CN102229989B - Method and kit for detecting ethambutol resistance mutation of Mycobacterium tuberculosis - Google Patents
Method and kit for detecting ethambutol resistance mutation of Mycobacterium tuberculosis Download PDFInfo
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Abstract
The invention relates to techniques for detecting drug resistance mutation of Mycobacterium tuberculosis and provides a method and kit for detecting ethambutol resistance mutation of Mycobacterium tuberculosis, which have the advantages of simple design, simplified operation, high analysis sensitivity and low cost. The method comprises the following steps of: extracting DNA of a Mycobacterium tuberculosis sample; designing primers and probes by using the primer design software PrimerPremier 5 according to the embB gene sequences of Mycobacterium tuberculosis, constructing a PCR (polymerase chain reaction) system, performing PCR, performing melting curve analysis and detection result judgment, and determining whether the mutation occurs in the regions which are covered by the probes according to the Tm change of the analyzed melting curve. By adopting three pairs of primers and four probes and constructing the double-tube double-color system, the purpose of detecting the mutations of six codons 306, 368. 378, 380, 406 and 497 on the embB gene is achieved.
Description
Technical field
The present invention relates to the detection technique of drug resistance of Mycobacterium tuberculosis sudden change, be particularly related to a kind of detection method and test kit of mycobacterium tuberculosis Tibutol medicament-resistant mutation, the method is a kind of probe melting curve analysis technology based on double-tagging self-quenching probe, for detection of mycobacterium tuberculosis Tibutol medicament-resistant mutation.
Background technology
Tibutol is one of phthisical medicine for the treatment of, and it is a kind of pectinose analogue, affects cell walls mycolic acid-arabogalactan-Bai glycan mixture and forms, and brings into play anti-mycobacterium effect.According to the result of national tuberculosis epidemiology investigation in 2000 as can be known, the average resistant rate of Tibutol is 1.3%.Its Drug Resistance Detection still need be cultivated with drug sensitive experiment and be confirmed at present, sense cycle is long, workload is large, and Tibutol drug sensitivity tests coincidence rate on the low side (1, Johnson, R., Jordaan, A.M., Pretorius, L., Engelke, E., van der Spuy, G., Kewley, C., et al.Ethambutol resistance testing by mutation detection.Int J Tuberc Lung Dis.2006,10 (1), 68-73), this utmost point is unfavorable for state of an illness treatment and epidemic situation control.So faster, more accurate, more reliable novel method of clinical need.In recent years, along with the development of Molecular Biology and technology, the molecular basis of resistance mechanism and resistance is progressively illustrated, for the foundation of the whole bag of tricks provides a new platform.
Studies show that recently the embABC operon gene that Tibutol resistance and regulation and control arabinofuranosyltransferase are expressed suddenlys change relevant, this operon is by embA, three genomic constitutions (2 of embB and embC, Telenti, A., Philipp, W.J., Sreevatsan, S., Bernasconi, C., Stockbauer, K.E, Wieles, B., et al.The emb operon, a gene cluster of Mycobacterium tuberculosis involved in resistance to ethambutol.1997, Nat Med, 3 (5), 567-570.), wherein the sudden change of embB gene is the main molecules mechanism of anti-Tibutol, about bacterial strain of anti-Tibutol of 47%～70% relevant with the embB transgenation (3, Lee, H.Y, Myoung, H.J., Bang, H.E., Bai, G.H., Kim, S.J., Kim, J.D., et al.Mutations in the embB locus among Korean clinical isolates of Mycobacterium tuberculosis resistant to ethambutol.2002, Yonsei Med J, 43 (1), 59-64.), and wherein 55% sudden change occurs on embB 306 codons, in addition embB 406 and embB 497 (4, Ramaswamy, S.V., Amin, A.G., Goksel, S., Stager, C.E, Dou, S.J., El Sahly, H., et al.Molecular genetic analysis of nucleotide polymorphisms associated with ethambutol resistance in human isolates of Mycobacterium tuberculosis.2000, Antimicrob Agents Chemother, 44 (2), 326-336.) also be common mutational site.The sudden change of current research embB 368, embB 378 and embB 380 and Tibutol resistance have certain contacting (5, Srivastava, S., Garg, A., Ayyagafi, A., Nyati, K.K., Dhole, T.N. ， ﹠amp; Dwivedi, S.K.Nucleotide polymorphism associated with ethambutol resistance in clinical isolates of Mycobacterium tuberculosis.2006, Curr Microbiol, 53 (5), 401-405.).
At present the gene test of Tibutol drug-resistant type comprised two large steps, namely nucleic acid amplification reaches the analysis to amplified production.According to the difference to the amplified production processing mode, can be divided into heterogeneous detection and homogeneous phase and detect.The former comprises that mainly dna sequencing, polymerase chain reaction-single strand conformation polymorphism (PCR-SSP), polymerase chain reaction-restriction fragment length polymorphism analysis (PCR-RFLP), linear probe hybridization technique, multiple binding rely on probe amplification (MLPA) technology, amplification refractory mutation system PCR (ARMS-PCR) technology, distortion high performance liquid chromatography (dHPLC) technology, gene chip etc.Because these methods all need the open pipe operation, so complex steps easily causes product pollution, sensitivity for analysis is not high simultaneously, experimental result can be subject to the impact of the objective factors such as outside temperature, expanding fragment length and sequence, therefore be not used widely (6, Johnson, R., Jordaan, A.M., Pretorius, L, Engelke, E, van der Spuy, G., Kewley, C., et al.Ethambutol resistance testing by mutation detection.Int J Tuberc Lung Dis.2006,10 (1), 68-73.).
