CN102559916A - Method for detecting multi-drug resistance of Mycobacterium tuberculosis - Google Patents
Method for detecting multi-drug resistance of Mycobacterium tuberculosis Download PDFInfo
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Abstract
The invention relates to a method for detecting multi-drug resistance of Mycobacterium tuberculosis, which aims at detecting the resistance of the Mycobacterium tuberculosis to isoniazid and rifampicin at the same time and has the characteristics of high specificity and sensitivity, quickness in detection, and easiness and convenience in operation. The technical scheme is as follows: the method comprises the following steps: A, establishing PCR (polymerase chain reaction) templates of an MTB clinical strain and a standard strain H37Rv; B, designing three pairs of primers of katG, inhA and rpoB gene fragments which are closely related to the isoniazid resistance and the rifampin resistance, performing mPCR amplification on katG, inhA and rpoB genes of the clinical strain, and performing PCR amplification on katG, inhA and rpoB genes of the clinical strain and the standard strain H37Rv; and C, detecting the mutation conditions of related INH-resistant and RFP-resistant 3 genes of the clinical strain at the same time by a single-strand conformation polymorphism detection method.
Description
Technical field
The invention belongs to biological technical field; Be specifically related to a kind ofly be used for detecting simultaneously mycobacterium tuberculosis (mycobacterium tuberculosis based on multiple polysaccharase formula reaction-single strand conformation polymorphism (mPCR-SSCP); Abbreviation MTB) method of the transgenation that the anti-vazadrine of clinical separation strain is relevant with Rifampin can be used for clinical mycobacterium tuberculosis vazadrine and the chemical sproof rapid detection of Rifampin simultaneously.
Background technology
China's tuberculosis patient numerical digit is in the whole world the second, and the resistance of Mycobacterium tuberculosis presents significant rising tendency in recent years, and China also is one of the highest country of resistance degree, and the appearance of resistance mycobacterium tuberculosis strain has become a serious public health issue.Vazadrine (INH), Rifampin (RFP), Streptomycin sulphate (SM) and Tibutol (EMB) are clinical line antitubercular agents.So-called anti-multiple medicines white plaque is meant at least to INH and two drug-fast white plaque of main antitubercular agent of RFP; The whole world has 4.3% tuberculosis case that anti-multiple medicines takes place approximately; The anti-multiple medicines of part hotspot is especially up to more than 10%; The particularly anti-multiple medicines rate of drug-fast rising rises year by year, becomes white plaque popular major cause day by day.Retrospective review is the result show, total resistant rate of Chinese Mycobacterium tuberculosis is 27.8%, and anti-multiple medicines rate is 10.7%.
MTB drug sensitive test result early is so that work out the key that personalized therapy program is tuberculosis control.Because MTB poor growth, classical drug sensitive test are the basis with the microbial culture, though the appearance of new liquid nutrient medium is arranged at present, consuming time still longer, can not in time obtain the drug sensitive test result; Not only influence result of treatment, and in time blocking propagation possibly promote also persister to form.Therefore, develop the detection method that a kind of rapid determination MTB resistance particularly detects anti-multiple medicines property simultaneously, for standardized drug, in time block the propagation of anti-tuberculosis strain, it is significant to improve the white plaque curative ratio.The experimental results shows that the transgenation of a plurality of drug effect target position is the major cause that causes anti-multiple medicines in the substance of medicines-resistant branched tubercle bacillus.Reported the molecular biology method of many detection tuberculosis drug resistance related gene sudden changes in recent years; Yet these methods often need high cost, the plant and instrument of specialty and extremely strong professional operating skill.Based on above reason, at present domesticly be used for the sudden change of MTB drug resistance related gene and detect and rest in research institution or the laboratory more, present also do not have a kind of the operation without the professional and technical personnel can be in the detection method of clinical application.
The PCR-SSCP technology is that Japanese Guan Gu laboratory in 1989 combines the PCR reaction and foundes with the single stranded DNA native polyacrylamide gel electrophoresis.Its ultimate principle is: dna fragmentation is the complex spatial folded conformation, and this three-dimensional arrangement is mainly kept by interaction force in its inner base pairing equimolecular; When single stranded DNA fragment base changes, influence its space conformation, conformation is changed; The discrepant single strand dna of space conformation is varied in size by exclusion in polyacrylamide gel; Pass through native polyacrylamide gel electrophoresis; Can very observantly discrepant molsep on the conformation be opened; Be so-called single strand conformation polymorphism (Single-Strand Conformation PolymorPhism, SSCP).The SSCP detection method is used to check the transgenation of pcr amplification product, and the PCR-SSCP technology of foundation can be used for detecting in the scientific research disappearance and the insertion of point mutation and short sequence, carries out the DNA quantitative analysis.This method is easy and simple to handle, and is the basis with the pcr amplification, can detect the change that the individual gene sudden change produces, and its sensitivity, specificity height also can be used for general clinical labororatory.Hong Kong Chan in 2007 etc. reported with PCR-SSCP and detected some transgenation of MTB and drug-fast dependency; Bao Hong etc. utilized PCR-SSCP to detect in the sputum specimen and Rifampin drug resistance related gene rpoB sudden change and drug-fast dependency in 2007, and specificity and sensitivity all have the good clinical meaning; But, carry out PCR-SSCP one by one and analyze still very trouble and time-consuming taking a lot of work because MTB drug resistant gene quantity is many; Domestic reports such as Cheng Xiaodong detect relevant three transgenations with the vazadrine resistance simultaneously with mPCC-SSCP, have solved the time-consuming defective of taking a lot of work of single PCR-SSCP, but have only detected the drug resistance related gene sudden change of single medicine vazadrine; Many resistant rate of the incompatible MTB that increases year by year.
