CN102229987A - Method and kit for detecting isoniazid resistance mutation of Mycobacterium tuberculosis - Google Patents
Method and kit for detecting isoniazid resistance mutation of Mycobacterium tuberculosis Download PDFInfo
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- CN102229987A CN102229987A CN2011101377923A CN201110137792A CN102229987A CN 102229987 A CN102229987 A CN 102229987A CN 2011101377923 A CN2011101377923 A CN 2011101377923A CN 201110137792 A CN201110137792 A CN 201110137792A CN 102229987 A CN102229987 A CN 102229987A
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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 isoniazid resistance mutation of Mycobacterium tuberculosis, which have low cost and high throughout and are simple and specific. The method comprises the following steps of: designing primers and probes by using the primer design software Primer Premier 5 according to the nucleic acid sequence of the H37Rv genome of Mycobacterium tuberculosis in the GenBank, extracting DNA of a Mycobacterium tuberculosis sample, constructing a PCR (polymerase chain reaction) system, analyzing PCR products by use of a melting curve after PCR is completed, and differentiating wild-type and mutation-type according to the differences in melting point and shape of the melting peaks. By adopting the real-time PCR melting curve, almost all the known drug resistance mutation types can be involved. Besides, the method and kit have the advantages of low cost, simplicity in operation, rapid response and high throughout.
Description
Technical field
The present invention relates to the detection technique of mycobacterium tuberculosis medicament-resistant mutation, relate in particular to a kind of mycobacterium tuberculosis vazadrine medicament-resistant mutation detection method and test kit, this method is that a kind of probe melting curve analytical technology based on double-tagging self-quenching probe is used to detect mycobacterium tuberculosis vazadrine medicament-resistant mutation.
Background technology
Since the eighties in 20th century, because acquired immune deficiency syndrome (AIDS) and appearance of multiple-drug resistance tuberculosis bacterium and floating population's increase, incidence of tuberculosis rises again, becomes the most serious transmissible disease of whole world harm gradually.According to the World Health Organization's report in 2009, at present the whole world have 1/3rd population promptly 2,000,000,000 people infect mycobacterium tuberculosis, annual newly-increased patient surpasses 9,000,000, wherein 420,000 is multidrug resistance tuberculosis, dead 2,000,000.China is one of the high burden of 22 tuberculosis country in the world, and number of patients is only second to India and occupies second in the whole world, and the resistance situation is serious, and resistant rate constitutes severe challenge for prevention lungy and treatment up to 27.8%.
Drug sensitive experiment excessive cycle (needing 6～8 time-of-weeks) becomes the major cause that multidrug resistance tuberculosis (MDR-TB) is propagated, so quick diagnosis is very necessary for the propagation of instructing clinical application and control MDR-TB rationally and effectively.Because the drug-fast generation of tubercule bacillus mainly is in the genomic dna due to the antitubercular agent effect mutant target gene, so gene test is more direct, is also comparatively fast developed in recent years.The primary process of DNA test is the drug resistance related gene that increases earlier, analysing amplified then product.
(isoniazid is to begin nineteen fifty-two to be used for clinical hydrazides class chemical synthetic drug INH), is a line core psychological treatment thing of tuberculotherapy, also is the choice drug of mycobacterium tuberculosis inapparent infection simultaneously in the vazadrine.But up to the present, the mechanism of target molecule, mechanism of action and the anti-vazadrine of mycobacterium tuberculosis of vazadrine effect is still not exclusively clear and definite.It is a kind of prerequisite medicine for more bibliographical information, in thalline, activated by the catalase of katG genes encoding-peroxidase, the enzyme that acts in the cell walls mycolic acid route of synthesis is and the biosynthesizing of interference mycolic acid finally to cause the damage of somatic cells wall and death.Resistance relevant sudden change in vazadrine mainly occurs in the KatG315 site, the inhA94 site, inhA promoter region and ahpC promoter region (removing-46 sites) (1.Hazbon, M.H., et al., Population genetics study of isoniazid resistance mutations and evolution of multidrug-resistant Mycobacterium tuberculosis.Antimicrob Agents Chemother, 2006.50 (8): 2640-2649.2.Luo, T., et al., Selection of mutations to detect multidrug-resistant Mycobacterium tuberculosis strains in Shanghai, China.Antimicrob Agents Chemother.54 (3): 1075-1081.3.Zhang, M., et al., Detection of mutations associated with isoniazid resistance in Mycobacterium tuberculosis isolates from China.J Clin Microbiol, 2005.43 (11): 5477-5482.).AhpC promoter region-46G → A is a pleomorphism site, at wild strain and mutant strain existence is arranged all, with medicament-resistant mutation irrelevant (4, Doustdar, F., et al., Molecular analysis of isoniazid resistance in different genotypes of Mycobacterium tuberculosis isolates from Iran.Microb Drug Resist, 2008.14 (4): 273-279.).
At present, use more tuberculosis medicament-resistant mutation analytical technology and comprise Sanger order-checking, the reverse hybridized method of linear probe and solid phase chip method etc.The Sanger order-checking is " gold standard " that sudden change detects, but on the tuberculosis Drug Resistance Detection, be only applicable to the Rifampin Drug Resistance Detection that mutantional hotspot is concentrated, and for resistance site, vazadrine dispersive situation, need carry out a plurality of sequencing reactions, waste time and energy and both expensive (5.Hazbon, M.H., Recent advances in molecular methods for early diagnosis of tuberculosis and drug-resistant tuberculosis.Biomedica, 2004.24 Supp 1:149-162.).