Developed recently rapidly real-time fluorescence PCR has plurality of advantages, owing to adopt stopped pipe to measure mode, eliminates the hidden danger of the product pollution of heterogeneous method.Simultaneously fluoroscopic examination has the effect that signal amplifies, therefore sensitivity for analysis, simplicity and etc. the aspect have more advantage.Real-time fluorescence PCR not only can for detection of the nucleic acid of pathogenic agent, can detect transgenation by design specificity fluorescent probe simultaneously in real time.The probe that has been used widely at present mainly comprises hydrolysis probes (TaqMan probe, the MGB probe), molecular beacon (Molecular probe) and FRET probe, hydrolysis probes designs specific probes with molecular beacon for different mutation types, only have when probe and template are mated fully and can send fluorescent signal on the annealing rank of pcr amplification, when studies show that these two kinds of probes for detection of the Drug Resistance for Tuberculosis transgenation, has very high specific (7, Garcia-Quintanilla, A., Gonzalez-Martin, J., Tudo, G., Espasa, M. ， ﹠amp; Jimenez de Anta, M.T.Simultaneous identification of Mycobacterium genus and Mycobacterium tuberculosis complex in clinical samples by 5 '-exonuclease fluorogenic PCR.2002, J Clin Microbiol, 40 (12), 4646-4651.), but because a probe can only detect a kind of mutation type, sudden change for polygene, multidigit point detects, and certain difficulty is arranged in design.Comparatively speaking, the FRET probe design of Luo Shi patent is comparatively simple, it is by two and masterplate is complementary and adjacent specific probe forms (apart from 1-5bp), 3 of upstream probe ' end mark donor fluorophor, 5 of adjacent downstream probe ' end mark Red 640 acceptor fluorescence groups.The FRET probe gathers fluorescence at the annealing stage of pcr amplification, also can melt by Tm simultaneously and carry out gene type, therefore as long as a kind of probe of design just can detect different mutation types simultaneously.To seal to avoid reaction owing to form two probe ends of FRET probe, so synthetic cost can be higher, also cumbersome.
Summary of the invention
One of purpose of the present invention is the problem that exists in the existing drug resistance of Mycobacterium tuberculosis sudden change detection method, a kind of simplicity of design is provided, simplified operation, improve the detection method of sensitivity for analysis, mycobacterium tuberculosis Tibutol medicament-resistant mutation with low cost, i.e. a kind of probe melting curve analysis technology based on double-tagging self-quenching probe.
Another object of the present invention is to provide a kind of test kit of detection method of mycobacterium tuberculosis Tibutol medicament-resistant mutation.
The detection method of described mycobacterium tuberculosis Tibutol medicament-resistant mutation may further comprise the steps:
1) DNA of extraction mycobacterium tuberculosis sample;
2) according to mycobacterium tuberculosis embB gene order, utilize primer-design software Primer Premier 5 design primer and probes;
3) make up the PCR reaction system, carry out PCR and detect;
4) carrying out melting curve analysis and detected result judges: change by the Tm that analyzes melting curve and determine whether this probe institute overlay area undergos mutation.If the Tm at testing sample melting curve peak is lower more than 2 ℃ than positive control, illustrate to have sudden change.
In step 2) in, the concrete grammar of described design of primers is:
A, design of primers is at the sudden change two ends of detecting, and probe covers the mutational site;
B, the fluorophor of probe can be selected any one among FAM, ROX, HEX, CY5, TET, the CAL-Fluor etc., and quenching group can be selected BHQ, Dabcyl etc., and probe can carry out various types of modifications (such as the LNA mark) etc.;
The concrete sequence of described primer and probe can be:
306-F：5′-GCGCGAATACGTCGGACAA-3′
306-R：5′-CAGCAGCGACCAGCACACTA-3′
368-406-F：5′-AGCGACGCCAGTCTGTGGAT-3′
368-406R：5′-TGGCTGTACCGCATGGACCTT-3′
497-F：5′-TTGCCGTTGGTGTCGCCGAT-3′
497-R：5′-CGGTGGGCAGGATGAGGTAGTAGT-3′
306-P：5′-FAM-TCGGCATGGCCCGAGTCGCCG-Dabcyl-3′
368-380-P：5′-TET-TTGCCGTTTGCTGGCCTCCACACCTCACGCGGCA-BHQ1-3′
406-P：5′-FAM-TGCGGCAGAGGGCATGATGCCG-BHQ1-3′
497-P：5′-TET-TGTGGGTGTTCCGCGACCAGACCCAC-BHQ1-3′。
In step 3) in, described PCR reaction system contains PCR reaction solution I or PCR reaction solution II, 2U Taq enzyme, and 5 μ L testing samples, pure water is as negative control after the sterilization, and wild plasmid is as positive control.
Contain among the described PCR reaction solution I primer to 1:306-F and 306-R, primer to 2:497-F and 497-R, 306-P, 497-P, 10 * buffer (500mM KCl, 100mM Tris-HCl (pH=8.6), glycerine 50% (V/V)), dNTP mixture and deionized water.306-P and 497-P detect respectively embB 306 and embB 497 transgenations.
