CN102229989A - 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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- CN102229989A CN102229989A CN2011101378199A CN201110137819A CN102229989A CN 102229989 A CN102229989 A CN 102229989A CN 2011101378199 A CN2011101378199 A CN 2011101378199A CN 201110137819 A CN201110137819 A CN 201110137819A CN 102229989 A CN102229989 A CN 102229989A
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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 mycobacterium tuberculosis medicament-resistant mutation, be particularly related to a kind of detection method and test kit of mycobacterium tuberculosis Tibutol medicament-resistant mutation, this method is a kind of probe melting curve analytical technology based on double-tagging self-quenching probe, is used to detect mycobacterium tuberculosis Tibutol medicament-resistant mutation.
Background technology
Tibutol is one of phthisical medicine of treatment, and it is a kind of pectinose analogue, influences 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%.At present its Drug Resistance Detection still need be cultivated and drug sensitive experiment confirms that sense cycle is long, and workload is big, 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 theory and technology, resistance mechanism and drug-fast molecular basis are 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 pectinose based transferase 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, embB 406 and embB 497 (4 in addition, 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 getting in touch (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.).
Gene test to the Tibutol drug-resistant type at present comprises two big steps, and promptly nucleic acid amplification reaches the analysis to amplified production.According to difference, can be divided into heterogeneous detection and homogeneous phase and detect the amplified production processing mode.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 subjected to the influence of 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 real-time fluorescence PCR rapidly has plurality of advantages, owing to adopt stopped pipe to measure mode, eliminates the hidden danger of the product pollution of heterogeneous method.Fluoroscopic examination simultaneously 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 be used to detect the nucleic acid of pathogenic agent, can detect transgenation in real time by design specificity fluorescent probe simultaneously.The probe that has been used widely at present mainly comprises hydrolysis probes (TaqMan probe, MGB probe), molecular beacon (Molecular probe) and FRET probe, hydrolysis probes designs specific probes with molecular beacon at different mutation types, have only when probe and template are mated fully and can send fluorescent signal on the annealing rank of pcr amplification, studies show that when these two kinds of probes are used to detect the tuberculosis drug-tolerant gene mutation, have 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, detect, certain degree of difficulty is arranged in design for polygene, many site mutation.Comparatively speaking, the FRET probe design of Luo Shi patent is comparatively simple, it 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 by two with masterplate complementation and adjacent specific probe.The FRET probe is gathered fluorescence at the annealing stage of pcr amplification, simultaneously also can be by Tm fusion carrying out gene type, therefore as long as a kind of probe of design just can detect different mutation types simultaneously.To seal avoiding reaction owing to form two probe ends of FRET probe, so it is the synthetic cost can be than higher, also cumbersome.
Summary of the invention
One of purpose of the present invention is the problem that exists in the existing mycobacterium tuberculosis medicament-resistant mutation 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, promptly a kind of probe melting curve analytical 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), utilize primer-design software Primer Premier 5 design primer and probes according to mycobacterium tuberculosis embB gene order;
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 being detected, 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 (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), described PCR reaction system contains PCR reaction solution I or PCR reaction solution II, 2U Taq enzyme, and 5 μ L testing samples, sterilization back pure water is as negative control, 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 embB 306 and embB 497 transgenations respectively.
Described PCR reaction system II is made up of 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)), dNTP mixture and sterilization back pure water.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 pre-sex change 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 mixed solution, 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 an 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 mixed solution 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 mixed solution 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.Being stored in-20 ℃ disposes until sample extraction.
2) sample extraction and application of sample---extract the district
1. the mycobacterium tuberculosis of 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. seal film and seal, 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 the pipe lid immediately completely.
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 for use.
2. program run finishes, and PCR thin-walled reaction tubes (stopped pipe) is taken out put into concavo-convex bag, will seal and obturage, and presses source of pollution and handles.
(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 provide T automatically when instrument occurring
mDuring the situation of value, can obtain T by the method for adjusting baseline or direct labor's interpretation
mValue.