Summary of the invention
The objective of the invention is to overcome the deficiency of above-mentioned background technology; Provide a kind of mycobacterium tuberculosis anti-multiple medicines detection method; This method should be able to be measured the resistance of mycobacterium tuberculosis to vazadrine and Rifampin simultaneously, have specificity and sensitivity higher, detect quick, easy to operation, be easy to standardize and cost is low and the characteristics that help popularizing in clinical labororatory.
Technical scheme provided by the invention is: the anti-multiple medicines detection method of a kind of mycobacterium tuberculosis the steps include:
A, set up the pcr template of MTB clinical strains and reference culture H37Rv;
Three pairs of primers of B, design and vazadrine and the closely related katG of Rifampin resistance, inhA and rpoB gene fragment, katG, inhA and the rpoB gene to clinical strains carries out the mPCR amplification simultaneously; And katG, inhA and the rpoB gene of clinical strains and reference culture H37Rv carried out pcr amplification respectively;
C, employing single strand conformation polymorphism detection method detect the sudden change situation of the anti-INH of clinical strains 3 genes relevant with RFP simultaneously;
MPCR primer and PCR primer among the said step B are: the upstream and downstream primer of amplification katG gene is respectively 5 '-AAG GAA GCC ACC TGG CTC GGC-3 ' and 5 '-GCC GAA CGG GTC CGG GAT GGT-3 ', and the size of amplified production expection is 510bp; The upstream and downstream primer of amplification inhA gene is respectively 5 '-GGC ATC CAC ATC TCG GCG-3 ' and 5 '-CAG CGC GCA CAC CGT CTT GGC-3 ', and the size of amplified production expection is 381bp; The upstream and downstream primer of amplification rpoB gene is respectively 5 '-ACA TCC GGC CGG TGG TCG CCG-3 ' and 5 '-TTT CGA TCG GGC ACA TCC GGC-3 ', and the size of amplified production expection is 207bp.
The reaction system of said mPCR amplification is: PCR TV 100 μ L include the primer 2 50mmol/L of upstream and downstream separately, 2.5U Ex-Taq archaeal dna polymerase (TaKaRa), 2 μ L templates, 1 * PCR damping fluid (pH8.3) of 2.5mol/L dNTP, katG, inhA and rpoB gene.
The parameter of said mPCR amplification is: 94 ℃ of 4min; 94 ℃ of 30s, 52 ℃ of 30s, 72 ℃ of 45s, circulation 35 is taken turns; 72 ℃ of 5min; Adopt 1 μ g/mL ethidium bromide to dye 2% agarose gel electrophoresis in advance and detect amplified production.
Only add the primer 2 50mmol/L of upstream and downstream separately of katG, inhA or rpoB gene in the reaction system of said pcr amplification, other composition of reaction system and PCR parameter are identical with mPCR.
Said single strand conformation polymorphism detection method is: each 10 μ L of PCR product of above-mentioned mPCR product and mycobacterium tuberculosis reference culture H37RvkatG, inhA and rpoB gene are mixed with the equal-volume denaturing agent respectively; Ice bath 2min behind the boiling water bath 5min; 4 ℃ of centrifugal 2min of 5000r/min; Water intaking is added to appearance hole on 8% non-denaturing polyacrylamide gel, and 120V electrophoresis 10h behind 13 ℃ of 400V electrophoresis 5min gets offset plate then and carries out argentation dyeing; Observations (single-gene PCR product forms 2 or 3 strands after sex change, present 1～3 band behind the native polyacrylamide gel electrophoresis); If the strand position is different with reference culture H37Rv strand position, be called the motion displacement, be judged to be transgenation.
Also katG, inhA and the rpoB gene to MTB clinical strains and reference culture H37Rv carries out the dna direct order-checking respectively, then sequencing result compared the safety of checking single strand conformation polymorphism detection method.
