CN102559916B - Method for detecting multi-drug resistance of Mycobacterium tuberculosis - Google Patents
Method for detecting multi-drug resistance of Mycobacterium tuberculosis Download PDFInfo
- Publication number
- CN102559916B CN102559916B CN201210043565.9A CN201210043565A CN102559916B CN 102559916 B CN102559916 B CN 102559916B CN 201210043565 A CN201210043565 A CN 201210043565A CN 102559916 B CN102559916 B CN 102559916B
- Authority
- CN
- China
- Prior art keywords
- inha
- katg
- pcr
- resistance
- gene
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Expired - Fee Related
Links
Images
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 of based on multiple polysaccharase formula reaction-single strand conformation polymorphism (mPCR-SSCP) for detect mycobacterium tuberculosis (mycobacterium tuberculosis simultaneously, be called for short MTB) method of the transgenation that the resistance to vazadrine of clinical separation strain is relevant with Rifampin, can be simultaneously for the rapid detection of clinical mycobacterium tuberculosis vazadrine and detection of rifampin resistant.
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 is also one of country that resistance degree is the highest, 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 resistance to multiple medicines tuberculosis refers at least tuberculosis to INH and two Main Antituberculosis Drugs thing resistances of RFP, the whole world approximately has 4.3% tuberculosis case that resistance to multiple medicines occurs, the resistance to multiple medicines of part hotspot is especially up to more than 10%, the rising of resistance particularly resistance to multiple medicines rate is risen year by year, becomes the major cause that tuberculosis is increasingly popular.Retrospective review result shows, total resistant rate of Chinese Mycobacterium tuberculosis is 27.8%, and resistance to multiple medicines rate is 10.7%.
MTB drug sensitive test result is early the key of Control to work out personalized therapy program.Due to MTB poor growth, classical drug sensitive test be take microbial culture as basis, although there is the appearance of new liquid nutrient medium at present, consuming time still longer, can not obtain in time drug sensitive test result; Not only affect result for the treatment of, and blocking propagation also may promote persister to form in time.Therefore, develop a kind of Fast Measurement MTB resistance and particularly detect the detection method of multiple drug-resistance simultaneously, for standardized drug, block in time the propagation of resistance to tuberculosis strain, improve tuberculosis curative ratio significant.The experimental results demonstration, in substance of medicines-resistant branched tubercle bacillus, the transgenation of a plurality of drug effect target position is the major cause that causes resistance to multiple medicines.Reported in recent years the molecular biology method of many detection Drug Resistance for Tuberculosis associated gene mutations; Yet these methods often need high cost, the plant and instrument of specialty and extremely strong professional operation skill.Based on above reason, at present domesticly for the sudden change of MTB drug resistance related gene, detect and rest in research institution or laboratory more, current also do not have a kind of the operation without professional and technical personnel can be in the detection method of clinical application.
PCR-SSCP technology is that Japanese Guan Gu laboratory in 1989 combines PCR reaction and found with single stranded DNA native polyacrylamide gel electrophoresis.Its ultimate principle is: DNA fragmentation is complicated space folding conformation, and this three-dimensional arrangement is mainly maintained by interaction force in its inner base pairing equimolecular; When single stranded DNA fragment base changes, affect 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 molecular separation in conformation be opened, be so-called single strand conformation polymorphism (Single-Strand Conformation PolymorPhism, SSCP).By SSCP detection method, for checking the transgenation of pcr amplification product, the PCR-SSCP technology of foundation, can be used for detecting in scientific research the deletion and insertion of point mutation and short sequence, carries out DNA quantitative analysis.The method is easy and simple to handle, and take pcr amplification as basis, the change that individual gene sudden change produces can be detected, and its sensitivity, specificity are high, also can be used for general clinical labororatory.Hong Kong Chan in 2007 etc. reported the dependency that detects some transgenation of MTB and resistance with PCR-SSCP, Bao Hong etc. utilize PCR-SSCP to detect in sputum specimen and the dependency of rifampin-resistance genes involved rpoB sudden change with resistance for 2007, and specificity and sensitivity have good clinical meaning; But because MTB drug resistant gene quantity is many, carry out one by one PCR-SSCP analysis and still very bother and time-consuming taking a lot of work; The domestic reports such as Cheng Xiaodong detect three transgenations relevant to Isoniazid-resistant with mPCC-SSCP simultaneously, have solved the time-consuming defect of taking a lot of work of single PCR-SSCP, but have only detected single medicine Isoniazid-resistant associated gene mutation; Many resistant rate of the inadaptable MTB increasing year by year.