World health organisation recommendations in 2008 uses the GenoType MTBDRplus assay test kit of German Hain company to detect tuberculosis multidrug resistance (MDR-TB).It adopts linear probe hybridization paper slip membrane technique, on a test strip, cover Rifampin rpoB core area and common mutations thereof with 8 wild-types and 4 mutant probes respectively, cover resistance katG315 site, vazadrine and inhA promoter region gene and common mutations thereof respectively with 3 wild-types and 6 mutant probes.GenoType MTBDRplus detects clinical separation strain and the acid-fast stain positive sample has higher sensitivity and specificity (6.Ling, D.I., A.A.Zwerling, and M Pai, GenoType MTBDR assays for the diagnosis of multidrug-resistant tuberculosis:a meta-analysis.Eur Respir J, 2008.32 (5): 1165-1174.).Rich mycobacterium tuberculosis drug resistant gene detection kit difficult to understand adopts the dna microarray chip method, can 13 kinds of sudden change of examination Rifampin resistance rpoB core area, and resistance katG315 site, vazadrine and 4 kinds of sudden changes of inhA promoter region.
The common feature of above-mentioned two kinds of test kits is all to adopt the solid-phase hybridization pattern that the PCR product is detected, and needs to come sentence read result by color reaction step or fluorescent signal.Whole process operation step is many, and the time is long, easily causes the pollution of PCR product to follow-up amplification.Especially, these two kinds of test kits all pass through the endpoint signal sentence read result, and no matter artificial interpretation still be the machine automatic interpretation, all can be because of the frequent heterogeneity hybridization signal of appearance and produce so-called " gray area " in the sample, and cause the result can't interpretation.In addition, when mutator gene or site become the type that test kit do not cover, in the result, owing to wild signal only occurs, and cause erroneous judgement.
Disperse at tuberculosis resistance site, characteristics such as mutation type complexity, also occur high-throughput examination technology in recent years and be applied to the report that the tuberculosis medicament-resistant mutation is analyzed, (QIAplex multiplex amplification associating Luminex xMAP platform detects (7.Gegia as liquid-phase chip, M., et al., Prevalence of and Molecular Basis for Tuberculosis Drug Resistance in the Republic of Georgia:Validation of a QIAplex System for Detection of Drug Resistance-Related Mutations.Antimicrob Agents Chemother, 2008.52 (2): 725-729.]), MLPA (8.Bergval, I.L., et al., Development of multiplex assay for rapid characterization of Mycobacterium tuberculosis.J Clin Microbiol, 2008.46 (2): 689-699.), tetra-sodium order-checking (9.Jureen, P., et al., Rapid detection of rifampin resistance in Mycobacterium tuberculosis by Pyrosequencing technology.J Clin Microbiol, 2006.44 (6): 1925-1929.) etc.These method recall rates are different with the cost height, but all need expensive specific equipment, and laboratory condition and operator are required height, particularly owing to need complicated PCR post-processing step, increased the probability of product pollution greatly.
It is rapid to be based upon the operational real time pcr development in recent years of complete stopped pipe, simple and efficient with it, with low cost Dengs significant advantage, widespread use clinically.High resolution melting curve technology (the High Resolution Melting of development in recent years, HRM) at tuberculosis Rifampin and the analytically existing report (10.Pietzka of vazadrine medicament-resistant mutation, A.T., et al., Rapid identification of multidrug-resistant Mycobacterium tuberculosis isolates by rpoB gene scanning using high-resolution melting curve PCR analysis.J Antimicrob Chemother, 2009.63 (6): 1121-1127.11.Ong, D.C., et al., Rapid detection of rifampicin-and isoniazid-resistant Mycobacterium tuberculosis by high-resolution melting analysis.J Clin Microbiol.48 (4): 1047-1054.).Yet the HRM technology is used non-specific chimeric dyestuff, is difficult to distinguish the special and non-specific amplification product of PCR.When especially increasing the tuberculosis group, be subjected to its average GC content up to 67% influence, non-specific amplification is difficult to avoid, and the sudden change separating capacity of HRM technology can be under some influence.And in order to improve the heterozygote recall rate, also need sample and reference material genomic dna or the mixing of PCR product, the liquation of increasing again, this has also increased Operating Complexity.In addition, the HRM somatotype requires also very high to dna profiling purity and sample room DNA concentration homogeneity.These have all limited the clinical application of HRM technology in the examination of tuberculosis medicament-resistant mutation.
Summary of the invention
One of purpose of the present invention is at the above-mentioned problems in the prior art, provide a kind of low cost, high-throughput, easy and special mycobacterium tuberculosis vazadrine medicament-resistant mutation detection method, promptly a kind of method of the probe melting curve analytical technology based on double-tagging self-quenching probe.
Another object of the present invention is to provide the test kit of a kind of mycobacterium tuberculosis vazadrine medicament-resistant mutation detection method.
Described mycobacterium tuberculosis vazadrine medicament-resistant mutation detection method may further comprise the steps:
1) design primer and probe: according to (the accession number: NC_000962), utilize primer-design software Primer Premier 5 design primer and probes of mycobacterium tuberculosis H37Rv genomic nucleic acid sequence among the GenBank;
2) DNA of extraction mycobacterium tuberculosis sample;
3) set up the PCR reaction system;
4) result judges: after the PCR reaction finished, product was analyzed by melting curve, according to the fusing point at fusion peak and the difference of shape, distinguished wild-type and mutant, promptly finished the detection to mycobacterium tuberculosis vazadrine medicament-resistant mutation.
