based and reverse dot blot hybridization technique is due to easy, direct, cheap, quick, and multiple medicines drug resistance information can be provided in single test, show one's talent in various molecular diagnosis method, the most promising drug resistance of Mycobacterium tuberculosis Molecular Detection means of it seems at present, be successfully applied to the rapid detection [ZhangSL of drug resistant gene at present, ShenJG, XuPH, SunQ, YangZQ, etal, JApplMicrobiol, 2007,103 (4): 1262-1271.].This probe array can obtain the abrupt information in several genes or site in a hybrid experiment simultaneously, greatly reduces time and the cost of test.But some test kit prices of external exploitation are still expensive, and the mutantional hotspot district that can only be used for rpoB gene is detected, catastrophe [the HillemannD of the drug resistant gene of resistance to INH and EMB of mycobacterium tuberculosis can not be detected simultaneously, WeizeneggerM, KubicaT, RichterE, NiemannS, JClinMicrobiol.2005,43:3699-3703.].

Not yet find that there is at present and can detect the technology of mycobacterium tuberculosis to the resistance of RFP, INH and EMB tri-kinds of medicines simultaneously and applied in practice or report.

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 method for Drug Resistance of Mycobacterium Tuberculosis, set up a kind of being based upon and multiplex PCR basis carries out resistance to Rifampin (RIF) to mycobacterium tuberculosis, the reverse serial probing procedure of the resistance rapid detection of vazadrine (INH) and Tibutol (EMB) medicine, can by detecting and RIF, the rpoB that EMB with INH resistance is relevant, katG, inhA generegulation district and embB gene base mutation judge the drug susceptibility of bacterial strain, and bacterial classification (MTC) qualification can be carried out to mycobacterium tuberculosis complex simultaneously, wherein contain pcr amplification primer sequence, hybridization probe sequence.

Solution of the present invention is PCR-based technology and reverse probe hybridization technology.

Particular content of the present invention is as follows:

Multiplex PCR-reverse dot blot hybridization technique detects a method for Drug Resistance of Mycobacterium Tuberculosis, is namely a kind of method that reverse dot blot hybridization technique based on multiplex PCR detects Drug Resistance of Mycobacterium Tuberculosis, it is characterized in that:

The reaction of A, multiplex PCR completes in a pipe, and disposable pcr amplification at least four DNA fragmentation to be detected;

Low density probe array described in B, reverse dot blot hybridization at least comprise 19 can detect simultaneously with

The rpoB that RFP, EMB are relevant with INH resistance, the base mutation of katG, inhA generegulation district and embB transgenation hot zone and M.tuberculosis complex is carried out to the probe of bacterium identification diagnosis;

C, the described Hybond membrane with carboxyl in negative electricity, with the oligonucleotide probe covalent attachment of amino labeled, form bridge architecture;

The vitamin H that primer in D, multiplex PCR adopts Streptavidin-alkaline phosphatase to combine marks, and reacted by catalytic substrate BCIP/NBT and form the positive and the feminine gender that coloured material judges hybridization, reflection PCR primer and probe are with or without sudden change.

The Tm value difference≤3 DEG C of many primers in multiplex PCR of the present invention, amplified production clip size difference 50-77 base pair.

Probe of the present invention is 8 oligonucleotide probes and 1 MTC group specificity probe, and described probe length is 20 ± 3 bases; The base position (mutational site) of mispairing is as far as possible arranged in the middle part of probe; The Tm value of each probe is as far as possible close, and difference answers≤3 DEG C.