Summary of the invention
The object of the invention is to overcome the deficiency of above-mentioned background technology, a kind of method for detecting multi-drug resistance of Mycobacterium tuberculosis is provided, the 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 standardization and cost low and be conducive to the universal feature in clinical labororatory.
Technical scheme provided by the invention is: a kind of method for detecting multi-drug resistance 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 the closely related katG of B, design and vazadrine and rifampin-resistance, inhA and rpoB gene fragment carry out mPCR amplification to the katG of clinical strains, inhA and rpoB gene simultaneously; And katG, the inhA of clinical strains and reference culture H37Rv and rpoB gene are carried out respectively to pcr amplification;
C, employing single strand conformation polymorphism detection method detect the sudden change situation of 3 genes that the resistance to INH of clinical strains is relevant with RFP simultaneously;
MPCR primer and PCR primer in described 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 described mPCR amplification is: PCR cumulative volume 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 described 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 in advance 2% agarose gel electrophoresis and detect amplified production.
The primer 2 50mmol/L of upstream and downstream separately that only adds katG, inhA or rpoB gene in the reaction system of described pcr amplification, other composition of reaction system and PCR parameter are identical with mPCR.
Described single strand conformation polymorphism detection method is: by above-mentioned mPCR product and mycobacterium tuberculosis reference culture H37RvkatG, each 10 μ L of the PCR product of inhA and rpoB gene mix with equal-volume denaturing agent respectively, ice bath 2min after boiling water bath 5min, 4 ℃ of centrifugal 2min of 5000r/min, water intaking is added to 8% non-denaturing polyacrylamide gel loading hole, 120V electrophoresis 10h after 13 ℃ of 400V electrophoresis 5min, then get offset plate and carry out argentation dyeing, (single-gene PCR product forms 2 or 3 strands to observations after sex change, after native polyacrylamide gel electrophoresis, present 1～3 band), if strand position is different from reference culture H37Rv strand position, be called motion displacement, be judged to be transgenation.
Also katG, the inhA of MTB clinical strains and reference culture H37Rv and rpoB gene are carried out respectively to DNA direct Sequencing, then sequencing result is compared, the reliability of checking single strand conformation polymorphism detection method.
H37Rv strain is set when MTB clinical strains is carried out to DNA sequencing and makes standard control; After the katG of clinical strains and H37Rv strain, inhA or rpoB gene amplification fragment use respectively PCR product purification test kit (BioColor) to purify, reclaim, order-checking.
Also MTB clinical strains and reference culture H37Rv are carried out to drug sensitivity test, according to drug sensitivity tests criterion, measure mycobacterium tuberculosis clinical strains to vazadrine and detection of rifampin resistant.
Described drug sensitivity test is: adopt Absolute concentration method to detect the resistance of MTB clinical strains to INH and RFP, in experiment, take to the reference culture H37Rv of INH and RFP sensitivity is Quality Control bacterial strain, drug level is low high two concentration, INH is 1 and 10 μ g/mL, RFP is 50 and 250 μ g/mL, and experiment is in triplicate to guarantee that result is correct.
Described drug sensitivity tests criterion is: when on without medicine control medium, tested strain growth is good, pastille medium slant is responsive without colony growth; Colony growth accounts for inclined-plane area 1/4 for resistance 1+; Colony growth accounts for inclined-plane area 1/2 resistance 2+; Colony growth accounts for inclined-plane area 3/4 for resistance 3+.Lower concentration pastille substratum colony growth 1+ point out resistance above.
In described steps A, the DNA of pcr template extracts from the MTB clinical strains through cultivating and reference culture H37RvDNA genome.
The DNA extraction method of described pcr template is: bacterial cultures is moved in 1.5ml centrifuge tube, and with physiological saline washing 3 times, 6000r/m, 4 ℃ of centrifugal 5min abandon supernatant, gets precipitation and extracts DNA with extracting DNA test kit.
The composition of described 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 three couples disposable detection vazadrine of the NC_000962 H37Rv gene order design that Genbank announces and the mPCR primer sequence of rifampin-resistance genes involved, can make all clinical strains all can amplify corresponding goal gene fragment.