In step 1), the concrete grammar of described design primer and probe is as follows:
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 general probe mark dyestuff, quenching group can be selected general probe mark quencher, described probe mark dyestuff can be series dyes such as FAM, ROX, HEX, CY5, CY3, TET, ALEX, CAL Fluor, and described probe mark quencher can be BHQ1, BHQ2 or DABCYL etc.;
The sequence of described primer and probe can be:
ahpC-F：5′-CGGCCGGCTAGCACCTCT-3′
ahpC-F：5′-CGGCCGGCTAGCACCTCT-3′
ahpC-R：5′-CCAATGGTTAGCAGTGGCATGACT-3′
inhA94-F：5′-ATCGGGGCGGGCAACAAG-3′
inhA94-R：5′-TAGGGCGCGTCGAAGAACGG-3′
inhA-F：5′-CGGAAATCGCAGCCACGTTAC-3′
inhA-R：5′-ACGGGATACGAATGGGGGTTTG-3′
katG-F：5′-GCGGTCACACTTTCGGTAAGAC-3′
katG-R：5′-CGTACAGGATCTCGAGGAAACTG-3′
ahpC?P1：5′-FAM-GTCAGGCATATATCACCTTTGCCTGAC-DABCYL-3′
ahpC?P2：5′-FAM-CGCTGCGGCACGATGGAATGTGCAGCG-DABCYL-3′
inhA94-P：5′-TET-GGTGCAACCCAATCGAATGCACC-DABCYL-3′
inhA-P：5′-FAM-CGAGGCCGACAACCTATCGTCTCCCTCG-DABCYL-3′
katG-P：5′-TET-CCGAGGCACCAGCGGCATCGACCTCGG-DABCYL-3′。
In step 2) in, the concrete grammar of the DNA of described extraction mycobacterium tuberculosis sample can adopt water-boiling method, phenol chloroform extraction method or magnetic bead extraction method etc.
In step 3), the described concrete grammar of setting up the PCR reaction system is as follows:
Adopt the substance PCR mode of a reaction of a pair of primer amplification, to collocation, carry out the PCR reaction with single probe and a pair of primer; Or
Adopt the multiplex PCR mode of the common amplification of at least two pairs of primers,, carry out the PCR reaction with the probe mix and match;
In step 4), the concrete grammar of described differentiation wild-type and mutant is as follows: the difference of comparative sample and wild-type contrast fusing point (Tm) value, if sample and wild-type then are judged to be wild-type to the Tm value that impinges upon four detection zones equal consistent (wild-type contrast Tm value ± 1 ℃); If the Tm value of the arbitrary detection zone of sample is lower than at least 2 ℃ of wild-type contrasts.
The test kit of described mycobacterium tuberculosis vazadrine medicament-resistant mutation detection method is provided with box body, amplifing reagent, contrast agents and extraction reagent, and amplifing reagent, contrast agents and extraction reagent are placed in the box body; Described amplifing reagent comprises INH PCR Mix A, INH PCR Mix B and TB enzyme mixed solution; Described contrast agents comprises INH positive control and TB negative control; Described extraction reagent is TB DNA extraction liquid;
Described INH PCR Mix A comprises 10mM Tris-HCl (pH8.5,25 ℃), 50mM KCl, 5% glycerine, 4.5mMMgCl
2, 200 μ M dNTPs/dUTP mix, primer ahpC-F 0.21 μ M, ahpC-R 2 μ M, inhA94-F 0.1 μ M, inhA94-R 1 μ M, probe ahpC-P1, ahpC-P2, each 0.2 μ M of inhA94-P.
Described INH PCR Mix B comprises 50mM Tris-HCl (pH8.5,25 ℃), 50mM KCl, 3.3% glycerine, 4.5mMMgCl
2, 160 μ M dNTPs/dUTP mix, primer inhA-F 0.2 μ M, inhA-R 2 μ M, katG-F 0.3 μ M, katG-R 3 μ M, each 0.2 μ M of probe inhA-P, katG-P.
Described TB enzyme mixed solution comprises 2U Taq, 0.01U UNG.
Described INH positive control is the INH wild 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 described mycobacterium tuberculosis vazadrine medicament-resistant mutation detection method may further comprise the steps:
1) reagent is prepared---the dosing district
1. at first with all reagent from refrigerator take out and balance to room temperature.PCR reaction solution dosing standard is: get n * 19.6 μ LINH 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 INH 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 5min;
Second step: 95 ℃ of 10s, 71 ℃ of 25s (each circulation reduces by 1 ℃), 75 ℃ of 30s, 10 circulations;
The 3rd step: 95 ℃ of 10s, 61 ℃ of 25s, 75 ℃ of 25s, 45 circulations;
The 4th step: 95 ℃ of 2min, 40 ℃ of 2min;
The 5th step: 40～80 ℃, collect fluorescent signal for per 1 ℃, 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)
The T of INH positive control in each system and each passage
mThe value scope is as follows:
The FAM passage has two continuous peaks among the reaction system A, detects ahpC promoter region (44～-30 and-15～3 site) respectively, T
mValue is respectively 58 ℃ ± 1 ℃ and 69 ℃ ± 1 ℃.The TET passage detects inhA94 codon, peak T
mValue is 66 ℃ ± 1 ℃.
The FAM passage detects inhA promoter region (17～-8) site, peak T among the reaction system B
mValue is 64 ℃ ± 1 ℃.The TET passage detects katG315 codon, peak T
mValue is 68 ℃ ± 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 vazadrine 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 vazadrine resistance.
INH reaction system AFAM passage, the sudden change of some type can cause that two peaks of this passage are fused into a peak, and be judged to be mutant this moment, and test strain is to the vazadrine resistance.
If the situation of INH reaction system B TET passage no signal occurs, then show katG genetically deficient, be judged to be mutant this moment, and test strain is to the vazadrine 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. (when plural peak appears in reaction system A FAM passage) when bimodal occur when sample, be the heterozygosis sample; 2. sample is one and merges the peak, and is consistent with the fusing point of positive control, but the peak type of peak type and positive control has than big-difference, 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 vazadrine 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.