The constructive method of multiple oligonucleotide probe array of the present invention is as follows: use 0.5mol/LNa ₂cO ₃/ NaHCO ₃damping fluid (pH8.4) dissolves probe, and weaker concn is 20 μMs, and fully after mixing, Refrigerator store is for subsequent use.BiodyneC nylon membrane is cut into a certain size, and uses pencil defined area.First apply 16% ethyl dimethylamino-propyl carbodiimide (EDAC) immersion treatment nylon membrane 10min with activated nylon membrane (this solution can be reused), then with distilled water cleaning, dry.Application micro sample adding appliance point 2 μ l probe solution is on BiodyneC nylon membrane corresponding position, and reaction 15min, makes the probe of amino labeled and the carboxyl covalent attachment of nylon membrane; Again with 0.1mol/LNaOH process 10min, distilled water cleans, and dries rear for subsequent use.On diaphragm, Fig. 2 is shown in the arrangement of each probe.Wild-type probe is generally put in the left side of nylon membrane, and the saltant type probe in corresponding site is put on the right.

Multiplexed PCR amplification of the present invention refers to increase in 1 amplification system rpoB, katG, inhA generegulation district and embB gene 4 kinds of fragments, makes a PCR react generation four drug resistant gene fragments.Each reaction system is got bacterial genomes DNA1 μ l(and is about 20ng), in 50 μ l reaction systems (containing 0.2mmol/LdNTPs, DNA profiling 100ng in 50 μ L reaction response systems, primer 0.2mmol/L, Taq enzyme 1.5U, 10 × PCR damping fluid of 1/10 volume, 1.5mmol/LMgCl ₂).Amplification program: 95 DEG C, 5min; 95 DEG C, 1min, 56 DEG C, 1min, and 72 DEG C, 1min, totally 40 circulations; Last 10min extension at 72 DEG C.

The method of reverse dot blot hybridization of the present invention is as follows: get biotin labeled pcr amplification product 40 μ L and add 1 × SSPE200 μ L in the EP pipe of 0.5mL, after 95 DEG C of 15min carry out sex change, put into rapidly ice and prevent PCR primer renaturation, then to learn from else's experience the PCR primer of sex change, add and place in advance and preheating nylon membrane and 3mL hybridization solution (5 × SSPE; 0.5%SDS) centrifuge tube, 57 DEG C of hybridization 30min.Take out film bar, move to and washing lotion (1 × SSPE is housed; 0.5%SDS) and through the centrifuge tube of 57 DEG C of preheatings, 57 DEG C of each 15min wash twice.All the other operating process are at room temperature carried out: hatch 15min in 3mL connecting fluid (including Streptavidin-alkaline phosphatase), then use washing lotion room temperature washing 2 times, each 10min.Use substrate buffer solution (PBS) to wash once again, then add 3mL substrate reactions liquid BCIP/NBT, under room temperature, jog 30min, washes with water diaphragm, dry, and namely visible blue spot manifests.

The reaction of multiplex PCR of the present invention completes in a pipe, and disposable pcr amplification is four DNA fragmentations to be detected.

Low density probe array described in reverse dot blot hybridization of the present invention comprises 19 can detect the rpoB relevant to RIF, EMB and INH resistance simultaneously, the base mutation of katG, inhA generegulation district and embB transgenation hot zone and M.tuberculosis complex is carried out to the probe of bacterium identification diagnosis.

The present invention is multiplex PCR and reverse hybridizedly combine, disposable pcr amplification four DNA fragmentations to be detected, by designing a novel low density probe array, being applied to the quick Drug Resistance Detection of tubercule bacillus, the catastrophe of multiple drug resistant gene can be detected like this in single test.The all Hybond membranes of the present invention are BiodyneC type nylon membrane, are produced by PallBioSupport company of the U.S., this nylon membrane because be negative electricity with carboxyl, can with the oligonucleotide probe covalent attachment of amino labeled, formation bridge architecture, can improve hybrid rate greatly.And primer vitamin H marks, Streptavidin-alkaline phosphatase can be used to combine, be reacted by catalytic substrate BCIP/NBT and form the positive and the feminine gender that coloured material judges hybridization, and then reflect that PCR primer and probe are with or without sudden change.