Described PCR reaction system II is by primer 3 (368-406-F and 368-406-R), 368-380-P, 406-P, 10 * buffer (500mM KCl, 100mM Tris-HCl (pH=8.6), glycerine 50% (V/V)), pure water forms after dNTP mixture and the sterilization.The 368-380-P probe of TET mark can detect all mutation types of embB 368, embB 378 and 380 3 codons of embB, and the 406-P probe examination of FAM mark occurs in the sudden change on the embB 406.
The program of described pcr amplification is: 50 ℃, and 2min, 95 ℃ of denaturation 10min, 95 ℃ of sex change 15s then, 58 ℃ of annealing 15s, 74 ℃ are extended 20s, altogether 55 circulation.
The test kit of the detection method of described mycobacterium tuberculosis Tibutol medicament-resistant mutation is provided with box body, amplifing reagent, contrast agents and extraction reagent, described amplifing reagent, contrast agents and extraction reagent are placed in the box body, described amplifing reagent comprises EMBPCR Mix A, EMB PCR Mix B and TB enzyme mixation, described contrast agents comprises EMB positive control and TB negative control, and described extraction reagent is TB DNA extraction liquid.
Described EMB PCR Mix A comprises 1 * buffer (50mM KCl, 10mM Tris-HCl (pH=8.6), glycerine 5% (V/V)), dNTP/dUTP Mix (dATP, dCTP, each 200 μ M of dGTP, dUTP 400 μ M), MgCl
2Solution 3mM, 0.4 μ M 306-F, 2 μ M 306-R, 0.2 μ M 306-P, 0.4 μ M 497-F, 2 μ M 497-R, 0.2 μ M 497-P.
Described EMB PCR Mix B comprises 1 * buffer (50mM KCl, 10mM Tris-HCl (pH=8.6), glycerine 5% (V/V)), MgCl
2Solution 3mM, dNTP/dUTP Mix (dATP, dCTP, each 0.2mM of dGTP, dUTP 0.4mM), 0.4 μ M 368-406-F, 2 μ M 368-406-R, 0.2 μ M 368-380-P, 0.2 μ M 406-P.
Described EMB positive control is EMB wild-type standard plasmid.
Described TB negative control can be TB DNA extraction liquid, H
2O, Tris or physiological saline etc.
The composition of described TB DNA extraction liquid is disodium ethylene diamine tetraacetate, Tris and TritonX-100.
The method of the test kit inspection specimen of the detection method of described mycobacterium tuberculosis Tibutol medicament-resistant mutation may further comprise the steps:
1) reagent is prepared---the dosing district
1. at first all reagent is taken out from refrigerator and balance to room temperature.PCR reaction solution dosing standard is: get n * 19.6 μ LEMB PCR Mix A and n * 0.4 μ L TB enzyme mixation and join in the 1.5mL centrifuge tube, vibration mixing several seconds, centrifugal several seconds of 3000rpm; Other gets n * 19.6 μ L EMB PCR Mix B and n * 0.4 μ L TB enzyme mixation joins in another 1.5mL centrifuge tube, vibration mixing several seconds, centrifugal several seconds of 3000rpm.The PCR reaction solution for preparing must be stored in-20 ℃ and use in 4h.
2. the packing of PCR reaction solution.PCR reaction solution A/B is sub-packed in PCR thin-walled reaction tubes with every pipe 20 μ L respectively.
3. the concavo-convex bag of the PCR reaction tubes for preparing being packed into is transferred between extraction.Be stored in-20 ℃ until sample extraction be disposed.
2) sample extraction and application of sample---extract the district
The mycobacterium tuberculosis of 1. growing on the solid medium is collected bacterium 1 ring with 22SWG standard inoculation ring, and is suspended in the 250 μ L TB DNA extraction liquid.The mycobacterium tuberculosis of growing in the liquid nutrient medium is got 1mL, and the centrifugal 15min of 10000rpm abandons supernatant and resuspended bacterium in 250 μ L TB DNA extraction liquid.
2. sealed membrane sealing, 99 ℃ of heating 20min.The centrifugal 10min of 14000rpm shifts supernatant to new 1.5mL centrifuge tube.Supernatant is the pcr amplification template.(template can be stored in-20 ℃, and finishes test in 1 month.Note not multigelation sample.)
3. in every PCR thin-walled reaction tubes, add corresponding sample or the moon/positive reference substance 5 μ L of extracting with micropipette.Cover tightly immediately the pipe lid.
The PCR thin-walled reaction tubes that 4. will add template is transferred to the pcr amplification district.
3) pcr amplification---amplification region
1. the program setting of instrument is as follows:
The first step: 50 ℃ of 2min, 95 ℃ of 10min;
Second step: 95 ℃ of 15s, 58 ℃ of 15s, 74 ℃ of 20s, 55 circulations, fluorescent signal is collected at 58 ℃ of 15s places;
The 3rd step: 95 ℃ of 1min, 35 ℃ of 5min;
The 4th step: 40～85 ℃, collect fluorescent signal for per 0.5 ℃, fluorescence channel is selected FAM and TET.
2. the program operation is complete, PCR thin-walled reaction tubes (stopped pipe) is taken out put into concavo-convex bag, and sealing is obturaged, and presses source of pollution and processes.