The H37Rv strain is set when the MTB clinical strains is carried out dna sequencing makes standard control; After the katG of clinical strains and H37Rv strain, inhA or rpoB gene amplification fragment use PCR product purification test kit (BioColor) to purify, reclaim respectively, order-checking.
Also MTB clinical strains and reference culture H37Rv are carried out drug sensitivity test,, measure the mycobacterium tuberculosis clinical strains vazadrine and Rifampin resistance according to drug sensitivity tests criterion.
Said drug sensitivity test is: adopt the absolute concentration method to detect the resistance of MTB clinical strains to INH and RFP; In the experiment being the Quality Control bacterial strain to the responsive reference culture H37Rv of INH and RFP; Drug level is low high two concentration; INH is 1 and 10 μ g/mL, and RFP is 50 and 250 μ g/mL, and the experiment triplicate is to guarantee that the result is correct.
Said drug sensitivity tests criterion is: when seized strain growth was good on no medicine control medium, the pastille medium slant is aseptic to fall to being grown to sensitivity; Colony growth accounts for inclined-plane area 1/4 and is resistance 1+; Colony growth accounts for inclined-plane area 1/2 resistance 2+; Colony growth accounts for inclined-plane area 3/4 and is resistance 3+.The above prompting of lower concentration pastille substratum colony growth 1+ resistance.
The DNA of pcr template is from through extracting the MTB clinical strains of cultivating and the reference culture H37RvDNA genome in the said steps A.
The DNA extraction method of said pcr template is: bacterial cultures is moved in the 1.5ml centrifuge tube, and with saline water washing 3 times, 6000r/m, 4 ℃ of centrifugal 5min abandon supernatant, get deposition and extract DNA with extracting the DNA test kit.
The composition of said denaturing agent is: the tetrabromophenol sulfonphthalein of the methane amide of 95% weight ratio, 10mmoL/LEDTA, 0.02% weight ratio, all the other are water.
The invention has the beneficial effects as follows:
1) the present invention utilizes the three couples disposable detection vazadrine of the NC_000962 H37Rv gene order design that Genbank announces and the mPCR primer sequence of Rifampin drug resistance related gene, can make all clinical strains all can amplify corresponding target gene fragment.
2) the present invention detects and Mycobacterium tuberculosis INH and the closely-related katG of RFP resistance, inhA and rpoB transgenation through mPCR-SSCP simultaneously; Compare with conventional absolute concentration method drug sensitive test; The specificity of method is respectively 100% and 97.8%, and sensitivity is 78.6% and 91.1%; Be higher than traditional mycobacterium tuberculosis drug sensitive test, show that the mPCR-SSCP that we invent can be used for the many resistances detections of clinical strains.
3) the present invention detects through the mPCR amplified production being carried out single strand conformation polymorphism (SSCP) (like MTB clinical strains katG, inhA and rpoB gene fragment existence sudden change; Then through whether existing different with the number and the position of H37Rv strain comparison dna strand behind the electrophoresis), filter out thus and have the goal gene mutant strain; Directly check order through PCR again and confirm the goal gene mutational site, (detecting proves: all INH sensitivity mycobacterium tuberculosis genes involveds are all consistent with PCR-DS result, and RFP sensitive organism coincidence rate is 98.9% further to have verified the safety of SSCP; The INH mPCR-SSCP of anti-bacterial strain detected result and PCR-DS coincidence rate are 91.7%).(RFP Resistant strain mPCR-SSCP detected result and PCR-DS coincidence rate are 95.3% also to have measured resistance through drug sensitivity test.The total coincidence rate of mPCR-SSCP and PCR-DS is at least 91.7%).The detection Mycobacterium tuberculosis INH of mPCR-SSCP and the safety of the closely-related katG of RFP resistance, inhA and rpoB transgenation have further been verified.
4) the present invention carries out a plurality of in same PCR reaction system and drug resistance related gene purpose fragment amplification through design of primers; In conjunction with the single stranded DNA native polyacrylamide gel electrophoresis; Not only utilized the highly sensitive of PCR; And set up mPCR three gene fragments relevant with the Rifampin resistance that increase simultaneously with the vazadrine through the design primer, simplify experimental implementation, reduced experimentation cost.
5) specificity of the present invention and sensitivity are higher; Can detect MTB, also can directly detect doubtful tuberculosis patient sputum specimen, can in 1～2 day, obtain experimental result fast through cultivating; Not only the MTB recall rate is higher than the smear positive rate; And because the present invention is easy and simple to handle, do not need expensive plant and instrument, the cost that is easy to standardize low again, help popularizing, will bring remarkable economic efficiency and social benefit in clinical labororatory.