The present invention is based on the reverse serial probe hybridization technology of PCR and reverse dot blot hybridization technique, there is easy, direct, cheap, feature fast, multiple medicines drug resistance information can be provided in single test, particularly can detect the resistance of mycobacterium tuberculosis to RIF, INH and EMB tri-kinds of medicines simultaneously, reduce time and the cost of test, result accurately, reliable, and without the need to the specialized equipment of costliness and reagent, be conducive to large-scale promotion application.

Materials and methods of the present invention comprises following content:

1. the Design & preparation of primer and oligonucleotide probe

Tubercule bacillus rpoB is searched for from GenBank, katG, inhA generegulation district and embB gene, and determine above-mentioned drug-tolerant gene mutation hot zone, apply PrimerPremier5.0 and Oligo6.0 software design four pairs of Auele Specific Primers respectively according to the conservative region on its side, in order to carry out multiplex PCR, the Tm value of four primers is close, amplified production clip size difference 50-77 base pair is beneficial to electrophoretic separation (table 1), and reverse primer all applies biotin labeling.

According to drug-tolerant gene mutation focus, the biosoftwares such as application PrimerPremier5.0 design 18 oligonucleotide probes altogether.In addition, according to rpoB sequences Design 1 MTC group specificity probe.Probe design principle is: probe length is about 20 bases; The base position (mutational site) of mispairing is as far as possible arranged in the middle part of probe; The Tm value of each probe is as far as possible close, differs about 3 DEG C.Shown in each probe specifying information table 2.All probe 5 ' ends carry out amido modified.Primer and probe synthesize by Invitrogen Shanghai company.

Table 1PCR amplification drug resistant gene rpoB, the primer of katG, inhA generegulation district and embB gene

2. multiple oligonucleotide probe array

Use 0.5mol/LNa ₂cO ₃/ NaHCO ₃damping fluid (pH8.4) dissolves probe, and weaker concn is 20 μMs, and fully after mixing, Refrigerator store is for subsequent use.BiodyneC nylon membrane is cut into a certain size, and uses pencil defined area.First apply 16% ethyl dimethylamino-propyl carbodiimide (EDAC) immersion treatment nylon membrane 10min with activated nylon membrane (this solution can be reused), then with distilled water cleaning, dry.Application micro sample adding appliance point 2 μ L probe solution is on BiodyneC nylon membrane corresponding position, and reaction 15min, makes the probe of amino labeled and the carboxyl covalent attachment of nylon membrane; Again with 0.1mol/LNaOH process 10min, distilled water cleans, and dries rear for subsequent use.On diaphragm, Fig. 2 is shown in the arrangement of each probe.Wild-type probe is generally put in the left side of nylon membrane, and the saltant type probe in corresponding site is put on the right.Half a year can be preserved at 4 DEG C with the nylon membrane of this method process.

Table 2 drug resistant gene rpoB, katG, inhA generegulation district and embB gene oligonucleotide probe

^aPR:rpoB； ^bK:katG； ^cEembB；

3.PCR increases

Amplification rpoB, katG, inhA generegulation district and embB gene 4 kinds of fragments increase at 1 amplification system, make a PCR react generation four drug resistant gene fragments.Each reaction system is got bacterial genomes DNA1 μ L(and is about 20ng), in 50 μ l reaction systems (containing 0.2mmol/LdNTPs, DNA profiling 100ng in 50 μ L reaction response systems, primer 0.2mmol/L, Taq enzyme 1.5U, 10 × PCR damping fluid of 1/10 volume, 1.5mmol/LMgCl ₂).Amplification program: 95 DEG C, 5min; 95 DEG C, 1min, 56 DEG C, 1min, and 72 DEG C, 1min, totally 40 circulations; Last 10min extension at 72 DEG C.