The present invention at first utilizes pcr amplification to produce a large amount of target sequences, target sequence and probe hybridization, and the hybridization product has specific fusing point, and it is lower to contain the hybridization product fusing point of the target sequence of sudden change and probe.According to variation of melting point, the judgement target sequence has or not undergos mutation.The melting curve analytical technology belongs to homogeneous phase and detects, and probe places in the pipe in advance, and pcr amplification step and liquation step can be finished with single program.With regard to the vazadrine medicament-resistant mutation detects, after finishing the liquation step, by the difference of comparative sample and wild-type contrast fusing point (Tm) value, if sample and wild-type then are judged to be wild-type to the Tm value that impinges upon four detection zones equal consistent (wild-type contrast Tm value ± 1 ℃); If the Tm value of the arbitrary detection zone of sample is lower than the wild-type contrast more than 2 ℃, then be judged to be mutant, the existence of resistant organism is then indicated in the existence of mutant.The present invention adopts PCR in real time melting curve method, not only can cover nearly all known medicament-resistant mutation type, and have that cost is low, easy and simple to handle, reaction fast, the flux advantages of higher.
Description of drawings
Fig. 1 is ahpC promoter region typical case detected result figure.In Fig. 1, X-coordinate is a temperature, and ordinate zou is that (dF/dT), curve 1 is a wild-type, and curve 2 is-30C → T that curve 3 is-10C → T curve 4 negative contrasts for the negative derivative of fluorescence intensity and temperature.
Fig. 2 is inhA94 site typical case's detected result figure.In Fig. 2, X-coordinate is a temperature, and ordinate zou is that (dF/dT), curve 1 is a wild-type, and curve 2 is S94A GCG, curve 3 negative contrasts for the negative derivative of fluorescence intensity and temperature.
Fig. 3 is inhA promoter region typical case detected result figure.In Fig. 3, X-coordinate is a temperature, and ordinate zou is that (dF/dT), curve 1 is a wild-type, and curve 2 is-15C → T that curve 3 is-8T → C curve 4 negative contrasts for the negative derivative of fluorescence intensity and temperature.
Fig. 4 is katG315 site typical case's detected result figure.In Fig. 4, X-coordinate is a temperature, and ordinate zou is that (dF/dT), curve 1 is a wild-type, and curve 2 is S315T ACC, and curve 3 is S315T ACG, curve 4 negative contrasts for the negative derivative of fluorescence intensity and temperature.
Heterogeneity resistance figure as a result when Fig. 5 is 1 ℃ of fusion of inhA promoter region-15C → Y.In Fig. 5, X-coordinate is a temperature, and ordinate zou is that (dF/dT), curve 1 is a wild-type, and curve 2 is inhA promoter region-15C → Y for the negative derivative of fluorescence intensity and temperature.
Heterogeneity resistance figure as a result when Fig. 6 is 0.5 ℃ of fusion of inhA promoter region-15C → Y.In Fig. 6, X-coordinate is a temperature, and ordinate zou is that (dF/dT), curve 1 is a wild-type, and curve 2 is inhA promoter region-15C → Y for the negative derivative of fluorescence intensity and temperature.
Heterogeneity resistance figure as a result when Fig. 7 is 1 ℃ of katG315AGC → ASC fusion.In Fig. 7, X-coordinate is a temperature, and ordinate zou is that (dF/dT), curve 1 is a wild-type, and curve 2 is katG315AGC → ASC for the negative derivative of fluorescence intensity and temperature.
Heterogeneity resistance figure as a result when Fig. 8 is 0.5 ℃ of katG315AGC → ASC fusion.In Fig. 8, X-coordinate is a temperature, and ordinate zou is that (dF/dT), curve 1 is a wild-type, and curve 2 is katG315AGC → ASC for the negative derivative of fluorescence intensity and temperature.
Fig. 9 is the sequencing result figure of inhA promoter region-15C → Y heterogeneity drug-resistant type sample.AGATGATA is a base, and sequencer map all has significantly wild and the medicament-resistant mutation heterozygosis is bimodal.
Figure 10 is the sequencing result figure of katG315AGC → ASC heterogeneity drug-resistant type sample.ACCACCGG is a base, and sequencer map all has significantly wild and the medicament-resistant mutation heterozygosis is bimodal.
Figure 11 is the structural representation of the test kit embodiment of mycobacterium tuberculosis of the present invention vazadrine medicament-resistant mutation detection method.
Embodiment
The present invention has designed 4 pairs of specific mycobacterium tuberculosis vazadrine drug resistant gene amplimers, carry out two double real-time PCR reactions, increase corresponding 4 tuberculosis vazadrine drug resistance related gene surveyed areas, i.e. ahpC promoter region (44～-30 and-15～3 site), inhA94 codon, inhA promoter region (17～-8) site and katG315 site.Adopt melting curve technology for detection mycobacterium tuberculosis vazadrine medicament-resistant mutation situation then.
Embodiment 1
The preparation of primer and probe:
According to (the accession number: NC_000962) design and synthesize primer and probe in the table 1 of mycobacterium tuberculosis H37Rv genomic nucleic acid sequence among the GenBank.Be used to detect ahpC promoter region (44～-30 and-15～3 site), inhA94 codon, inhA promoter region (17～-8) site, katG315 codon, determine resistance the vazadrine.
Primer and probe design adopt design softwares such as Clustal X (1.8), Primer Premier5.0, Oligo6.0 and Tm Utility v1.3 to assist and finish.Probe 5 ' a kind of fluorophor of end mark (FAM or TET), the 3 ' end Biao Ji temper group DABCYL that goes out.Because the ahpC promoter region is longer, a probe can't effectively cover, so the scheme that adopts two probes to cover.Primer that designs and probe all carry out the homology comparison to guarantee its specificity by BLAST.Primer and probe are given birth to worker's biotechnology company limited by Shanghai and are synthesized.The concrete sequence of mycobacterium tuberculosis vazadrine medicament-resistant mutation detection architecture primer and probe is referring to table 1.