In addition, the present invention can be used for sputum sample and directly detects; Use the amount of expectoration restriction owing to extract MTB, recall rate is starkly lower than and directly detects with the MTB sample; The present invention has detected doubtful tuberculosis patient sputum sample katG relevant with the INH resistance and inhA transgenation simultaneously with mPCR-SSCP, and the rpoB transgenation relevant with the RFP resistance, and method steps is that the mPCR-SSCP of sample is identical with cultivating MTB; Reducing though find sensitivity and specificity, is direct detected object with patient's sputum, and detected result can provide in 24h; Take for 2～March than the very big shortening time with traditional MTB susceptibility, for the propagation of blocking-up MTB Resistant strain in time provides strong technical support.
Description of drawings
Fig. 1 is a detection method synoptic diagram of the present invention.
Fig. 2 is each the gene upstream and downstream primer sequence design section synoptic diagram that is used for the mPCR amplification among the present invention.
Fig. 3 is the gel electrophoresis figure of mycobacterium tuberculosis katG, inhA and rpoB gene mPCR amplified production.Wherein: swimming lane M:DNA Marker; Swimming lane 1:H37Rv strain amplified band; Swimming lane 2: to INH and the responsive clinical strains of RFP; Swimming lane 3 and 4: only to the drug-fast clinical strains of INH; Swimming lane 5 and 6: only to the drug-fast clinical strains of RFP; Swimming lane 7 and 8: to INH and all drug-fast clinical strains of RFP.
Fig. 4 is mycobacterium tuberculosis katG, inhA and rpoB gene mPCR-SSCP analyzing and testing figure.Wherein: swimming lane MK, MI and M R represent katG, inhA and the rpoB gene PCR-SSCP electrophoresis result of mycobacterium tuberculosis H37Rv strain respectively; Swimming lane 1 and 5:H37Rv strain; Swimming lane 2:katG displacement clinical strains electrophoresis result; Swimming lane 3:inhA displacement clinical strains electrophoresis result; Swimming lane 4:rpoB displacement clinical strains electrophoresis result; Swimming lane 6:katG+inhA displacement clinical strains electrophoresis result; Swimming lane 7:katG+rpoB displacement clinical strains electrophoresis result; Swimming lane 8:inhA+rpoB displacement clinical strains electrophoresis result; Swimming lane 9:katG+inhA+rpoB displacement clinical strains electrophoresis result.
Embodiment
The sequence table explanation
Utilization of the present invention is to cause this resistance mechanism of the drug-fast major cause of some bacterium of tuberculosis branch according to transgenation, sets up the mPCR-SSCP of the sudden change of detection and many drug resistance related genes through molecular biology method.
The foundation of gene Selection: so-called anti-multiple medicines white plaque is meant at least to INH and two drug-fast white plaque of main antitubercular agent of RFP; The experimental results shows; The transgenation of a plurality of drug effect target position is to cause multidrug resistant major cause in the substance of medicines-resistant branched tubercle bacillus, and wherein the sudden change of mycobacterium tuberculosis multidrug-resistance genes involved katG, inhA and rpoB and bacterium are closely related to INH and RFP resistance.Sporting of katG, inhA and rpoB is clinical to provide the mycobacterium tuberculosis clinical strains anti-multiple medicines characteristics so through detecting; We have made up the sudden change that the mPCR-SSCP that can detect the said gene sudden change simultaneously is used to detect the corresponding gene of MTB; The result shows that the sudden change recall rate reaches more than 90%; And detection method sensitivity, specificity height are a kind of simple and easy to do, sensitive, special detection methods.
The present invention adopts the reason of mPCR-SSCP to be: transgenation is to cause the drug-fast major cause of mycobacterium tuberculosis (transgenation of a plurality of drug effect target position is to cause multidrug resistant major cause in the substance of medicines-resistant branched tubercle bacillus); Detecting with the term single gene sudden change at present is the existing many research reports of PCR-SSCP of purpose, along with the continuous rising of anti-multiple medicines rate, usually need detect the resistance of clinical strains to multiple medicine simultaneously; If use a plurality of PCR-SSCP to detect a plurality of drug-tolerant gene mutations, not only improve and detect cost, and operating process is complicated and increased the experimental error incidence; Through mPCR (multiplex amplification) can a plurality of genes of disposable detection the purpose fragment, simplified experimental implementation, reduced experimentation cost.
Detect the mPCR-SSCP of the sudden change of the mycobacterium tuberculosis genes involved of anti-multiple medicines katG, inhA and rpoB, comprise following step:
1) sets up the pcr template (simultaneously as the mPCR amplification template) of MTB clinical strains and reference culture H37Rv
2) the upstream and downstream primer of design and vazadrine and closely related each gene amplification of Rifampin resistance;
3) adopt mPCR in same reaction system, increase simultaneously clinical strains katG, inhA and rpoB target gene fragment;
4) katG, inhA and rpoB goal gene mPCR product filter out goal gene mutant strain (through screening with the reference culture contrast) through SSCP;
5) PCR katG, inhA and the rpoB target gene fragment of clinical strains and reference culture H37Rv that increase respectively expands
Carry out dna sequencing after the raising the output thing purifying and recovering.