2) the present invention detects and Mycobacterium tuberculosis INH and the closely-related katG of RFP resistance, inhA and rpoB transgenation by mPCR-SSCP simultaneously, with conventional Absolute concentration method drug sensitive test comparison, the specificity of method is respectively 100% and 97.8%, and sensitivity is 78.6% and 91.1%; Higher than traditional drug susceptibility test of mycobacterium tuberculosis, show that the mPCR-SSCP that we invent can detect for clinical strains Multidrug resistance.
3) the present invention detects by mPCR amplified production being carried out to single strand conformation polymorphism (SSCP) (as MTB clinical strains katG, inhA and rpoB gene fragment existence sudden change, by whether existing different from number and the position of H37Rv strain comparison dna strand after electrophoresis), filter out thus and have goal gene mutant strain; By PCR direct Sequencing, determine goal gene mutational site again, further verified that the reliability of SSCP (detects proof: the responsive mycobacterium tuberculosis genes involved of all INH is all consistent with PCR-DS result, and RFP sensitive organism coincidence rate is 98.9%; The INH mPCR-SSCP of resistance to 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 by drug sensitivity test, to have measured resistance.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 reliability 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 and drug resistance related gene object fragment amplification in same PCR reaction system by design of primers, in conjunction with single stranded DNA native polyacrylamide gel electrophoresis, not only utilized the highly sensitive of PCR, and set up mPCR three gene fragments relevant to vazadrine and rifampin-resistance that simultaneously increase by design primer, simplify experimental implementation, reduced experimentation cost.
5) specificity of the present invention and sensitivity are higher, can detect the MTB by cultivating, also can direct-detection Tuberculosis people sputum specimen, can in 1～2 day, obtain fast experimental result, not only MTB recall rate is higher than 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 is low again, is conducive to popularize in clinical labororatory, will bring significant economic benefit and social benefit.
In addition, the present invention can be used for sputum sample direct-detection; Owing to extracting MTB, use amount of expectoration restriction, recall rate is starkly lower than and directly with MTB sample, detects; The present invention has detected katG and the inhA transgenation relevant to INH resistance of Tuberculosis people sputum sample with mPCR-SSCP simultaneously, and the rpoB transgenation relevant to RFP resistance, and method steps is identical with the mPCR-SSCP that cultivation MTB is sample; Although find that sensitivity and specificity reduce, take patient's sputum as direct-detection object, 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.
Accompanying drawing explanation
Fig. 1 is detection method schematic diagram of the present invention.
Fig. 2 is each gene upstream and downstream primer sequence design section schematic diagram increasing for mPCR in 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: the clinical strains to INH and RFP sensitivity; Swimming lane 3 and 4: the clinical strains to INH resistance only; Swimming lane 5 and 6: the clinical strains to RFP resistance only; Swimming lane 7 and 8: the clinical strains to INH and the equal resistance 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 respectively katG, inhA and rpoB gene PCR-SSCP electrophoresis result of Mycobacterium tuberculosis H37Rv strain; 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
Sequence table explanation
Sequence 1 is according to the upstream primer of the amplification mycobacterium tuberculosis katG gene object fragment of H37Rv gene order (Accession:NC_000962) design of GenBank (DNA sequence data storehouse) announcement, is nucleotide sequence.
Sequence 2 is according to the downstream primer of the amplification mycobacterium tuberculosis katG gene object fragment of H37Rv gene order (Accession:NC_000962) design of GenBank announcement, is nucleotide sequence.
Sequence 3 is according to the upstream primer of the amplification mycobacterium tuberculosis inhA gene object fragment of H37Rv gene order (Accession:NC_000962) design of GenBank announcement, is nucleotide sequence.
Sequence 4 is according to the downstream primer of the amplification mycobacterium tuberculosis inhA gene object fragment of H37Rv gene order (Accession:NC_000962) design of GenBank announcement, is nucleotide sequence.
Sequence 5 is according to the upstream primer of the amplification mycobacterium tuberculosis rpoB gene object fragment of H37Rv gene order (Accession:NC_000962) design of GenBank announcement, is nucleotide sequence.