(3) reference value of test kit (term of reference)
Positive control, the i.e. T of wild-type in each system and each passage
mThe value scope is as follows: the Tm value at FAM passage wild-type contrast peak is 66.5 ℃ ± 1 ℃ among the reaction system A; The Tm value at TET passage wild-type contrast peak is 61.5 ℃ ± 1 ℃.The Tm value at FAM passage wild-type contrast peak is 64.5 ℃ ± 1 ℃ among the reaction system B; The Tm value at TET passage wild-type contrast peak is 67.5 ℃ ± 1 ℃.
Each T wherein
mValue is the modal value of gained on particular B io-Rad CFX96 instrument, as a reference.When using Other Instruments, T
mValue may slightly change, with the T when inferior test positive control (wild-type) gained
mValue is as the criterion.
T
mValue is as the criterion with instrument automatic interpretation gained, when instrument provides an above T
mDuring value, the peak type that please refer to positive control and negative control is selected effective T
mValue.Can't automatically provide T when instrument occurring
mDuring the situation of value, can obtain T by the method for adjusting baseline or direct labor's interpretation
mValue.
(4) as a result interpretation
By comparing the fusing point (T of melting curve between institute's test sample and the positive control
mWhether diversity judgement sample value) undergos mutation.When the fusing point of the fusing point of sample in four passages and positive control all is judged to be wild-type when consistent (error is no more than 1 ℃), test strain is responsive to Tibutol; The fusing point of sample is lower than 2 ℃ of positive controls and (Δ T when above in four arbitrary passages of passage
m〉=2 ℃) be judged to be mutant, test strain is to the Tibutol resistance.
Interpretation about the heterozygosis sample result: when arbitrary passage melting curve in four passages occurs bimodal or merges the peak, be the heterozygosis sample, minute 3 kinds of situations: 1. occur being the heterozygosis sample when bimodal when sample; 2. sample is one and merges the peak, and is consistent with the fusing point of positive control, but the peak type has larger difference with the peak type of positive control, small peak or projection occur in the position at the peak that might occur suddenling change, and is the heterozygosis sample; 3. sample is a fusion peak, is the sudden change fusing point, but small peak or projection occurs in the position at wild peak, is the heterozygosis sample.The heterozygosis sample is to the Tibutol resistance, when the heterozygosis sample appears in suggestion with the sample duplicate detection, to determine the sample resistance.
False positive results appears in laboratory environment pollution, reagent contamination, sample crossed contamination meeting; Reagent transportation, preserve improper or reagent is prepared inaccurate meeting and caused that reagent detects usefulness and descends, false negative occurs or detect inaccurate result.
Technical scheme of the present invention comprises the asymmetric PCR amplification of testing sample and amplified production is carried out melting curve analysis.Thereby the present invention adopts 4 probes of 3 pairs of primers to set up two-tube double-colored system system and realizes being positioned at 6 codons 306 on the embB gene, 368,378,380,406 and 497 sudden change detection.This system the design of primers scheme comprise: (1) primer to 1 (306-F and 306-R), primer to 2 (497-F and 497-R) and primer to 3 (368-406-F and 368-406-R) be respectively applied to increase embB 306 genes, embB 497 genes and embB 368-embB 406 genes.Wherein primer to 1 and primer form a double PCR system to 2, experimental result shows that two target sequences in the dual system are all well increased.The probe design scheme that adopts of the present invention is as follows: (1) is for a single-stranded probe that mates fully with wild-type of site design that will detect, and 5 ' end mark fluorescent group, 3 ' end mark quenching group.Probe and the difference of different target sequence and bonding force cause that melting curve Tm's is different.Probe and wild-type template are mated fully, and bonding force is the strongest, so Tm is the highest, exists between probe and the sudden change template and do not mate base, bonding force a little less than, Tm also decreases.By relatively the Tm of testing sample and the Tm of wild contrast just can determine whether this template exists sudden change.When the Tm of probe and wildtype target sequence is higher more than 3 ℃ the time than the Tm of mutant, just possesses good site mutation detectivity.
Description of drawings
Fig. 1 is the typical consequence figure of 306-P probe in detecting single gene mutation.In Fig. 1, X-coordinate be temperature (℃), ordinate zou is fluorescence intensity, curve 1 represents embB 306 ATG＞GTG, curve 2 and represents that embB 306 ATG＞ATA, curve 3 represent embB 306 ATG＞CTG, curve 4 represents wild-type.
Fig. 2 is the typical consequence figure of 497-P probe in detecting single gene mutation.In Fig. 2, X-coordinate be temperature (℃), ordinate zou is fluorescence intensity, curve 1 represents that embB 497 CAG＞CGG, curve 2 represent embB 497 CAG＞AAG, curve 3 represents wild-type.
Fig. 3 is the typical consequence figure of 368-380-P probe in detecting single gene mutation.In Fig. 3, X-coordinate be temperature (℃), ordinate zou is fluorescence intensity, curve 1 represents embB 378 GAG＞GCG, curve 2 represents wild-type.
Fig. 4 is the typical consequence figure of 406-P probe in detecting single gene mutation.In Fig. 4, X-coordinate be temperature (℃), ordinate zou is fluorescence intensity, curve 1 represents embB 406 GGC＞GAC, 2 and represents wild-type.