Above-mentioned detection method is applicable to each application units (hospital, institute, disease prevention and control center etc.).
The present invention has also comprised following content:
1) clinical strains and reference culture are carried out the drug sensitive test of absolute concentration method, relatively confirm mPCR-SSCP method specificity and sensitivity;
2), confirm that the mPCR-SSCP method detects the recall rate of transgenation with the PCR comparison of directly checking order.
Further specify below in conjunction with embodiment.
Embodiment:
This source of present embodiment acceptance of the bid:, also can be the sputum specimen of doubtful tuberculosis patient for mycobacterium tuberculosis clinical strains through cultivating and identifying.
Reagent source in the present embodiment: used all experiments all can be from domestic and international related industries company or home sale shipping agency of offshore company buy separately with material and reagent and relevant device in the present embodiment.The professional of technical fields such as microbiology, molecular biology, all can repeat the preparation method in the present embodiment and try out.
1.DNA genome extracts: from DNA genome, extract through cultivation and 134 routine MTB clinical strains of identifying and reference culture H37Rv; Specifically: bacterial cultures is moved in the 1.5ml centrifuge tube, and with saline water washing 3 times, the centrifugal 5min of 6000r/m (4 ℃) abandons supernatant, gets deposition and extracts DNA with extracting the DNA test kit, as the template of mPCR and pcr amplification.
2.mPCR design of primers: according to the H37Rv gene order (Accession:NC_000962) of GenBank announcement; Containing 85% above drug-tolerant gene mutation site as far as possible by amplification region is principle, selects the suitable amplification region of katG, inhA and rpoB gene and adopts Blast software design amplimer (the design section synoptic diagram is seen accompanying drawing 2).The upstream and downstream primer of amplification katG gene is respectively 5 '-AAG GAA GCC ACC TGG CTC GGC-3 ' and 5 '-GCC GAA CGG GTC CGG GAT GGT-3 ', and the size of amplified production expection is 510bp; The upstream and downstream primer of amplification inhA gene is respectively 5 '-GGC ATC CAC ATC TCG GCG-3 ' and 5 '-CAG CGC GCA CAC CGT CTT GGC-3 ', and the size of amplified production expection is 381bp; The upstream and downstream primer of amplification rpoB gene is respectively 5 '-ACA TCC GGC CGG TGG TCG CCG-3 ' and 5 '-TTT CGA TCG GGC ACA TCC GGC-3 ', and the size of amplified production expection is 207bp.
3.mPCR reaction system: reaction system TV 100 μ L include the primer 2 50mmol/L of upstream and downstream separately, 2.5U Ex-Taq archaeal dna polymerase (TaKaRa), 2 μ L templates, 1 * PCR damping fluid (pH8.3) of 2.5mol/L dNTP, katG, inhA and rpoB gene.
4.mPCR parameter: 94 ℃ of 4min; 94 ℃ of 30s, 52 ℃ of 30s, 72 ℃ of 45s, circulation 35 is taken turns; 72 ℃ of 5min.2% agarose gel electrophoresis that adopts 1 μ g/mL ethidium bromide to dye in advance detects amplified production.
5.PCR amplification: only add the primer 2 50mmol/L of upstream and downstream separately of katG, inhA or rpoB gene in the reaction system of pcr amplification, other composition of reaction system and PCR parameter are identical with mPCR.
6.SSCP: (totally 4 parts of each 10 μ L of the PCR product of above-mentioned mPCR product and reference culture katG, inhA and rpoB gene; Wherein the mPCR product is 1 part, each 1 part of the PCR product of katG, inhA and rpoB gene) and the equal-volume denaturing agent (composition and weight ratio are: 95% methane amide, 10mmoL/LEDTA, 0.02% tetrabromophenol sulfonphthalein, all the other are water) mix; Ice bath 2min behind the boiling water bath 5min; 5000r/min, 4 ℃ of centrifugal 2min, water intaking is added to appearance hole on 8% non-denaturing polyacrylamide gel, 120V electrophoresis 10h behind 13 ℃ of 400V electrophoresis 5min; Get offset plate then and carry out argentation dyeing, observations.Single-gene PCR product forms 2 or 3 strands after sex change, present 1～3 band behind the native polyacrylamide gel electrophoresis.If the strand position different with reference culture H37Rv strand position (being called the motion displacement) of clinical strains is judged to be transgenation.