Sequence 6 is according to the downstream primer of the amplification mycobacterium tuberculosis rpoB gene object fragment of H37Rv gene order (Accession:NC_000962) design of GenBank announcement, is nucleotide sequence.
Utilization of the present invention is this resistance mechanism of major cause that causes some bacterium resistance of tuberculosis branch according to transgenation, sets up the mPCR-SSCP of the sudden change of detection and many drug resistance related genes by molecular biology method.
The foundation of gene Selection: so-called resistance to multiple medicines tuberculosis refers at least tuberculosis to INH and two Main Antituberculosis Drugs thing resistances of RFP, the experimental results shows, in substance of medicines-resistant branched tubercle bacillus, the transgenation of a plurality of drug effect target position is to cause multidrug resistant major cause, 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.So by detecting, sporting of katG, inhA and rpoB is clinical provides mycobacterium tuberculosis clinical strains resistance to multiple medicines feature, we have built and can detect the mPCR-SSCP of said gene sudden change for detection of the sudden change of the corresponding gene of MTB simultaneously, the result recall rate that shows to suddenly change reaches more than 90%, and detection method sensitivity, specificity are high, 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 the major cause (in substance of medicines-resistant branched tubercle bacillus, the transgenation of a plurality of drug effect target position is to cause multidrug resistant major cause) that causes drug resistance of Mycobacterium tuberculosis; The term single gene sudden change of take at present detects the existing many research reports of PCR-SSCP as object, along with the continuous rising of resistance to multiple medicines rate, usually needs to detect the resistance of clinical strains to multi-medicament simultaneously; If use a plurality of PCR-SSCP to detect a plurality of drug-tolerant gene mutations, not only improve testing cost, and operating process is complicated and increased experimental error incidence; Object fragment that can a plurality of genes of disposable detection by mPCR (multiplex amplification), has simplified experimental implementation, has reduced experimentation cost.
The mPCR-SSCP that detects the sudden change of multi-drug resistance of Mycobacterium tuberculosis genes involved katG, inhA and rpoB, comprises following step:
1) set up the pcr template (simultaneously as 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 clinical strains katG, inhA and rpoB goal gene fragment simultaneously;
4) katG, inhA and rpoB goal gene mPCR product filter out goal gene mutant strain (by screening with reference culture contrast) through SSCP;
5) PCR katG, inhA and the rpoB goal gene fragment of clinical strains and reference culture H37Rv that increase respectively, expands
Increase after product purification reclaims and carry out DNA sequencing.
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 to Absolute concentration method drug sensitive test, relatively determine mPCR-SSCP method specificity and sensitivity;
2), with the comparison of PCR direct Sequencing, determine that mPCR-SSCP method detects the recall rate of transgenation.
Below in conjunction with embodiment, further illustrate.
Embodiment:
Specimen origin in the present embodiment: being the mycobacterium tuberculosis clinical strains through cultivating and identifying, can be also Tuberculosis people's sputum specimen.
Reagent source in the present embodiment: 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.The professional of the 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 the DNA genome of the 134 routine MTB clinical strains through cultivating and identifying and reference culture H37Rv, extract; Specifically: bacterial cultures is moved in 1.5ml centrifuge tube, and with physiological saline washing 3 times, the centrifugal 5min of 6000r/m (4 ℃) abandons supernatant, get precipitation and extract DNA with extracting DNA test kit, as the template of mPCR and pcr amplification.
2.mPCR design of primers: the H37Rv gene order (Accession:NC_000962) of announcing according to GenBank, by amplification region, containing as far as possible 85% above drug-tolerant gene mutation site is principle, selects the amplification region that katG, inhA and rpoB gene are suitable and adopts Blast software design amplimer (accompanying drawing 2 is shown in by design section schematic diagram).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 cumulative volume 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.Adopt 2% agarose gel electrophoresis that 1 μ g/mL ethidium bromide dyes in advance to detect 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 mPCR product is 1 part, each 1 part of the PCR product of katG, inhA and rpoB gene) (composition and weight ratio are: 95% methane amide, 10mmoL/LEDTA, 0.02% tetrabromophenol sulfonphthalein with equal-volume denaturing agent, all the other are water) mix, ice bath 2min after boiling water bath 5min, 5000r/min, 4 ℃ of centrifugal 2min, water intaking is added to 8% non-denaturing polyacrylamide gel loading hole, 120V electrophoresis 10h after 13 ℃ of 400V electrophoresis 5min, then get offset plate and carry out argentation dyeing, observations.Single-gene PCR product forms 2 or 3 strands after sex change, presents 1～3 band after native polyacrylamide gel electrophoresis.If the strand position of clinical strains different from reference culture H37Rv strand position (being called motion displacement), is judged to be transgenation.