Fig. 5 is the structural representation of test kit embodiment of the detection method of mycobacterium tuberculosis Tibutol medicament-resistant mutation of the present invention.
Embodiment
The present invention adopts 4 probes of 3 pairs of primers to set up two-tube double-colored system and realizes 6 password sudden changes relevant with the Tibutol resistance that are positioned on the embB gene are detected.These 6 mutational sites and mutation type are respectively embB 306 (ATG＞CTG, ATG＞TTG, ATG＞GTG, ATG＞ATA, ATG＞ATT, ATG＞ATC, ATG＞ACG), embB 368 (GAG＞CAG, GAG＞GCG, GAG＞GAT, GAG＞GAA), embB 378 (GAG＞GCG, GAG＞AAA), embB 380 (AGC＞AAC, AGC＞CGT), embB 406 (GGC＞GAC, GGC＞AGC, GGC＞TGC, GGC＞GCC, GGC＞GCG) and embB 497 (CAG＞CGG, CAG＞AAG).
The design of embodiment 1 primer and probe is with synthetic
Extract the mycobacterium tuberculosis sample DNA by the conventional DNA extraction method in laboratory.According to mycobacterium tuberculosis embB gene order, design 4 probes of 3 pairs of primers, set up two-tube double-colored detection system, detection is positioned at 6 codons 306 on the embB gene, 368,378,380,406 and 497 sudden change, and the concrete sequence of described primer is as follows:
Primer and probe are given birth to the biological company limited of worker by Shanghai and are synthesized.Primer of the present invention and probe can also for comprise above 6 primer sequences and 4 probes derive come, difference is not more than the oligonucleotide sequence of 8 bases and the antisense complementary sequence of each sequence, or their variant, or their partial sequence.
Embodiment 2EMB medicament-resistant mutation detection kit detects the EMB medicament-resistant mutation
Detect minute two reaction systems and carry out dual double-colored detection:
Every part of EMB PCR MIX A is 19.6 μ l, contains 1 * buffer (50mM KCl, 10mM Tris-HCl (pH=8.6) in every part of reaction solution, glycerine 5% (V/V)), dNTP/dUTP Mix (dATP, dCTP, each 200 μ M of dGTP, dUTP400 μ M), MgCl
2Solution 3mM, 306-F 0.4 μ M, 306-R 2 μ M, 306-P 0.2 μ M, 497-F 0.4 μ M, 497-R 2 μ M, 497-P 0.2 μ M), adding 2U enzyme mixation (0.4 μ l) before detecting, template add-on to be measured is 5 μ l.
Every part of EMB PCR MIX B is 19.6 μ l, contains 1 * buffer (50mM KCl, 10mM Tris-HCl (pH=8.6), glycerine 5% (V/V)) in every part of reaction solution, MgCl
2Solution 3mM, dNTP/dUTP Mix (dATP, dCTP, each 0.2mM of dGTP, dUTP 0.4mM), 368-406-F 0.4 μ M, 368-406-R 2 μ M, 368-380-P 0.2 μ M, 406-P 0.2 μ M, add 2U enzyme mixation (0.4 μ l) before detecting, template add-on to be measured is 5 μ l.
EMB wild-type standard plasmid (10
3Copy/μ L) as positive control, TB DNA extraction liquid is as negative control.
Reaction conditions: at the enterprising performing PCR of Bio-rad CFX96 real-time fluorescence PCR instrument, the PCR response procedures is:
50 ℃, 2min, 95 ℃ of denaturation 10min; Then 95 ℃ of sex change 15s, 58 ℃ of annealing 15s, 74 ℃ are extended 20s, altogether 55 circulation; 95 ℃ of denaturation 1min, 35 ℃ of insulation 5min, then with the temperature rise rate of 0.5 ℃/step from 40 ℃ or increase to 85 ℃, gather simultaneously the fluorescence of FAM and TET passage in this process.Experimental result such as Fig. 1～shown in Figure 4, all mutation types can both separate with the wild-type check plot.
As a result interpretation:
FAM passage wild-type contrast peak Tm value is 66.5 ℃ ± 1 ℃ among the reaction system A; TET passage wild-type contrast peak Tm value is 61.5 ℃ ± 1 ℃.FAM passage wild-type contrast peak Tm value is 64.5 ℃ ± 1 ℃ among the reaction system B; TET passage wild-type contrast peak Tm value is 67.5 ℃ ± 1 ℃.
By comparing the fusing point (T of melting curve between institute's test sample and the positive control
mWhether diversity judgement sample value) undergos mutation.When the fusing point of the fusing point of sample in four passages and positive control all is judged to be wild-type when consistent (error is no more than 1 ℃), test strain is responsive to Tibutol; The fusing point of sample is lower than 2 ℃ of positive controls and (Δ T when above in four arbitrary passages of passage
m〉=2 ℃) be judged to be mutant, test strain is to the Tibutol resistance.
Fig. 5 provides the structural representation of test kit embodiment of the detection method of mycobacterium tuberculosis Tibutol medicament-resistant mutation of the present invention, be provided with box body 1, amplifing reagent (comprising EMB PCR Mix A2, EMB PCR Mix B3 and TB enzyme mixation 4), contrast agents (comprising EMB positive control 7 and TB negative control 6) and extract reagent (TB DNA extraction liquid 5), described amplifing reagent, contrast agents and extraction reagent are placed in the box body 1.