7.PCR directly order-checking: be the safety of verifying that above-mentioned mPCR-SSCP detects, also katG, inhA and the rpoB gene of clinical strains and reference culture carried out PCR directly check order (PCR-DS) respectively; The same mPCR of pcr amplification primer of the individual gene that is used to check order is the primer 2 50mmol/L of upstream and downstream separately that only adds katG, inhA and rpoB gene in the reaction system, and other composition of reaction system is identical with mPCR, and the PCR parameter is also identical with mPCR; The H37Rv strain is set makes standard control, after the katG of clinical strains and reference culture H37Rv, inhA and rpoB gene amplification fragment are purified, are reclaimed with PCR product purification test kit (BioColor), order-checking.
8. drug sensitivity test:, adopt the absolute concentration method to detect the resistance of mycobacterium tuberculosis clinical strains to INH and RFP for verifying resistance that above-mentioned mPCR-SSCP detects coincidence rate as a result; To be the Quality Control bacterial strain to INH and the responsive reference culture H37Rv of RFP, drug level is low high two concentration in the experiment, and INH is 1 and 10 μ g/mL, and RFP is 50 and 250 μ g/mL, and the experiment triplicate is to guarantee that the result is correct.
9. drug sensitivity tests criterion: when seized strain growth was good on no medicine control medium, the pastille medium slant is aseptic to fall to being grown to sensitivity; Colony growth accounts for inclined-plane area 1/4 and is resistance 1+; Colony growth accounts for inclined-plane area 1/2 resistance 2+; Colony growth accounts for inclined-plane area 3/4 and is resistance 3+.The above prompting of lower concentration pastille substratum colony growth 1+ resistance.
The present embodiment result
1. drug sensitive test result: in the 134 strain clinical strains, 52 strains are to INH or to the RFP resistance, and total resistant rate is 38.8%; 35 strains are anti-multiple medicines to INH and RFP resistance simultaneously, and anti-multiple medicines rate is 26.1%; 7 and 10 strains are arranged only to INH or RFP resistance respectively.
2. detected result: the katG gene of 510bp, the inhA gene of 381bp and the rpoB gene amplification fragment (Fig. 3) of 207bp all occur behind all (134 strain) mycobacterium tuberculosis clinical strains mPCR and the H37Rv strain PCR.
3.PCR-DS result's (referring to table 1 and table 2): the katG of 134 strain clinical separation strains, inhA and rpoB gene fragment sequencing result and H37Rv strain corresponding gene sequences (Accession:NC_000962) comparative result are seen table 2.Wherein 82 strains are in INH and the equal sensitive strain of RFP, have KatG and the inhA gene fragment order of 81 strains and H37Rv strain corresponding gene sequences identical, and the L511M sudden change takes place 1 strain rpoB gene; 10 strains are only only identical to rpoB gene fragment and the H37Rv strain corresponding sequence of RFP sensitive strain to the katG of INH sensitive strain and inhA gene fragment, 7 strains; PCR-DS detects clinical strains INH and the drug-fast specificity of RFP is respectively 100% and 98.9%.42 strains are in the drug-fast clinical strains of INH, katG and/or inhA transgenation 36 strains, and PCR-DS detects bacterial strain, and drug-fast sensitivity is 85.7% to INH.In the INH Resistant strain, katG genetic testing zone has 8 amino acid that 10 types replacement sudden change takes place, and is the most common with 315 silk amino acid sudden changes; Wherein the S315N/T/I sudden change takes place in 22 strains, and 4 strains are two sudden changes in other site of S315 sudden change companion.InhA genetic testing zone, 11 replacement sudden changes take place in 7 amino acid sites.Detect bacterial strain 43 strains of rpoB transgenation in the 45 strain RFP Resistant strain, PCR-DS detects clinical strains, and drug-fast sensitivity is 95.6% to RFP, and modal mutational site is the nucleotide sequence of coding 526,531 and 516 amino acids; Its mutation frequency H526D/Y/R/L (48.8%; 21/43)＞S531L (39.5%, 17/43)＞D516V/Y (9.3%, 4/43)＞L533P (7.0%; 3/43), wherein two each 1 strain of sudden change of D516V+H526D and H526L+S531L.