7.PCR direct Sequencing: for the reliability of verifying that above-mentioned mPCR-SSCP detects, also the katG of clinical strains and reference culture, inhA and rpoB gene are carried out respectively to PCR direct Sequencing (PCR-DS); For the same mPCR of pcr amplification primer of the individual gene that checks order, just in reaction system, only add the primer 2 50mmol/L of upstream and downstream separately of katG, inhA and rpoB gene, other composition of reaction system is identical with mPCR, and PCR parameter is also identical with mPCR; H37Rv strain is set and makes standard control, after katG, the inhA of clinical strains and reference culture H37Rv and rpoB gene amplification fragment are purified, are reclaimed with PCR product purification test kit (BioColor), order-checking.
8. drug sensitivity test: be the resistance result coincidence rate of verifying that above-mentioned mPCR-SSCP detects, adopt Absolute concentration method to detect the resistance of mycobacterium tuberculosis clinical strains to INH and RFP; In experiment, take to the reference culture H37Rv of INH and RFP sensitivity is 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 experiment is in triplicate to guarantee that result is correct.
9. drug sensitivity tests criterion: when tested strain growth is good on without medicine control medium, pastille medium slant is responsive without colony growth; Colony growth accounts for inclined-plane area 1/4 for resistance 1+; Colony growth accounts for inclined-plane area 1/2 resistance 2+; Colony growth accounts for inclined-plane area 3/4 for resistance 3+.Lower concentration pastille substratum colony growth 1+ point out resistance above.
The present embodiment result
1. drug sensitive test result: in 134 strain clinical strains, 52 strains are to INH or to RFP resistance, and total resistant rate is 38.8%; 35 strains are resistance to multiple medicines to INH and RFP resistance simultaneously, and resistance to multiple medicines rate is 26.1%; There are respectively 7 and 10 strains only to INH or RFP resistance.
2. detected result: the rpoB gene amplification fragment (Fig. 3) that all occurs the katG gene of 510bp, the inhA gene of 381bp and 207bp after all (134 strain) mycobacterium tuberculosis clinical strains mPCR and H37Rv strain PCR.
3.PCR-DS result (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 in Table 2.Wherein 82 strains, in INH and the equal sensitive strain of RFP, have the KatG of 81 strains identical with H37Rv strain corresponding gene sequences with inhA gene fragment order, and L511M sudden change occurs 1 strain rpoB gene; 10 strains are only only identical with H37Rv strain corresponding sequence to the rpoB gene fragment of RFP sensitive strain with inhA gene fragment, 7 strains to the katG of INH sensitive strain; PCR-DS detects clinical strains the specificity of INH and RFP resistance is respectively to 100% and 98.9%.42 strains are in the clinical strains of INH resistance, katG and/or inhA transgenation 36 strains, and it is 85.7% to the sensitivity of INH resistance that PCR-DS detects bacterial strain.In INH Resistant strain, katG genetic testing region has 8 amino acid that the Substitution of 10 types occurs, the most common with 315 silk amino acid sudden changes; There is S315N/T/I sudden change in 22 strains wherein, and 4 strains are two sudden changes in other site of S315 sudden change companion.InhA genetic testing region, there are 11 Substitutions in 7 amino acid sites.Bacterial strain 43 strains of rpoB transgenation in 45 strain RFP Resistant strain, detected, it is 95.6% to the sensitivity of RFP resistance that PCR-DS detects clinical strains, 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 strains of sudden change of D516V+H526D and H526L+S531L.