Table 1
The extraction of mycobacterium tuberculosis sample:
Sputum specimen extracting method: add the 1M NaOH of 1～3 times of volume of sputum specimen, liquefaction sample 15min.The NaOH dosage is looked the sputum specimen viscosity, and what viscosity was low adds 1～2 times, high adds 2～3 times of volumes, is entirely standard with liquefaction.
Sucking-off 1mL liquefaction back sample is put the 1.5mL centrifuge tube, and the centrifugal 10min of 13500rpm removes supernatant.Add 800 μ l TE washingss (10mM Tris-Cl 8.5,0.5mM EDTA), the centrifugal 10min of 13000rpm removes supernatant.
(10mM Tris-Cl 8.0,0.1%mM EDTA 1%TX-100), seal film and seal to add 300 μ l lysates.99 ℃ of heating 20min.The centrifugal 10min of 13500rpm shifts supernatant to the 1.5mL centrifuge tube.Supernatant is the pcr amplification template, is stored in-20 ℃.
Solid culture sample extracting method: with transfering loop collect the mycobacterium tuberculosis bacterium colony of an amount of 2～3 all cell ages and be suspended in 300 μ l lysates (10mM Tris-Cl 8.0,0.1%mM EDTA, 1%TX-100) in, seal film and seal.99 ℃ of heating 20min.The centrifugal 10min of 13500rpm shifts supernatant to the 1.5mL centrifuge tube.Supernatant is the pcr amplification template, is stored in-20 ℃.
Liquid culture sample extracting method: directly draw 1 milliliter of bacterium liquid, the centrifugal 15min of 13500rpm removes supernatant, and (10mM Tris-Cl 8.0,0.1%mM EDTA 1%TX-100), seal film and seal to add 300 μ l lysates.99 ℃ of heating 20min.The centrifugal 10min of 13500rpm shifts supernatant to the 1.5mL centrifuge tube.Supernatant is the pcr amplification template, is stored in-20 ℃.
Embodiment 3
The preparation of mycobacterium tuberculosis vazadrine medicament-resistant mutation detection reaction liquid (INH PCR MIX):
Detect and divide two reaction systems to carry out dual double-colored detection:
INHPCRMIXA is 19.6 μ l for every part, contains 10mM Tris-HCl (pH8.5,25 ℃), 50mMKCl, 3.3% glycerine, 4.5mM MgCl in every part of reaction solution
2, 200 μ M dNTPs/dUTP mix, primer ahpC-F 0.21 μ M, ahpC-R 2 μ M, inhA94-F 0.1 μ M, inhA94-R 1 μ M, probe ahpC-P1, ahpC-P2, each 0.2 μ M of inhA94-P.Add 2U enzyme mixed solution (0.4 μ l) before detecting, template add-on to be measured is 5 μ l.
INH PCR MIX B is 19.6 μ l for every part, contains 10mM Tris-HCl (pH8.5,25 ℃), 50mMKCl, 3.3% glycerine, 4.5mM MgCl in every part of reaction solution 2
2, 160 μ M dNTPs/dUTPmix, primer inhA-F 0.2 μ M, inhA-R2 μ M, katG-F 0.3 μ M, katG-R 3 μ M, probe is each 0.2 μ M of inhA-P, katG-P.Add 2U enzyme mixed solution (0.4 μ l) before detecting, template add-on to be measured is 5 μ l.
Reaction conditions: at the enterprising performing PCR of Bio-rad CFX96 real-time fluorescence PCR instrument, the PCR response procedures is:
95 ℃ of 5min; 95 ℃ of 10s, 71 ℃ of 25s (1 ℃ of each cycle down), 75 ℃ of 30s (10 circulations); 95 ℃ of 10s, 61 ℃ of 25s, 75 ℃ of 25s (45 circulations); 95 ℃ of 2min, 40 ℃ of 2min, 40 ℃ of-80 ℃ of liquations detect FAM and TET passage fluorescence.
The result judges:
1) the FAM passage has two peaks among the system A, detects ahpC promoter region (44～-30 and-15～3 site) respectively, and wild-type contrast peak Tm value is respectively 58 ± 1 ℃ and 69 ± 1 ℃.The TET passage detects the inhA94 codon, and wild-type contrast peak Tm value is 66 ± 1 ℃.
2) the FAM passage detects inhA promoter region (17～-8) site among the system B, and wild-type contrast peak Tm value is 64 ± 1 ℃.The TET passage detects the katG315 codon, and wild-type contrast peak Tm value is 68 ± 1 ℃.
3) among system A and the system B, wild-type contrast FAM and TET passage must all have the melting curve peak, and peak Tm value is in above-mentioned scope.Otherwise experimental result is considered as invalid.
4) then be judged to be wild-type during the melting curve peak value that produced of the melting curve that test sample produced and wild-type contrast consistent (Tm value ± 1 ℃).Except that system A FAM passage,, then be judged as drug-resistant type if the fusion peak that test sample produced is lower than more than 2 ℃ of wild-type contrast Tm values reach.
5) in system A FAM passage, if the melting curve that test sample produced and wild-type contrast consistent (two peak be Tm value ± 1 ℃) then is judged as wild-type.If peak type that test sample produced and Tm can not be in full accord with wild-type, then be judged as mutant.
6) except that system A FAM passage, if two peaks appear in the melting curve that test sample produces, one consistent with wild-type (Tm value ± 1 ℃), one is lower than wild-type and contrasts more than 2 ℃ of Tm values reach, and then is judged as the heterogeneity drug-resistant type.