(4) interpretation as a result
By comparing the fusing point (T of melting curve between institute's test sample and the positive control
mWhether difference 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 to the Tibutol sensitivity; 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, divide 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 than big-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 the 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 detecting being positioned at 6 codons 306,368,378,380,406 on the embB gene and 497 sudden change.This system the design of primers scheme comprise: (1) primer is respectively applied for amplification embB 306 genes, embB 497 genes and embB 368-embB 406 genes to 2 (497-F and 497-R) and primer to 3 (368-406-F and 368-406-R) to 1 (306-F and 306-R), primer.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 at 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, have the base that do not match between probe and the sudden change template, 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 a fluorescence intensity, on behalf of embB 306 ATG＞GTG, curve 2, curve 1 represent embB 306 ATG＞ATA, curve 3 to represent embB 306 ATG＞CTG, curve 4 to represent 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 a fluorescence intensity, on behalf of embB 497 CAG＞CGG, curve 2, curve 1 represent embB 497 CAG＞AAG, curve 3 to represent 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 a fluorescence intensity, on behalf of embB 378 GAG＞GCG, curve 2, curve 1 represent 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 a fluorescence intensity, on behalf of embB 406 GGC＞GAC, 2, curve 1 represent 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 architecture, detect 6 codons 306,368,378,380,406 be positioned on the embB gene and 497 sudden change, 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 and divide two reaction systems to carry out dual double-colored detection:
EMB PCR MIX A is 19.6 μ l for every part, contains 1 * buffer (50mM KCl, 10mM Tris-HCl (pH=8.6), glycerine 5% (V/V)) in every part of reaction solution, 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 mixed solution (0.4 μ l) before detecting, template add-on to be measured is 5 μ l.
EMB PCR MIX B is 19.6 μ l for every part, 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 adds 2U enzyme mixed solution (0.4 μ l) before detecting, and 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 pre-sex change 10min; 95 ℃ of sex change 15s then, 58 ℃ of annealing 15s, 74 ℃ are extended 20s, altogether 55 circulation; 95 ℃ of pre-sex change 1min, 35 ℃ of insulation 5min, then with the temperature rise rate of 0.5 ℃/step from 40 ℃ or increase to 85 ℃, gather the fluorescence of FAM and TET passage simultaneously 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.
Interpretation as a result:
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 difference 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 to the Tibutol sensitivity; 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 mixed solution 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 an 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 mixed solution 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 mixed solution 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.Being stored in-20 ℃ disposes until sample extraction.
2) sample extraction and application of sample---extract the district
1. the mycobacterium tuberculosis of 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. seal film and seal, 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 the pipe lid immediately completely.
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 for use.
2. program run finishes, and PCR thin-walled reaction tubes (stopped pipe) is taken out put into concavo-convex bag, will seal and obturage, and presses source of pollution and handles.
(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 provide T automatically when instrument occurring
mDuring the situation of value, can obtain T by the method for adjusting baseline or direct labor's interpretation
mValue.
(4) interpretation as a result
By comparing the fusing point (T of melting curve between institute's test sample and the positive control
mWhether difference 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 to the Tibutol sensitivity; 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, divide 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 than big-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 (10)
1. the detection method of a mycobacterium tuberculosis Tibutol medicament-resistant mutation is characterized in that may further comprise the steps:
1) DNA of extraction mycobacterium tuberculosis sample;
2), utilize primer-design software Primer Premier 5 design primer and probes according to mycobacterium tuberculosis embB gene order;
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.
2. the detection method of a kind of mycobacterium tuberculosis Tibutol medicament-resistant mutation as claimed in claim 1 is characterized in that 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 being detected, and probe covers the mutational site;
B, the fluorophor of probe is selected any one among FAM, ROX, HEX, CY5, TET, the CAL-Fluor, and quenching group is selected BHQ, Dabcyl.