Described EMB PCR Mix A comprises 1 * buffer (50mM KCl, 10mM Tris-HCl (pH=8.6), glycerine 5% (V/V)), dNTP/dUTP Mix (dATP, dCTP, each 200 μ M of dGTP, dUTP 400 μ M), MgCl
2Solution 3mM, 0.4 μ M 306-F, 2 μ M 306-R, 0.2 μ M 306-P, 0.4 μ M 497-F, 2 μ M 497-R, 0.2 μ M 497-P.
Described EMB PCR Mix B comprises 1 * buffer (50mM KCl, 10mM Tris-HCl (pH=8.6), glycerine 5% (V/V)), MgCl
2Solution 3mM, dNTP/dUTP Mix (dATP, dCTP, each 0.2mM of dGTP, dUTP 0.4mM), 0.4 μ M 368-406-F, 2 μ M 368-406-R, 0.2 μ M 368-380-P, 0.2 μ M 406-P.
Described EMB positive control is EMB wild-type standard plasmid.
Described TB negative control can be TB DNA extraction liquid, H
2O, Tris or physiological saline etc.
The composition of described TB DNA extraction liquid is disodium ethylene diamine tetraacetate, Tris and TritonX-100.
The method of the test kit inspection specimen of the detection method of described mycobacterium tuberculosis Tibutol medicament-resistant mutation may further comprise the steps:
1) reagent is prepared---the dosing district
1. at first all reagent is taken out from refrigerator and balance to room temperature.PCR reaction solution dosing standard is: get n * 19.6 μ LEMB PCR Mix A and n * 0.4 μ L TB enzyme mixation and join in the 1.5mL centrifuge tube, vibration mixing several seconds, centrifugal several seconds of 3000rpm; Other gets n * 19.6 μ L EMB PCRMix B and n * 0.4 μ L TB enzyme mixation joins in another 1.5mL centrifuge tube, vibration mixing several seconds, centrifugal several seconds of 3000rpm.The PCR reaction solution for preparing must be stored in-20 ℃ and use in 4h.
2. the packing of PCR reaction solution.PCR reaction solution A/B is sub-packed in PCR thin-walled reaction tubes with every pipe 20 μ L respectively.
3. the concavo-convex bag of the PCR reaction tubes for preparing being packed into is transferred between extraction.Be stored in-20 ℃ until sample extraction be disposed.
2) sample extraction and application of sample---extract the district
The mycobacterium tuberculosis of 1. growing on the solid medium is collected bacterium 1 ring with 22SWG standard inoculation ring, and is suspended in the 250 μ L TB DNA extraction liquid.The mycobacterium tuberculosis of growing in the liquid nutrient medium is got 1mL, and the centrifugal 15min of 10000rpm abandons supernatant and resuspended bacterium in 250 μ L TB DNA extraction liquid.
2. sealed membrane sealing, 99 ℃ of heating 20min.The centrifugal 10min of 14000rpm shifts supernatant to new 1.5mL centrifuge tube.Supernatant is the pcr amplification template.(template can be stored in-20 ℃, and finishes test in 1 month.Note not multigelation sample.)
3. in every PCR thin-walled reaction tubes, add corresponding sample or the moon/positive reference substance 5 μ L of extracting with micropipette.Cover tightly immediately the pipe lid.
The PCR thin-walled reaction tubes that 4. will add template is transferred to the pcr amplification district.
3) pcr amplification---amplification region
1. the program setting of instrument is as follows:
The first step: 50 ℃ of 2min, 95 ℃ of 10min;
Second step: 95 ℃ of 15s, 58 ℃ of 15s, 74 ℃ of 20s, 55 circulations, fluorescent signal is collected at 58 ℃ of 15s places;
The 3rd step: 95 ℃ of 1min, 35 ℃ of 5min;
The 4th step: 40～85 ℃, collect fluorescent signal for per 0.5 ℃, fluorescence channel is selected FAM and TET.
2. the program operation is complete, PCR thin-walled reaction tubes (stopped pipe) is taken out put into concavo-convex bag, and sealing is obturaged, and presses source of pollution and processes.
(3) reference value of test kit (term of reference)
Positive control, the i.e. T of wild-type in each system and each passage
mThe value scope is as follows: the Tm value at FAM passage wild-type contrast peak is 66.5 ℃ ± 1 ℃ among the reaction system A; The Tm value at TET passage wild-type contrast peak is 61.5 ℃ ± 1 ℃.The Tm value at FAM passage wild-type contrast peak is 64.5 ℃ ± 1 ℃ among the reaction system B; The Tm value at TET passage wild-type contrast peak is 67.5 ℃ ± 1 ℃.
Each T wherein
mValue is the modal value of gained on particular B io-Rad CFX96 instrument, as a reference.When using Other Instruments, T
mValue may slightly change, with the T when inferior test positive control (wild-type) gained
mValue is as the criterion.
T
mValue is as the criterion with instrument automatic interpretation gained, when instrument provides an above T
mDuring value, the peak type that please refer to positive control and negative control is selected effective T
mValue.Can't automatically provide T when instrument occurring
mDuring the situation of value, can obtain T by the method for adjusting baseline or direct labor's interpretation
mValue.