4.SSCP result (referring to table 1, table 2 and Fig. 4): tri-goal gene SSCP electrophoretograms of reference standard bacterial strain H37Rv, 82 strains are to INH and all mycobacterium tuberculosis clinical strains SSCP result demonstrations of sensitivity 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 showed no extremely RFP or INH sensitive strain SSCP; SSCP detects mycobacterium tuberculosis the specificity of INH and RFP resistance is respectively to 100% and 97.8%.SSCP detects 42 strain INH resistance clinical strains katG and inhA transgenation situation, detect katG and/or 33 strains of inhA gene electrophoresis dyskinesia, it is 78.6% (33/42) to the sensitivity of INH resistance that SSCP detects clinical strains, katG gene and inhA gene mPCR-SSCP show as electrophoresis motion displacement bacterial strain number and are respectively 27 strains and 10 strains, wherein the displacement simultaneously of two gene mPCR-SSCP electrophoresis motion of 4 strain katG and inhA.SSCP detects 45 strain RFP resistance clinical strains rpoB transgenation situations, find 43 strain RFP Resistant strain rpoB gene electrophoresis motion displacements, it is 95.6% (43/45) to the sensitivity of RFP resistance that SSCP detects clinical strains, wherein 21 strain companion katG gene electrophoresis motion displacements, 4 strain companion inhA gene electrophoresis motion displacements, there are katG, inhA and tri-gene motion displacements simultaneously of rpoB in 2 strains.
Table 1 52 strain Drug-Resistant Mycobacterium tuberculosis mPCR-SSCP and PCR-DS detected result
Note: drug sensitivity tests R represents resistance, S represents sensitivity; Sequencing result S, N, H, D, R, A, G, L, C, V, I, T, Y, P and W represent respectively Serine, l-asparagine, Histidine, aspartic acid, arginine, L-Ala, glycine, leucine, halfcystine, α-amino-isovaleric acid, Isoleucine, Threonine, tyrosine, proline(Pro) and tryptophane
Sensitivity and the specificity of table 2mPCR-SSCP and PCR-DS detection method
Note: INH-R, INH+RFP-R, INH-S and RFP-R, INH+RFP-R, RFP-S represent respectively to Isoniazid-resistant, simultaneously to Isoniazid-resistant and rifampin-resistance, to vazadrine sensitive strain and to rifampin-resistance, simultaneously to Isoniazid-resistant and rifampin-resistance, to Rifampin sensitive strain.
Sequence table
<110> Sun Aihua, Yan Jie, Li Zhaodong
<120> 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
The upstream primer of <221> mycobacterium tuberculosis katG gene
<400>1
aaggaagcca?cctggctcgg?c
<210>2
<211> Nucleotide 21
<212>DNA
<213> synthetic
The downstream primer of <221> mycobacterium tuberculosis katG gene
<400>1
gccgaacggg?tccgggatgg?t
<210>3
<211> Nucleotide 18
<212>DNA
<213> synthetic
The upstream primer of <221> mycobacterium tuberculosis inhA gene
<400>1
ggcatccaca?tctcggcg
<210>4
<211> Nucleotide 21
<212>DNA
<213> synthetic
The downstream primer of <221> mycobacterium tuberculosis inhA gene
<400>1
cagcgcgcac?accgtcttgg?c
<210>5
<211> Nucleotide 21
<212>DNA
<213> synthetic
The upstream primer of <221> mycobacterium tuberculosis rpoB gene
<400>1
acatccggcc?ggtggtcgcc?g
<210>6
<211> Nucleotide 21
<212>DNA
<213> synthetic
The downstream primer of <221> mycobacterium tuberculosis rpoB gene
<400>1
tttcgatcgg?gcacatccgg?c
Claims (2)
1. the multi-drug resistance of Mycobacterium tuberculosis of non-diagnostic purpose detects katG, inhA and a rpoB gene fragment pcr amplification method of using, and the steps include:
A, set up MTB clinical strains and H
37the pcr template of Rv reference culture;
Three pairs of primers of the closely related katG of B, design and vazadrine and rifampin-resistance, inhA and rpoB gene fragment, carry out mPCR amplification to the katG of clinical strains, inhA and rpoB gene; And to clinical strains and H
37the katG of Rv reference culture, inhA and rpoB gene carry out respectively pcr amplification;
C, employing single strand conformation polymorphism detection method detect the sudden change situation of 3 genes that the resistance to INH of clinical strains is relevant with RFP simultaneously;
It is characterized in that: the PCR primer in described step B is: 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 described mPCR amplification is: PCR cumulative volume 100 μ L, include the primer 2 50mmol/L of upstream and downstream separately, 2.5U Ex-Taq archaeal dna polymerase, the 2 μ L templates of 2.5mol/L dNTP, katG, inhA and rpoB gene, 1 * PCR damping fluid of pH8.3;
The parameter of described 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 in advance 2% agarose gel electrophoresis and detect amplified production;
Described single strand conformation polymorphism detection method is: by 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 equal-volume denaturing agent respectively, ice bath 2min after boiling water bath 5min, 5000r/min4 ℃ of centrifugal 2min, water intaking is added to 8% non-denaturing polyacrylamide gel loading hole, 120V electrophoresis 10h after 13 ℃ of 400V electrophoresis 5min, then get offset plate and carry out argentation dyeing, observations; If strand position is different from H37Rv reference culture strand position, be called motion displacement, be judged to be transgenation;
The primer 2 50mmol/L of upstream and downstream separately that only adds katG, inhA or rpoB gene in the reaction system of described pcr amplification, other composition of reaction system and PCR parameter are identical with mPCR.