3. the detection method of a kind of mycobacterium tuberculosis Tibutol medicament-resistant mutation as claimed in claim 1 is characterized in that in step 2) in, the concrete sequence of described primer and probe 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′。
4. the detection method of a kind of mycobacterium tuberculosis Tibutol medicament-resistant mutation as claimed in claim 1, it is characterized in that in step 3), described PCR reaction system contains PCR reaction solution I or PCR reaction solution II, 2U Taq enzyme, 5 μ L testing samples, sterilization back pure water is as negative control, and wild plasmid is as positive control.
5. the detection method of a kind of mycobacterium tuberculosis Tibutol medicament-resistant mutation as claimed in claim 3, it is characterized in that containing 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, dNTP mixture and deionized water, 306-P and 497-P detect embB 306 and embB 497 transgenations respectively;
Described PCR reaction system II is by primer 3,368-380-P, 406-P, 10 * buffer, glycerine 50% (V/V)), dNTP mixture and sterilization back pure water are formed, the 368-380-P probe of TET mark can detect all mutation types of embB 368, embB 378 and three codons of embB380, and the 406-P probe examination of FAM mark occurs in the sudden change on the embB 406.
6. the detection method of a kind of mycobacterium tuberculosis Tibutol medicament-resistant mutation as claimed in claim 1, it is characterized in that in step 3), the program of described pcr amplification is: 50 ℃, 2min, 95 ℃ of pre-sex change 3min, 95 ℃ of sex change 15s then, 58 ℃ of annealing 15s, 74 ℃ are extended 20s, altogether 55 circulation.
7. the test kit of the detection method of a kind of mycobacterium tuberculosis Tibutol medicament-resistant mutation as claimed in claim 1, 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 Mix A, EMB PCR Mix B and TB enzyme mixed solution, described contrast agents comprises EMB positive control and TB negative control, and described extraction reagent is TB DNA extraction liquid.
8. the test kit of the detection method of a kind of mycobacterium tuberculosis Tibutol medicament-resistant mutation as claimed in claim 7 is characterized in that described EMB PCR Mix A comprises 1 * buffer, glycerine 5%, dNTP/dUTP Mix, 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 1 * buffer is 50mM KCl, 10mM Tris-HCl, pH=8.6; Described dNTP/dUTP Mix is dATP, dCTP, each 0.2mM of dGTP, dUTP 0.4mM;
Described EMB PCR Mix A comprises 1 * buffer, glycerine 5%, dNTP/dUTP Mix, 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 1 * buffer is 50mM KCl, 10mM Tris-HCl, pH=8.6; Described dNTP/dUTP Mix is dATP, dCTP, each 0.2mM of dGTP, dUTP 0.4mM.
9. the test kit of the detection method of a kind of mycobacterium tuberculosis Tibutol medicament-resistant mutation as claimed in claim 7 is characterized in that described EMB positive control is an 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.
10. the method for the test kit inspection specimen of the detection method of a kind of mycobacterium tuberculosis Tibutol medicament-resistant mutation as claimed in claim 7 is characterized in that 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 mixed solution 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 L EMB PCR Mix B and n * 0.4 μ L TB enzyme mixed solution 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, is stored in-20 ℃ and disposes until sample extraction;
2) sample extraction and application of sample---extract the district
1. the mycobacterium tuberculosis of 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. seal film and seal, 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 the pipe lid immediately completely;
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 for use;
2. program run finishes, and PCR thin-walled reaction tubes is taken out put into concavo-convex bag, will seal and obturage, and presses source of pollution and handles;
(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 provide T automatically 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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| CN102559868A (en) * | 2011-11-28 | 2012-07-11 | 厦门大学 | Method for qualitative and quantitative detection of multiple target nucleotide sequences with single tube |
| CN103060464A (en) * | 2013-01-23 | 2013-04-24 | 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 |
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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 |
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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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| CN102559868A (en) * | 2011-11-28 | 2012-07-11 | 厦门大学 | Method for qualitative and quantitative detection of multiple target nucleotide sequences with single tube |
| 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 |
| CN103060464A (en) * | 2013-01-23 | 2013-04-24 | 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 |
| 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 |
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| 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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