7) in the system A FAM passage, if the fusion peak that test sample produces greater than two, the peak except two consistent with wild-type (Tm value ± 1 ℃) is lower than wild-type in addition and contrasts 2 ℃ of Tm values and above peak, then is judged as the heterogeneity drug-resistant type.
The heterogeneity drug-resistant type of some mutation type wild peak may occur and the sudden change peak is fused into a unusual broad peak.For the sample that is not easy to judge whether into the heterogeneity drug-resistant type, suggestion is with the 0.5 degree alternating temperature speed melting curve that tries again, so that heterozygosis is bimodal obvious.
Use mycobacterium tuberculosis of the present invention vazadrine medicament-resistant mutation detection kit (fluorescent PCR melting curve method) that 1096 strain mycobacterium tuberculosis clinical samples are carried out vazadrine medicament-resistant mutation examination.Compare with clinical drug sensitivity tests, clinical sensitivity of the present invention can reach 90.2%, and clinical specificity can reach 96.4%, and the positive prediction rate is 94.3%, and negative prediction rate is 93.7%, and resultant accuracy is 93.9%.The present invention detects four passage type specimens of mycobacterium tuberculosis vazadrine medicament-resistant mutation system detected result and sees Fig. 1～4.The wild-type contrast is the wild-type standard plasmid, and negative control is a TB DNA extraction liquid.The result shows by Tm value of relatively fusing the peak and peak shape can distinguish wild-type (wild-type contrast Tm value ± 1 ℃) and mutant (being lower than at least 2 ℃ of wild-type contrast Tm values), detects the resistance sample.
The heterogeneity Resistant strain can adopt the fusion speed of 1 ℃/step, and the part bacterial strain can fuse so that the sudden change peak shape in the heterogeneity Resistant strain is more obvious with 0.5 ℃/step, the results are shown in Figure 5～8.The wild-type contrast is the wild-type standard plasmid, and negative control is a TB DNA extraction liquid.When fusion speed was 1 ℃/step, the broad peak of wild-type standard plasmid appearred being different from inhA promoter region-15C → Y and katG315 AGC → ASC heterogeneity persister.When fusion speed was 0.5 ℃/step, heterogeneity persister peak shape changed more obvious, the acromion that fusing point is lower than wild-type occurs.This acromion is formed by a small amount of mutant template that exists in the sample amplified production.The result shows can distinguish wild-type and heterogeneity medicament-resistant mutation type by comparison peak Tm value and peak shape, detects part heterogeneity drug-resistant type sample.Fig. 9 is the sequencing result figure of inhA promoter region-15C → Y heterogeneity drug-resistant type sample, and sequencer map all has significantly wild and the medicament-resistant mutation heterozygosis is bimodal.The sequencing result figure of Figure 10 katG315AGC → ASC heterogeneity drug-resistant type sample, sequencer map all have significantly wild and the medicament-resistant mutation heterozygosis is bimodal.
Figure 11 provides the structural representation of the test kit embodiment of described mycobacterium tuberculosis vazadrine medicament-resistant mutation detection method, be provided with box body 1, amplifing reagent (comprising INH PCR Mix A2, INH PCR Mix B3 and TB enzyme mixed solution 4), contrast agents (comprising INH positive control 7 and TB negative control 6) and extract reagent (TB DNA extraction liquid 5), amplifing reagent, contrast agents and extraction reagent are placed in the box body.
Described INH PCR Mix A comprises 10mM Tris-HCl (pH8.5,25 ℃), 50mMKCl, 5% glycerine, 4.5mMMgCl
2, 200 μ M dNTPs/dUTP mix, primer ahpC-F 0.21 μ M, ahpC-R 2 μ M, inhA94-F 0.1 μ M, inhA94-R 1 μ M, probe ahpC-P1, ahpC-P2, each 0.2 μ M of inhA94-P.
Described INH PCR Mix B comprises 50mM Tris-HCl (pH8.5,25 ℃), 50mM KCl, 3.3% glycerine, 4.5mMMgCl
2, 160 μ M dNTPs/dUTP mix, primer inhA-F 0.2 μ M, inhA-R 2 μ M, katG-F 0.3 μ M, katG-R 3 μ M, each 0.2 μ M of probe inhA-P, katG-P.
Described TB enzyme mixed solution comprises 2U Taq, 0.01U UNG.
Described INH positive control is the INH wild 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 described mycobacterium tuberculosis vazadrine medicament-resistant mutation detection method may further comprise the steps:
1) reagent is prepared---the dosing district
1. at first with all reagent from refrigerator take out and balance to room temperature.PCR reaction solution dosing standard is: get n * 19.6 μ LINH 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 INH 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 5min;
Second step: 95 ℃ of 10s, 71 ℃ of 25s (each circulation reduces by 1 ℃), 75 ℃ of 30s, 10 circulations;
The 3rd step: 95 ℃ of 10s, 61 ℃ of 25s, 75 ℃ of 25s, 45 circulations;
The 4th step: 95 ℃ of 2min, 40 ℃ of 2min;
The 5th step: 40～80 ℃, collect fluorescent signal for per 1 ℃, 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)
The T of INH positive control in each system and each passage
mThe value scope is as follows:
The FAM passage has two continuous peaks among the reaction system A, detects ahpC promoter region (44～-30 and-15～3 site) respectively, T
mValue is respectively 58 ℃ ± 1 ℃ and 69 ℃ ± 1 ℃.The TET passage detects inhA94 codon, peak T
mValue is 66 ℃ ± 1 ℃.
The FAM passage detects inhA promoter region (17～-8) site, peak T among the reaction system B
mValue is 64 ℃ ± 1 ℃.The TET passage detects katG315 codon, peak T
mValue is 68 ℃ ± 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 vazadrine 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 vazadrine resistance.