4. oppositely film hybridization

Get biotin labeled pcr amplification product 40 μ L and add 1 × SSPE200 μ L in the EP pipe of 0.5mL, after 95 DEG C of 15min carry out sex change, put into rapidly ice and prevent PCR primer renaturation, then to learn from else's experience the PCR primer of sex change, add and place in advance and preheating nylon membrane and 3ml hybridization solution (5 × SSPE; 0.5%SDS) centrifuge tube, 57 DEG C of hybridization 30min.Take out film bar, move to and washing lotion (1 × SSPE is housed; 0.5%SDS) and through the centrifuge tube of 57 DEG C of preheatings, 57 DEG C of each 15min wash twice.All the other operating process are at room temperature carried out: hatch 15min in 3ml connecting fluid (including Streptavidin-alkaline phosphatase), then use washing lotion room temperature washing 2 times, each 10min.Use substrate buffer solution (PBS) to wash once again, then add 3ml substrate reactions liquid BCIP/NBT, under room temperature, jog 30min, washes with water diaphragm, dry, and namely visible blue spot manifests.

5. result judges

, there is blue hybridization spot in the method that result judges: the judgement detected result that puts in order of probe on control film bar, is judged to be that this probe hybridization is positive, otherwise then hybridizes feminine gender.

Verification method:

1, bacterium source: 99 strains from Chengdu tuberculosis prevention & care inst, have the clinical separation strain of different resistance pattern and 20 strains to Rimactazid and Tibutol equal responsive clinical separation strain comprising 79 strains for examination mycobacterium tuberculosis strain isolateds.The code that all Culture Collection, qualification and preservation are all formulated with WHO is carried out, mycobacterium cultivation, strain identification and drug sensitive test [Chinese anti-tuberculosis association is carried out with reference to Laboratory Science Procedure of Diagnostic Bacteriology in Tuberculosis, middle national defence consumptive disease magazine, 1996,18:28-31.].

2, medicine and reagent: Taq DNA polymerase: purchased from the precious biotech firm in Dalian; DNTPs: purchased from Chengdu Bo Ruike biotech firm; PCR primer reclaims test kit: U.S. Qiagen Products; DNA molecular amount standard DL-2000: precious biotechnology (Dalian) company limited product; Nylon membrane: BiodyneC type film, PallBioSupport company of the U.S.; EDAC(ethyl dimethylamino-propyl carbodiimide): purchased from purchased from Shanghai Sangon Biological Engineering Technology And Service Co., Ltd; 20 × SSPE:3.6MNaCl, 0.2MNa ₂pO ₄, 0.02MEDTA (pH7.5); Hybridization solution: 5 × SSPE, 0.5%SDS; Washing lotion: 1 × SSPE, 0.5%SDS; Streptavidin-alkaline phosphatase: purchased from Shanghai Sangon Biological Engineering Technology And Service Co., Ltd; Phosphoric acid buffer (PBS): 50mM sodium phosphate salt, 200mMNaCl(pH7.4); 5-bromo-4-chloro-3-indolylphosphate salt (5-bromo-4-chloro-3-indoylphosphate, BCIP): German BoehringerMannheim Products; Chlorination nitro tetrazole (nitrobluetetrazolium, NBT): German BoehringerMannheim Products.Other agents useful for same is domestic analytical reagent; Water is redistilled water.Cultivation and the drug sensitive test of Mycobacterium tuberculosis all adopt modified Russell medium.

3, pcr amplification and detection: to the rpoB gene of 99 strain mycobacterium tuberculosis clinical separation strains, and the katG of part bacterial strain, in inhA regulatory region and embB, go to increase in interval, get PCR primer 5 μ L respectively, at 3%(w/v) sepharose carries out electrophoresis, strength of electric field 5V/cm, time 60min, be soaked in after electrophoresis terminates in 0.5 μ g/mL ethidium bromide and dye.Observe gel under ultraviolet lamp with or without object amplified band, and with DNA molecular amount standard comparing, if object pillar location correctly can be judged to amplification positive.

4, PCR primer carries out DNA sequencing: send Hua Da gene to check order PCR primer.