4.SSCP result (referring to table 1, table 2 and Fig. 4): three goal gene SSCP of reference standard bacterial strain H37Rv electrophoretogram; 82 strains show INH and all responsive mycobacterium tuberculosis clinical strains SSCP result of RFP; KatG and inhA gene electrophoresis no abnormality seen; RpoB gene electrophoresis has detected 2 strains and has moved displacement, and 17 strains are only to RFP or the equal no abnormality seen of INH sensitive strain SSCP; SSCP detects mycobacterium tuberculosis INH and the drug-fast specificity of RFP is respectively 100% and 97.8%.SSCP detects 42 strain INH resistance clinical strains katG and inhA transgenation situation; Detect unusual 33 strains of katG and/or inhA gene electrophoresis motion; SSCP detects clinical strains, and drug-fast sensitivity is 78.6% (33/42) to INH; KatG gene and inhA gene mPCR-SSCP show as electrophoresis motion displacement bacterial strain number and are respectively 27 strains and 10 strains, wherein 4 strain katG and two gene mPCR-SSCP of inhA electrophoresis motion displacement simultaneously.SSCP detects 45 strain RFP resistance clinical strains rpoB transgenation situation; Find 43 strain RFP Resistant strain rpoB gene electrophoresis motion displacements; SSCP detects clinical strains, and drug-fast sensitivity is 95.6% (43/45) to RFP; Wherein 21 strains companion katG gene electrophoresis motion displacement, the displacement of 4 strains companion inhA gene electrophoresis motion, katG, inhA and three gene motion displacements simultaneously of rpoB appear in 2 strains.
Table 1 52 strain resistance mycobacterium tuberculosis mPCR-SSCP and PCR-DS detected results
Annotate: drug sensitivity tests R representes resistance, and S representes sensitivity; Sequencing result S, N, H, D, R, A, G, L, C, V, I, T, Y, P and W represent Serine, l-asparagine, Histidine, aspartic acid, l-arginine, L-Ala, glycocoll, leucine, halfcystine, Xie Ansuan, Isoleucine, Threonine, tyrosine, proline(Pro) and tryptophane respectively
The sensitivity and the specificity of table 2mPCR-SSCP and PCR-DS detection method
Annotate: INH-R, INH+RFP-R, INH-S and RFP-R, INH+RFP-R, RFP-S represent respectively to the vazadrine resistance, simultaneously to vazadrine resistance and Rifampin resistance, to the vazadrine sensitive strain and to the Rifampin resistance, simultaneously to vazadrine resistance and Rifampin resistance, to the Rifampin sensitive strain.
Sequence table
< 110>Sun Aihua, Yan Jie, Li Zhaodong
< 120>a kind of many Drug Resistance Detection of mycobacterium tuberculosis method
<160>6
<170>WinBlast?v.0.2.0
<210>1
< 211>Nucleotide 21
<212>DNA
< 213>synthetic
< 221>upstream primer of mycobacterium tuberculosis katG gene
<400>1
aaggaagcca?cctggctcgg?c
<210>2
< 211>Nucleotide 21
<212>DNA
< 213>synthetic
< 221>downstream primer of mycobacterium tuberculosis katG gene
<400>1
gccgaacggg?tccgggatgg?t
<210>3
< 211>Nucleotide 18
<212>DNA
< 213>synthetic
< 221>upstream primer of mycobacterium tuberculosis inhA gene
<400>1
ggcatccaca?tctcggcg
<210>4
< 211>Nucleotide 21
<212>DNA
< 213>synthetic
< 221>downstream primer of mycobacterium tuberculosis inhA gene
<400>1
cagcgcgcac?accgtcttgg?c
<210>5
< 211>Nucleotide 21
<212>DNA
< 213>synthetic
< 221>upstream primer of mycobacterium tuberculosis rpoB gene
<400>1
acatccggcc?ggtggtcgcc?g
<210>6
< 211>Nucleotide 21
<212>DNA
< 213>synthetic
< 221>downstream primer of mycobacterium tuberculosis rpoB gene
<400>1
tttcgatcgg?gcacatccgg?c
Claims (10)
1. the anti-multiple medicines detection method of mycobacterium tuberculosis the steps include:
A, set up MTB clinical strains and H
37The pcr template of Rv reference culture;
Three pairs of primers of B, design and vazadrine and the closely related katG of Rifampin resistance, inhA and rpoB gene fragment, katG, inhA and the rpoB gene to clinical strains carries out the mPCR amplification simultaneously; And to clinical strains and H
37The Rv reference culture katG, inhA and rpoB gene carry out pcr amplification respectively;
C, employing single strand conformation polymorphism detection method detect the sudden change situation of the anti-INH of clinical strains 3 genes relevant with RFP simultaneously;
MPCR primer and PCR primer among the said step B are: the upstream and downstream primer of amplification katG gene is respectively 5 '-AAG GAA GCC ACC TGG CTC GGC-3 ' and 5 '-GCC GAA CGG GTC CGG GAT GGT-3 ', and the size of amplified production expection is 510bp; The upstream and downstream primer of amplification inhA gene is respectively 5 '-GGC ATC CAC ATC TCG GCG-3 ' and 5 '-CAG CGC GCA CAC CGT CTT GGC-3 ', and the size of amplified production expection is 381bp; The upstream and downstream primer of amplification rpoB gene is respectively 5 '-ACA TCC GGC CGG TGG TCG CCG-3 ' and 5 '-TTT CGA TCG GGC ACA TCC GGC-3 ', and the size of amplified production expection is 207bp.