INH reaction system AFAM passage, the sudden change of some type can cause that two peaks of this passage are fused into a peak, and be judged to be mutant this moment, and test strain is to the vazadrine resistance.
If the situation of INH reaction system B TET passage no signal occurs, then show katG genetically deficient, be judged to be mutant this moment, and test strain is to the vazadrine 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. (when plural peak appears in reaction system A FAM passage) when bimodal occur when sample, be the heterozygosis sample; 2. sample is one and merges the peak, and is consistent with the fusing point of positive control, but the peak type of peak type and positive control has than big-difference, 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 vazadrine 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. mycobacterium tuberculosis vazadrine medicament-resistant mutation detection method is characterized in that may further comprise the steps:
1) design primer and probe:, utilize primer-design software Primer Premier 5 design primer and probes according to mycobacterium tuberculosis H37Rv genomic nucleic acid sequence among the GenBank;
2) DNA of extraction mycobacterium tuberculosis sample;
3) set up the PCR reaction system;
4) result judges: after the PCR reaction finished, product was analyzed by melting curve, according to the fusing point at fusion peak and the difference of shape, distinguished wild-type and mutant, promptly finished the detection to mycobacterium tuberculosis vazadrine medicament-resistant mutation.
2. a kind of mycobacterium tuberculosis as claimed in claim 1 vazadrine medicament-resistant mutation detection method is characterized in that in step 1) the concrete grammar of described design primer and probe is as follows:
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 general probe mark dyestuff, quenching group is selected general probe mark quencher, described probe mark dyestuff is FAM, ROX, HEX, CY5, CY3, TET, ALEX, CAL Fluor series dyes, and described probe mark quencher is BHQ1, BHQ2 or DABCYL.
3. a kind of mycobacterium tuberculosis as claimed in claim 1 vazadrine medicament-resistant mutation detection method is characterized in that in step 1) the sequence of described primer and probe is:
ahpC-F：5′-CGGCCGGCTAGCACCTCT-3′
ahpC-F：5′-CGGCCGGCTAGCACCTCT-3′
ahpC-R：5′-CCAATGGTTAGCAGTGGCATGACT-3′
inhA94-F：5′-ATCGGGGCGGGCAACAAG-3′
inhA94-R：5′-TAGGGCGCGTCGAAGAACGG-3′
inhA-F：5′-CGGAAATCGCAGCCACGTTAC-3′
inhA-R：5′-ACGGGATACGAATGGGGGTTTG-3′
katG-F：5′-GCGGTCACACTTTCGGTAAGAC-3′
katG-R：5′-CGTACAGGATCTCGAGGAAACTG-3′
ahpC?P1：5′-FAM-GTCAGGCATATATCACCTTTGCCTGAC-DABCYL-3′
ahpC?P2：5′-FAM-CGCTGCGGCACGATGGAATGTGCAGCG-DABCYL-3′
inhA94-P：5′-TET-GGTGCAACCCAATCGAATGCACC-DABCYL-3′
inhA-P：5′-FAM-CGAGGCCGACAACCTATCGTCTCCCTCG-DABCYL-3′
katG-P：5′-TET-CCGAGGCACCAGCGGCATCGACCTCGG-DABCYL-3′。
4. a kind of mycobacterium tuberculosis as claimed in claim 1 vazadrine medicament-resistant mutation detection method is characterized in that in step 2) in, the concrete grammar of the DNA of described extraction mycobacterium tuberculosis sample adopts water-boiling method, phenol chloroform extraction method or magnetic bead extraction method.
5. a kind of mycobacterium tuberculosis as claimed in claim 1 vazadrine medicament-resistant mutation detection method is characterized in that in step 3), and the described concrete grammar of setting up the PCR reaction system is as follows:
Adopt the substance PCR mode of a reaction of a pair of primer amplification, to collocation, carry out the PCR reaction with single probe and a pair of primer; Or
Adopt the multiplex PCR mode of the common amplification of at least two pairs of primers,, carry out the PCR reaction with the probe mix and match.
6. a kind of mycobacterium tuberculosis as claimed in claim 1 vazadrine medicament-resistant mutation detection method, it is characterized in that in step 4), the concrete grammar of described differentiation wild-type and mutant is as follows: the difference of comparative sample and wild-type contrast melting point values, if sample is all consistent to the Tm value that impinges upon four detection zones with wild-type, then be judged to be wild-type; If the Tm value of the arbitrary detection zone of sample is lower than at least 2 ℃ of wild-type contrasts.
7. the test kit of a kind of mycobacterium tuberculosis as claimed in claim 1 vazadrine medicament-resistant mutation detection method is characterized in that being provided with box body, amplifing reagent, contrast agents and extraction reagent, and amplifing reagent, contrast agents and extraction reagent are placed in the box body; Described amplifing reagent comprises INH PCR Mix A, INH PCR Mix B and TB enzyme mixed solution; Described contrast agents comprises INH positive control and TB negative control; Described extraction reagent is TB DNA extraction liquid.
8. the test kit of a kind of mycobacterium tuberculosis as claimed in claim 7 vazadrine medicament-resistant mutation detection method is characterized in that described INH PCR Mix A comprises 10mM Tris-HCl, pH8.5,25 ℃, 50mM KCl, 5% glycerine, 4.5mM MgCl
2, 200 μ M dNTPs/dUTP mix, primer ahpC-F 021 μ M, ahpC-R 2 μ M, inhA94-F 0.1 μ M, inhA94-R 1 μ M, probe ahpC-P1, ahpC-P2, each 0.2 μ M of inhA94-P;
Described INH PCR Mix B comprises 50mM Tris-HCl, pH8.5,25 ℃, 50mM KCl, 3.3% glycerine, 4.5mMMgCl
2, 160 μ M dNTPs/dUTP mix, primer inhA-F 0.2 μ M, inhA-R 2 μ M, katG-F 0.3 μ M, katG-R 3 μ M, each 0.2 μ M of probe inhA-P, katG-P.