The result:

79 examples have different resistance type strain probe array detected result and contrast with the sequencing result of PCR primer, and as shown in table 3, nearly all probe in detecting result can obtain the support of PCR primer DNA sequence analysis result.And for 20 routine medicaments insensitive strains, sequencing result also shows their corresponding amplification region and all not suddenly change generation, all hybridize the positive with wild-type probe, support the detected result of probe array.

Table 3 probe array and DNA sequence analysis detect the Comparative result of substance of medicines-resistant branched tubercle bacillus sudden change

The mutantional hotspot district of the rpoB of the routine bacterial strain of resistance to Rifampin of application probe array detection 61,53 strains (86.9%) detect sudden change.49 strains are shown as the negative and correctly corresponding together saltant type probe hybridization of wild-type probe hybridization, 4 strain display wild-type probe are lost, also feminine gender is hybridized at the mutant probe listed, therefore can judge that this 4 strain bacterial strain is Resistant strain, but its emergent properties be can not determine, because do not place the probe of corresponding mutation type, so do not have mutant probe to hybridize with it.Modal mutation type is S531L (TCG → TTG), detects 34 examples (55.8%) in the 61 routine bacterial strains of resistance to RIF altogether.In addition, common mutation type also comprises 511(CTG → CCG), 516(GAC → GGC) and 526(GAC → TAC) etc. site.Compared with the Phenotypic examination result of routine, the susceptibility that this probe array detects Sichuan province crowd's mycobacterium tuberculosis RIF resistance is 86.9%.

Result shows, in the 52 routine bacterial strains of resistance to vazadrine, 30 examples (57.7%) detect sudden change in katG codon 315 site, there is S315T (AGC → ACC), only 2 examples present wild-type probe KW and saltant type probe KM and all hybridize negative pattern, this probe array do not design corresponding saltant type probe.Prompting katG codon 315 is undergone mutation, but the particular type that can not clearly suddenly change, their emergent properties needs to be determined by further DNA sequencing.Meanwhile, detect 4 examples (7.7%, 4/52) Resistant strain, in-15 sites of inhA regulatory region, (C → T) sudden change occurs, wherein only there is inhA sudden change in 2 examples, not with katG315 codon mutation; And another 2 routine inhA sudden changes are simultaneously with katG315 codon mutation.Generally, in the 52 routine bacterial strains of resistance to vazadrine, 34 examples (65.4%) at least detect a kind of sudden change and occur on katG315 bit codon or inhA-15 site.Phenotypic resistance detection method with routine is compared, and the susceptibility that this probe array detects INH resistance is 65.4%.

In the 47 strain bacterial strains of resistance to Tibutol, reverse hybridized display 23 strain finds sudden change in embB gene 306 site, and ATG → GTG, ATG → ATA, the sudden change of ATG → ATC type are 14,4,3 strains respectively, and ratio is 29.8%, 8.5%, 6.4% respectively.Susceptibility is 48.9%.Two strains are that wild probe, saltant type probe are all negative, and meaning is other mutation type.

Result shows, the present invention can to mycobacterium tuberculosis rpoB gene medicament-resistant mutation hot zone, katG315 site, inhA generegulation district and embB gene major part mutational site, substantially reduce the susceptibility detection time of mycobacterium tuberculosis, cheap, simple to operate, therefore likely develop into and be 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 multi-drug resistance tuberculosis control and prevent Drug-fast case bacterial strain propagate play an important role.

Accompanying drawing explanation

Fig. 1 is the PCR primer electrophorogram of each drug resistant gene.Wherein, M:Marker; 1:katGgene; 2:embBgene; 3:rpoBgene; 4:inhAgene

Show application rpoB, katG, inhA generegulation district and embB gene 4 pairs of primer amplified tubercule bacillus strain isolateds, result produces the DNA fragmentation of expection, and all without non-specific amplification band.Each primer carries out separately the result of pcr amplification.Create the DNA fragmentation in the mutantional hotspot district comprising each drug resistant gene respectively.