(4) as a result interpretation
By comparing the fusing point (T of melting curve between institute's test sample and the positive control
mWhether diversity judgement sample value) undergos mutation.When the fusing point of the fusing point of sample in four passages and positive control all is judged to be wild-type when consistent (error is no more than 1 ℃), test strain is responsive to Tibutol; The fusing point of sample is lower than 2 ℃ of positive controls and (Δ T when above in four arbitrary passages of passage
m〉=2 ℃) be judged to be mutant, test strain is to the Tibutol resistance.
Interpretation about the heterozygosis sample result: when arbitrary passage melting curve in four passages occurs bimodal or merges the peak, be the heterozygosis sample, minute 3 kinds of situations: 1. occur being the heterozygosis sample when bimodal when sample; 2. sample is one and merges the peak, and is consistent with the fusing point of positive control, but the peak type has larger difference with the peak type of positive control, small peak or projection occur in the position at the peak that might occur suddenling change, and is the heterozygosis sample; 3. sample is a fusion peak, is the sudden change fusing point, but small peak or projection occurs in the position at wild peak, is the heterozygosis sample.The heterozygosis sample is to the Tibutol resistance, when the heterozygosis sample appears in suggestion with the sample duplicate detection, to determine the sample resistance.
False positive results appears in laboratory environment pollution, reagent contamination, sample crossed contamination meeting; Reagent transportation, preserve improper or reagent is prepared inaccurate meeting and caused that reagent detects usefulness and descends, false negative occurs or detect inaccurate result.
Claims (2)
1. the detection kit of a mycobacterium tuberculosis Tibutol medicament-resistant mutation, it is characterized in that being provided with box body, amplifing reagent, contrast agents and extraction reagent, described amplifing reagent, contrast agents and extraction reagent are placed in the box body, described amplifing reagent comprises EMB PCR MixA, EMB PCR Mix B and TB enzyme mixation, described contrast agents comprises EMB positive control and TB negative control, and described extraction reagent is TB DNA extraction liquid;
Described EMB PCR Mix A comprises 1 * buffer, glycerine 5%, dNTP/dUTP Mix, MgCl
2Solution 3mM, 0.4 μ M306-F, 2 μ M306-R, 0.2 μ M306-P, 0.4 μ M497-F, 2 μ M497-R, 0.2 μ M497-P; Described 1 * buffer is 50mM KCl, 10mM Tris-HCl, pH=8.6; Described dNTP/dUTP Mix is dATP, dCTP, each 0.2mM of dGTP, dUTP0.4mM;
Described EMB PCR Mix B comprises 1 * buffer, glycerine 5%, MgCl
2Solution 3mM, dNTP/dUTP Mix, 0.4 μ M368-406-F, 2 μ M368-406-R, 0.2 μ M368-380-P, 0.2 μ M406-P; Described 1 * buffer is 50mM KCl, 10mM Tris-HCl, pH=8.6; Described dNTP/dUTP Mix is dATP, dCTP, each 0.2mM of dGTP, dUTP0.4mM;
Described EMB positive control is EMB wild-type standard plasmid; Described TB negative control is TB DNA extraction liquid, H
2O, Tris or physiological saline; The composition of described TB DNA extraction liquid is disodium ethylene diamine tetraacetate, Tris and TritonX-100;
The concrete sequence of 306-F, 306-R, 368-406-F, 368-406R, 497-F, 497-R, 306-P, 368-380-P, 406-P, 497-P is:
306-F：5′-GCGCGAATACGTCGGACAA-3′
306-R：5′-CAGCAGCGACCAGCACACTA-3′
368-406-F：5′-AGCGACGCCAGTCTGTGGAT-3′
368-406R：5′-TGGCTGTACCGCATGGACCTT-3′
497-F：5′-TTGCCGTTGGTGTCGCCGAT-3′
497-R：5′-CGGTGGGCAGGATGAGGTAGTAGT-3′
306-P：5′-FAM-TCGGCATGGCCCGAGTCGCCG-Dabcyl-3′
368-380-P：5′-TET-TTGCCGTTTGCTGGCCTCCACACCTCACGCGGCA-BHQ1-3′
406-P：5′-FAM-TGCGGCAGAGGGCATGATGCCG-BHQ1-3′
497-P：5′-TET-TGTGGGTGTTCCGCGACCAGACCCAC-BHQ1-3′。
2. method that is used for the inspection specimen of non-diagnosis and therapeutic purpose is characterized in that adopting the detection kit of a kind of mycobacterium tuberculosis Tibutol medicament-resistant mutation as claimed in claim 1, said method comprising the steps of:
1) reagent is prepared---the dosing district
1. at first all reagent is taken out from refrigerator and balance to room temperature, PCR reaction solution dosing standard is: get n * 19.6 μ LEMB PCR Mix A and n * 0.4 μ LTB enzyme mixation and join in the 1.5mL centrifuge tube, the vibration mixing several seconds, the centrifugal several seconds of 3000rpm; Other gets n * 19.6 μ LEMB PCR Mix B and n * 0.4 μ LTB enzyme mixation joins in another 1.5mL centrifuge tube, and the vibration mixing several seconds, the centrifugal several seconds of 3000rpm, the PCR reaction solution for preparing must be stored in-20 ℃ and use in 4h;
2. the packing of PCR reaction solution, PCR reaction solution A/B is sub-packed in PCR thin-walled reaction tubes with every pipe 20 μ L respectively;