9. the test kit of a kind of mycobacterium tuberculosis as claimed in claim 7 vazadrine medicament-resistant mutation detection method is characterized in that described TB enzyme mixed solution comprises 2U Taq, 0.01U UNG; Described INH positive control is the INH wild 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 a kind of mycobacterium tuberculosis as claimed in claim 7 vazadrine medicament-resistant mutation detection method is characterized in that may further comprise the steps:
1) reagent is prepared---the dosing district
1. at first also balance is to room temperature from the refrigerator taking-up with all reagent, and PCR reaction solution dosing standard is: get n * 19.6 μ LINH 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 INH 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 5min;
Second step: 95 ℃ of 10s, 71 ℃ of 25s, each circulation reduces by 1 ℃, 75 ℃ of 30s, 10 circulations;
The 3rd step: 95 ℃ of 10s, 61 ℃ of 25s, 75 ℃ of 25s, 45 circulations;
The 4th step: 95 ℃ of 2min, 40 ℃ of 2min;
The 5th step: 40～80 ℃, collect fluorescent signal for per 1 ℃, 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
The T of INH positive control in each system and each passage
mThe value scope is as follows:
The FAM passage has two continuous peaks among the reaction system A, detects the ahpC promoter region respectively, T
mValue is respectively 58 ℃ ± 1 ℃ and 69 ℃ ± 1 ℃, and the TET passage detects inhA94 codon, peak T
mValue is 66 ℃ ± 1 ℃;
The FAM passage detects-17～-8 sites of inhA promoter region, peak T among the reaction system B
mValue is 64 ℃ ± 1 ℃, and the TET passage detects katG315 codon, peak T
mValue is 68 ℃ ± 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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Cited By (11)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104164507A (en) * | 2014-08-18 | 2014-11-26 | 东北制药集团辽宁生物医药有限公司 | Mycobacterium tuberculosis (TB) nucleic acid detection kit |
| CN104651529A (en) * | 2015-03-24 | 2015-05-27 | 厦门大学 | Method for detecting deletion mutation of nucleic acid molecule |
| CN105734135A (en) * | 2016-03-25 | 2016-07-06 | 苏州达麦迪生物医学科技有限公司 | Molecular beacon probe and kit for fast detecting isoniazide drug-resistant mycobacterium tuberculosis |
| CN104164507B (en) * | 2014-08-18 | 2017-01-04 | 东北制药集团辽宁生物医药有限公司 | A kind of mycobacterium tuberculosis (TB) kit for detecting nucleic acid |
| CN106367528A (en) * | 2016-08-26 | 2017-02-01 | 厦门大学 | Detection method of hepatitis B virus drug-resistant mutation |
| CN107739756A (en) * | 2016-08-12 | 2018-02-27 | 嘉兴允英医学检验有限公司 | A kind of high-flux detection method in tubercle bacillus gene resistance site |
| CN110499353A (en) * | 2019-08-09 | 2019-11-26 | 佛山市第四人民医院(佛山市结核病防治所) | A kind of microbial markers and its application for Drug Resistance for Tuberculosis diagnosis |
| CN110791577A (en) * | 2019-10-25 | 2020-02-14 | 中山大学达安基因股份有限公司 | Kit and method for detecting mycobacterium tuberculosis isoniazid drug-resistant mutant gene |
| CN112941210A (en) * | 2021-02-07 | 2021-06-11 | 中山大学达安基因股份有限公司 | Kit and method for detecting drug-resistant mutation of mycobacterium tuberculosis rifampicin and isoniazid |
| CN114480691A (en) * | 2022-01-24 | 2022-05-13 | 广州迪澳基因科技有限公司 | Method and kit for detecting mycobacterium tuberculosis complex flora based on melting curve |
| CN117210590A (en) * | 2023-09-27 | 2023-12-12 | 北京岱美仪器有限公司 | Molecular beacon probe for detecting isoniazid-resistant mycobacterium tuberculosis and kit thereof |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| 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) |
-
2011
- 2011-05-25 CN CN2011101377923A patent/CN102229987A/en active Pending
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| 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) |
Non-Patent Citations (4)
| Title |
|---|
| GENBANK: "NC_000962", 《GENBANK》 * |
| HERNANDO HAZBO´N ET AL.: "Population Genetics Study of Isoniazid Resistance Mutations and Evolution of Multidrug-Resistant Mycobacterium tuberculosis", 《ANTIMICROBIAL AGENTS AND CHEMOTHERAPY》 * |
| 张明河 等: "荧光置换探针多重实时PCR检测结核杆菌3种耐药突变", 《海峡预防医学杂志》 * |
| 徐亚军: "分子信标荧光定量PCR快速检测结核分枝杆菌技术及产品的开发", 《中国优秀硕士学位论文全文数据库医药卫生科技辑》 * |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
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| CN104164507B (en) * | 2014-08-18 | 2017-01-04 | 东北制药集团辽宁生物医药有限公司 | A kind of mycobacterium tuberculosis (TB) kit for detecting nucleic acid |
| CN104651529A (en) * | 2015-03-24 | 2015-05-27 | 厦门大学 | Method for detecting deletion mutation of nucleic acid molecule |
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| CN106367528A (en) * | 2016-08-26 | 2017-02-01 | 厦门大学 | Detection method of hepatitis B virus drug-resistant mutation |
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| CN117210590A (en) * | 2023-09-27 | 2023-12-12 | 北京岱美仪器有限公司 | Molecular beacon probe for detecting isoniazid-resistant mycobacterium tuberculosis and kit thereof |
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