2. the multi-drug resistance of Mycobacterium tuberculosis of a kind of non-diagnostic purpose according to claim 1 detects katG, inhA and the rpoB gene fragment pcr amplification method of using, the DNA extraction method that it is characterized in that described pcr template is: bacterial cultures is moved in 1.5ml centrifuge tube, with physiological saline washing 3 times, 6000r/m, 4 ℃ of centrifugal 5min abandon supernatant, get precipitation and extract DNA with extracting DNA test kit.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201210043565.9A CN102559916B (en) | 2012-02-24 | 2012-02-24 | Method for detecting multi-drug resistance of Mycobacterium tuberculosis |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201210043565.9A CN102559916B (en) | 2012-02-24 | 2012-02-24 | Method for detecting multi-drug resistance of Mycobacterium tuberculosis |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN102559916A CN102559916A (en) | 2012-07-11 |
| CN102559916B true CN102559916B (en) | 2014-04-16 |
Family
ID=46406515
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201210043565.9A Expired - Fee Related CN102559916B (en) | 2012-02-24 | 2012-02-24 | Method for detecting multi-drug resistance of Mycobacterium tuberculosis |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN102559916B (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2016097291A1 (en) * | 2014-12-18 | 2016-06-23 | Roche Diagnostics Gmbh | Compositions and methods for detection of drug resistant mycobacterium tuberculosis |
Families Citing this family (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103555828B (en) * | 2013-10-10 | 2016-04-06 | 上海市肺科医院 | Mycobacterium quick medicine-sensitive detection method and detection target |
| CN107002148B (en) | 2014-10-10 | 2023-02-17 | 新泽西鲁特格斯州立大学 | Polymerase chain reaction primer and probe for mycobacterium tuberculosis |
| CN106048019A (en) * | 2016-06-13 | 2016-10-26 | 遵义医学院附属医院 | Antituberculous drug drug-resistance gene and screening method thereof |
| CN105907878A (en) * | 2016-06-24 | 2016-08-31 | 凯杰生物工程（深圳）有限公司 | Drug resistance gene mutation detection of tuberculous bacillus based on high-resolution melting curve technology |
| CN106404831B (en) * | 2016-12-20 | 2020-01-14 | 西北大学 | Rapid detection method for sensitivity of beta-lactam antibiotics |
| WO2020109600A1 (en) * | 2018-11-29 | 2020-06-04 | Universität Zürich | Tuberculosis resistance prediction method |
| CN110790676B (en) * | 2019-11-21 | 2022-03-22 | 西南石油大学 | Salt-tolerant temperature-resistant zwitterion viscoelastic surfactant and preparation method of reservoir transformation working solution |
| CN112921107A (en) * | 2020-12-31 | 2021-06-08 | 深圳市慢性病防治中心(深圳市皮肤病防治研究所、深圳市肺部疾病防治研究所) | Heterogeneous drug resistance detection method, kit and application of mycobacterium tuberculosis |
| CN117187425B (en) * | 2023-11-03 | 2024-02-20 | 迪飞医学科技(南京)有限公司 | AS-RAPID tuberculosis drug-resistant duplex detection primer probe group, kit and application |
-
2012
- 2012-02-24 CN CN201210043565.9A patent/CN102559916B/en not_active Expired - Fee Related
Non-Patent Citations (2)
| Title |
|---|
| A new Multi-PCR-SSCP assay for simultaneous detection of isoniazid and rifampin resistance in Mycobacterium tuberculosis;Xiaodong Cheng et al.;《Journal of Microbiological Methods》;20070510;第70卷;第301-305页 * |
| Xiaodong Cheng et al..A new Multi-PCR-SSCP assay for simultaneous detection of isoniazid and rifampin resistance in Mycobacterium tuberculosis.《Journal of Microbiological Methods》.2007,第70卷第301-305页. |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2016097291A1 (en) * | 2014-12-18 | 2016-06-23 | Roche Diagnostics Gmbh | Compositions and methods for detection of drug resistant mycobacterium tuberculosis |
Also Published As