3. the concavo-convex bag of the PCR reaction tubes for preparing being packed into is transferred between extraction, be stored in-20 ℃ until sample extraction be disposed;
2) sample extraction and application of sample---extract the district
The mycobacterium tuberculosis of 1. growing on the solid medium, collect bacterium 1 ring with 22SWG standard inoculation ring, and be suspended in the 250 μ L TB DNA extraction liquid, the mycobacterium tuberculosis of growing in the liquid nutrient medium is got 1mL, the centrifugal 15min of 10000rpm abandons supernatant and resuspended bacterium in 250 μ L TB DNA extraction liquid;
2. sealed membrane sealing, 99 ℃ of heating 20min, the centrifugal 10min of 14000rpm shifts supernatant to new 1.5mL centrifuge tube, and supernatant is the pcr amplification template;
3. in every PCR thin-walled reaction tubes, add corresponding sample or the moon/positive reference substance 5 μ L of extracting with micropipette, cover tightly immediately the pipe lid;
The PCR thin-walled reaction tubes that 4. will add template is transferred to the pcr amplification district;
3) pcr amplification---amplification region
1. the program setting of instrument is as follows:
The first step: 50 ℃ of 2min, 95 ℃ of 10min;
Second step: 95 ℃ of 15s, 58 ℃ of 15s, 74 ℃ of 20s, 55 circulations, fluorescent signal is collected at 58 ℃ of 15s places;
The 3rd step: 95 ℃ of 1min, 35 ℃ of 5min;
The 4th step: 40～85 ℃, collect fluorescent signal for per 0.5 ℃, fluorescence channel is selected FAM and TET;
2. the program operation is complete, PCR thin-walled reaction tubes is taken out put into concavo-convex bag, and sealing is obturaged, and presses source of pollution and processes;
(3) reference value of test kit
Positive control, the i.e. T of wild-type in each system and each passage
mThe value scope is as follows: the Tm value at FAM passage wild-type contrast peak is 66.5 ℃ ± 1 ℃ among the reaction system A; The Tm value at TET passage wild-type contrast peak is 61.5 ℃ ± 1 ℃; The Tm value at FAM passage wild-type contrast peak is 64.5 ℃ ± 1 ℃ among the reaction system B; The Tm value at TET passage wild-type contrast peak is 67.5 ℃ ± 1 ℃;
Each T wherein
mValue is the modal value of gained on particular B io-Rad CFX96 instrument, as a reference, when using Other Instruments, T
mValue may slightly change, with the T when inferior test positive control gained
mValue is as the criterion;
T
mValue is as the criterion with instrument automatic interpretation gained, when instrument provides an above T
mDuring value, the peak type that please refer to positive control and negative control is selected effective T
mValue; Can't automatically provide T when instrument occurring
mDuring the situation of value, obtain T by the method for adjusting baseline or direct labor's interpretation
mValue.
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| CN102559868B (en) * | 2011-11-28 | 2014-11-05 | 厦门大学 | Method for qualitative and quantitative detection of multiple target nucleotide sequences with single tube |
| CN103060464B (en) * | 2013-01-23 | 2014-06-25 | 中国医学科学院病原生物学研究所 | Kit for detecting or assisting in detecting ethambutol resistance of multiple drugs resistant bacillus tuberculosis |
| CN105779596A (en) * | 2016-03-25 | 2016-07-20 | 苏州达麦迪生物医学科技有限公司 | Molecular beacon probes and kit for rapidly assaying ethambutol-resistant mycobacterium tuberculosis |
| CN106011306A (en) * | 2016-08-11 | 2016-10-12 | 上海默礼生物医药科技有限公司 | Kit for detecting drug-resistance gene of mycobacterium tuberculosis and relative application thereof |
| CN106367528A (en) * | 2016-08-26 | 2017-02-01 | 厦门大学 | Detection method of hepatitis B virus drug-resistant mutation |
| CN107488715A (en) * | 2017-08-14 | 2017-12-19 | 福建医科大学孟超肝胆医院 | PNPLA3 kit for detecting susceptibility genes and method based on self-quenching probe melting curve |
| CN109900888B (en) * | 2019-04-15 | 2020-01-14 | 德阳市人民医院 | Specific metabolite for acute pancreatitis and application thereof |
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| CN1635159A (en) * | 2004-10-19 | 2005-07-06 | 中国人民解放军第三0九医院 | M.tuberculosis drug resistant gene detection reagent kit and process for preparation |
| CN101363799A (en) * | 2007-08-09 | 2009-02-11 | 上海主健生物工程有限公司 | Kit for evaluating medicine for tuberculosis |
| CN101845503A (en) * | 2010-06-10 | 2010-09-29 | 无锡锐奇基因生物科技有限公司 | Method and kit for detecting multi-drug resistant mycobacterium tuberculosis (MDR-TB) |
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| CN1635159A (en) * | 2004-10-19 | 2005-07-06 | 中国人民解放军第三0九医院 | M.tuberculosis drug resistant gene detection reagent kit and process for preparation |
| CN101363799A (en) * | 2007-08-09 | 2009-02-11 | 上海主健生物工程有限公司 | Kit for evaluating medicine for tuberculosis |
| CN101845503A (en) * | 2010-06-10 | 2010-09-29 | 无锡锐奇基因生物科技有限公司 | Method and kit for detecting multi-drug resistant mycobacterium tuberculosis (MDR-TB) |
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