Fig. 2 is the result figure of multiplex PCR.Four kinds of primers are in same PCR reaction system, and produce four specific bands, be rpoB, katG, inhA generegulation district and embB mutantional hotspot district simultaneously respectively.

Fig. 3 is oligonucleotide probe array mode chart and part tubercule bacillus strain isolated hybridization check schematic diagram.Wherein, (A) positive control (B) negativecontrol; (C) wildtype; (D) rpoBL533P, CTG → CCG; KatG315, AGC → ACC; (E) rpoBS531L, TCG → TTG, embB306:ATG → GTG:katG315AGC → ACC; (F) rpoB511:CTG → CCG, 516:GAC → GGC, embB306:ATG → GTG.

Claims

1. one group is detected the biochemical preparation of Drug Resistance of Mycobacterium Tuberculosis for the preparation of multiplex PCR-reverse dot blot hybridization technique, it is characterized in that described biochemical preparation comprises following reagent:

Multiple oligonucleotide probe array, multiplex PCR system, reverse dot blot hybridization system;

Wherein, the constructive method of multiple oligonucleotide probe array is: use 0.5mol/LNa ₂cO ₃/ NaHCO ₃buffer solution probe, weaker concn is 20 μMs, and fully after mixing, Refrigerator store is for subsequent use, and the pH of damping fluid is 8.4; BiodyneC nylon membrane is cut into a certain size, and uses pencil defined area; First apply 16% ethyl dimethylamino-propyl carbodiimide EDAC immersion treatment nylon membrane 10min with activated nylon membrane, then with distilled water cleaning, dry; Application micro sample adding appliance point 2 μ l probe solution is on BiodyneC nylon membrane corresponding position, and reaction 15min, makes the probe of amino labeled and the carboxyl covalent attachment of nylon membrane; Again with 0.1mol/LNaoH process 10min, distilled water cleans, and dries rear for subsequent use; Wild-type probe is generally put in the left side of nylon membrane, and the saltant type probe in corresponding site is put on the right;

Multiplex PCR system 50 μ l consists of: 0.2mmol/LdNTPs, DNA profiling 100ng, primer 0.2mmol/L, Taq enzyme 1.5U, 10 × PCR damping fluid of 1/10 volume, 1.5mmol/LMgCl ₂;

Consisting of of reverse dot blot hybridization system: vitamin H, pcr amplification product 40 μ L, 1 × SSPE200 μ L, preheating nylon membrane, the hybridization solution 3ml of 5 × SSPE, 0.5%SDS, include the connecting fluid 3ml of Streptavidin-alkaline phosphatase, substrate buffer solution PBS, 3ml substrate reactions liquid BCIP/NBT;

Described multiple oligonucleotide probe array is used for the rpoB relevant to RIF, EMB and INH resistance, and katG, inhA transgenation sequence is hybridized;

The drug resistant gene rpoB of described multiplex PCR system for increasing relevant to RIF, EMB and INH resistance, katG, inhA;

Described reverse dot blot hybridization system is carried out hybridization for drug resistant gene PCR primer and multiple oligonucleotide probe array and is developed the color, to determine its sudden change and resistant type;

Described multiple oligonucleotide probe array middle probe sequence is:

MTC5’-NH2-CTGTCGGGGTTGACCCAC-3’，

PW15’-NH2-CAGCCAGCTGAGCCAATT-3’

PM15’-NH2-AGCCAGCCGAGCCAATT-3

PW25’-NH2-CCAATTCATGGACCAGAACAA-3

PM25’-NH2-AATTCATGGGCCAGAACAAC-3

PW35’-NH2-GTTGACCCACAAGCGCC-3’

PM35’-NH2-TGACCTACAAGCGCCGA-3

PW45’-NH2-GACTGTCGGCGCTGGG-3’

PM45’-NH2-GCCGACTGTTGGCGCT-3’

PW55’-NH2-GACTGTCGGCGCTGGG-3’