| Publication number | Publication date |
|---|---|
| CN102559916A (en) | 2012-07-11 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN102559916B (en) | Method for detecting multi-drug resistance of Mycobacterium tuberculosis | |
| Xiao et al. | A novel method based on ligase detection reaction for low abundant YIDD mutants detection in hepatitis B virus | |
| CN1995380B (en) | Method for detecting and identifying mycobacterium tuberculosis strain and its dedicated reagent kit | |
| Kong et al. | secA1 gene sequence polymorphisms for species identification of Nocardia species and recognition of intraspecies genetic diversity | |
| CN105803099A (en) | Kit for simultaneously detecting SLCO1B1, APOE and LDLR gene multisite mutation | |
| CN102312001B (en) | Multiplex PCR-reverse dot blot hybridization technique detects the method for Drug Resistance of Mycobacterium Tuberculosis | |
| CN102816851A (en) | Primer, probe, reagent kit and method for detecting mutation of BRAF gene V600E | |
| CN103074434A (en) | CYP2C19 gene polymorphyism detection kit and detection method thereof | |
| CN101979659A (en) | Method for quickly detecting prolificacy FecB gene in sheep | |
| Hoshide et al. | Geographical differences associated with single-nucleotide polymorphisms (SNPs) in nine gene targets among resistant clinical isolates of Mycobacterium tuberculosis | |
| CN102229989B (en) | Method and kit for detecting ethambutol resistance mutation of Mycobacterium tuberculosis | |
| CN110004222A (en) | A kind of multiple gene detection kit and its application method for antipsychotics medication guide | |
| CN107513578A (en) | A kind of nucleic acid Mass Spectrometry detection method early sieved for lung cancer driving gene and tumor susceptibility gene | |
| CN110699446B (en) | SNP marker rs3174298 related to non-syndrome cleft lip and palate diagnosis and application thereof | |
| CN101993952A (en) | Mutation screening probe of SLC25A13 gene and application thereof | |
| CN103045717B (en) | Pyrosequencing techniques detects the method for mycobacterium tuberculosis detection of rifampin resistant | |
| CN101736081A (en) | Kit for rapidly detecting isothermal gene amplification of Mycobacterium tuberculosis and detection method | |
| Shewmaker et al. | Reevaluation of the taxonomic status of recently described species of Enterococcus: evidence that E. thailandicus is a senior subjective synonym of “E. sanguinicola” and confirmation of E. caccae as a species distinct from E. silesiacus | |
| CN101182585A (en) | Method for identifying HBV gene mutation type, special chip and reagent kit | |
| CN102286616A (en) | Method and kit for detecting mycobacterium tuberculosis isoniazide drug resistant gene mutation | |
| CN102719537A (en) | Multidrug-resistant mycobacterium tuberculosis non-fluorescent DNA (deoxyribonucleic acid) microarray detection method and kit | |
| CN101812537B (en) | Method and reagent kit for simultaneously detecting resistance site of three nucleotide analogues of hepatitis B virus | |
| CN104830991A (en) | Primer and kit for detecting PDGFRA (platelet-derived growth factor receptor alpha) gene D842V polymorphic sites and PCR (polymerase chain reaction) method of primer and kit | |
| CN103045716B (en) | Method for detecting isoniazid drug resistance of mycobacterium tuberculosis by using pyrosequencing technology | |
| Wang et al. | Rapid method for identification of six common species of mycobacteria based on multiplex SNP analysis |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant | ||
| CF01 | Termination of patent right due to non-payment of annual fee |
Granted publication date: 20140416 Termination date: 20180224 |
|
| CF01 | Termination of patent right due to non-payment of annual fee |