2. the anti-multiple medicines detection method of a kind of mycobacterium tuberculosis according to claim 1; The reaction system that it is characterized in that said mPCR amplification is: PCR TV 100 μ L include the primer 2 50mmol/L of upstream and downstream separately, 2.5UEx-TaqDNA polysaccharase, the 2 μ L templates of 2.5mol/L dNTP, katG, inhA and rpoB gene, 1 * PCR damping fluid of pH8.3.
3. the anti-multiple medicines detection method of a kind of mycobacterium tuberculosis according to claim 2 is characterized in that the parameter of said mPCR amplification is: 94 ℃ of 4min; 94 ℃ of 30s, 52 ℃ of 30s, 72 ℃ of 45s, circulation 35 is taken turns; 72 ℃ of 5min; Adopt 1 μ g/mL ethidium bromide to dye 2% agarose gel electrophoresis in advance and detect amplified production.
4. according to claim 2 or the anti-multiple medicines detection method of 3 described a kind of mycobacterium tuberculosis, it is characterized in that said single strand conformation polymorphism detection method is: with above-mentioned mPCR product and mycobacterium tuberculosis H
37Each 10 μ L of the PCR product of Rv reference culture katG, inhA and rpoB gene mix with the equal-volume denaturing agent respectively; Ice bath 2min behind the boiling water bath 5min; 5000r/min4 ℃ of centrifugal 2min, water intaking is added to appearance hole on 8% non-denaturing polyacrylamide gel, 120V electrophoresis 10h behind 13 ℃ of 400V electrophoresis 5min; Get offset plate then and carry out argentation dyeing, observations; If the strand position is different with H37Rv reference culture strand position, be called the motion displacement, be judged to be transgenation.
5. the anti-multiple medicines detection method of a kind of mycobacterium tuberculosis according to claim 4; It is characterized in that only adding in the reaction system of said pcr amplification the primer 2 50mmol/L of upstream and downstream separately of katG, inhA or rpoB gene, other composition of reaction system and PCR parameter are identical with mPCR.
6. the anti-multiple medicines detection method of a kind of mycobacterium tuberculosis according to claim 5 is characterized in that: also to MTB clinical strains and H
37The katG of Rv reference culture, inhA and rpoB gene carry out the dna direct order-checking respectively, then sequencing result are compared the safety of checking single strand conformation polymorphism detection method;
The H37Rv strain is set when the MTB clinical strains is carried out dna sequencing makes standard control; After the katG of clinical strains and H37Rv strain, inhA or rpoB gene amplification fragment are purified, are reclaimed with PCR product purification test kit respectively, order-checking.
7. the anti-multiple medicines detection method of a kind of mycobacterium tuberculosis according to claim 6 is characterized in that also to MTB clinical strains and H
37The Rv reference culture carries out drug sensitivity test, according to drug sensitivity tests criterion, measures the mycobacterium tuberculosis clinical strains to vazadrine and Rifampin resistance;
Said drug sensitivity test is: adopt the absolute concentration method to detect the resistance of MTB clinical strains to INH and RFP, in the experiment with H to INH and RFP sensitivity
37The Rv reference culture is the Quality Control bacterial strain, and drug level is low high two concentration, and INH is 1 and 10 μ g/mL, and RFP is 50 and 250 μ g/mL, and the experiment triplicate is to guarantee that the result is correct;
Said drug sensitivity tests criterion is: when seized strain growth was good on no medicine control medium, the pastille medium slant is aseptic to fall to being grown to sensitivity; Colony growth accounts for inclined-plane area 1/4 and is resistance 1+; Colony growth accounts for inclined-plane area 1/2 resistance 2+; Colony growth accounts for inclined-plane area 3/4 and is resistance 3+.The above prompting of lower concentration pastille substratum colony growth 1+ resistance.
8. the anti-multiple medicines detection method of a kind of mycobacterium tuberculosis according to claim 7, the DNA that it is characterized in that pcr template in the said steps A is from MTB clinical strains and H through cultivating
37Extract in the Rv reference culture DNA genome.
9. the anti-multiple medicines detection method of a kind of mycobacterium tuberculosis according to claim 8; The DNA extraction method that it is characterized in that said pcr template is: bacterial cultures is moved in the 1.5ml centrifuge tube; With saline water washing 3 times; 6000r/m, 4 ℃ of centrifugal 5min abandon supernatant, get deposition and extract DNA with extracting the DNA test kit.
10. the anti-multiple medicines detection method of a kind of mycobacterium tuberculosis according to claim 9 is characterized in that the composition of said denaturing agent is: the tetrabromophenol sulfonphthalein of the methane amide of 95% weight ratio, 10mmoL/LEDTA, 0.02% weight ratio, all the other are water.
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