PM55’-NH2-ACTGTCGGCGCCGGG-3

KW5’-NH2-ATCACCAGCGGCATCG-3’

KM5’-NH2-ATCACCACCGGCATCG-3’

EW5’-NH2-TACATCCTGGGCATGGCC-3’

EM15’-NH2-TACATCCTGGGCATCGC-3’

EM25’-NH2-TACATCCTGGGCATAGC-3’

EM35’-NH2-TACATCCTGGGCGTGGC-3’

IW5’-NH2-CGGCGAGATGATAGGTTGTC-3’

IM5’-NH2-CGGCGAGATGATAGGTTGTC-3’,

In described multiplex PCR system, primer sequence is:

rpoB-F5'-CGATCACACCGCAGACGTTG-3'

rpoB-R5'-CGTTTCGATGAACCCGAAC-3'

katG-F5'-ACACTTTCGGTAAGACCCATG-3’

katG-R5’-CGAGGAAACTGTTGTCCCAT-3’

inhA-F5’-GAAAGGGATCCGTCATGG-3

inhA-R5’-GCTCTTCTACCGCCGTGA-3’

embB-F5’-GCAAGCTGGCGCACCTTCAC-3’

embB-R5’-GCGCATCCACAGACTGGCGTC-3’。

2. one group of biochemical preparation for the preparation of multiplex PCR-reverse dot blot hybridization technique detection Drug Resistance of Mycobacterium Tuberculosis according to claim 1, it is characterized in that: wherein, the constructive method of multiple oligonucleotide probe array is: use 0.5mol/LNa ₂cO ₃/ NaHCO ₃pH of buffer 8.4 dissolves probe, and weaker concn is 20 μMs, and fully after mixing, Refrigerator store is for subsequent use; BiodyneC nylon membrane is cut into a certain size, and uses pencil defined area; First apply 16% ethyl dimethylamino-propyl carbodiimide EDAC immersion treatment nylon membrane 10min with activated nylon membrane, then with distilled water cleaning, dry; Application micro sample adding appliance point 2 μ l probe solution is on BiodyneC nylon membrane corresponding position, and reaction 15min, makes the probe of amino labeled and the carboxyl covalent attachment of nylon membrane; Again with 0.1mol/LNaoH process 10min, distilled water cleans, and dries rear for subsequent use; Wild-type probe is generally put in the left side of nylon membrane, and the saltant type probe in corresponding site is put on the right; Multiplex PCR system 50 μ l consists of: 0.2mmol/LdNTPs, DNA profiling 100ng, primer 0.2mmol/L, Taq enzyme 1.5U, 10 × PCR damping fluid of 1/10 volume, 1.5mmol/LMgCl ₂; Amplification program: 95 DEG C, 5min; 95 DEG C, 1min, 56 DEG C, 1min, and 72 DEG C, 1min, totally 40 circulations; Last 10min extension at 72 DEG C; The constructive method of reverse dot blot hybridization system is: get biotin labeled pcr amplification product 40 μ L and add 1 × SSPE200 μ L in the EP pipe of 0.5mL, after 95 DEG C of 15min carry out sex change, put into rapidly ice and prevent PCR primer renaturation, then to learn from else's experience the PCR primer of sex change, add and place in advance and preheating nylon membrane and 5 × SSPE; The hybridization solution 3ml centrifuge tube of 0.5%SDS, 57 DEG C of hybridization 30min; Take out film bar, move to and 1 × SSPE is housed; The washing lotion of 0.5%SDS and through the centrifuge tube of 57 DEG C of preheatings, 57 DEG C of each 15min wash twice; All the other operating process are at room temperature carried out: 3ml includes in the connecting fluid of Streptavidin-alkaline phosphatase and hatches 15min, then use washing lotion room temperature washing 2 times, each 10min; Wash once with substrate buffer solution PBS, then add 3ml substrate reactions liquid BCIP/NBT, under room temperature, jog 30min, washes with water diaphragm again, dry, and namely visible blue spot manifests.