CN102229991A - Method and kit for detecting streptomycin medicine resistant mutation of Mycobacterium tuberculosis - Google Patents
Method and kit for detecting streptomycin medicine resistant mutation of Mycobacterium tuberculosis Download PDFInfo
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Abstract
The invention discloses a method and a kit for detecting the streptomycin medicine resistant mutation of Mycobacterium tuberculosis. The invention relates to medicine resistant mutation detection technique and provides a method for detecting streptomycin medicine resistant mutation of Mycobacterium tuberculosis, which effectively improves sensitivity and specificity, and is simple and convenient in operation and short in period. The method comprises: designing primers and probes according to the complete sequence of the Mycobacterium tuberculosis and the gene sequences of the genomes rpsL and rrs of the Mycobacterium tuberculosis; extracting the DNA of a sample of the Mycobacterium tuberculosis; constructing a polymerase chain reaction (PCR) reaction system; and performing PCR amplification and analysis on a fusion curve. In the method, experiments are performed in two tubes respectively by using the specific primers and probes, and the amplification of the nucleic acid fragment of a target nucleotide sequence and subsequent analysis on the fusion curve are realized by using heat-resistance DNA polymerase, four kinds of nucleotide monomers and other components and by using real-time PCR technique. A fluorescent PCR fusion curve method with high specificity can quickly and accurately detects common medicine-resistance mutation of Mycobacterium tuberculosis and is expected to be directly used for medicine-resistance detection of a clinic Mycobacterium tuberculosis sample.
Description
Technical field
The present invention relates to the detection technique of medicament-resistant mutation, particularly a kind of probe melting curve analytical technology based on double-tagging self-quenching probe is used to detect mycobacterium tuberculosis Streptomycin sulphate medicament-resistant mutation.
Background technology
Tuberculosis is the chronic infectious disease that is caused by m tuberculosis infection.Tubercule bacillus may be invaded the various organs of human body whole body, but mainly invades lungs, is called pulmonary tuberculosis.To the later stage eighties 20th century, because factors such as poverty, acquired immune deficiency syndrome (AIDS), movement of population and resistances, quick rise appears in incidence of tuberculosis and mortality ratio in the world, becomes the global harm the most serious transmissible disease arranged side by side with acquired immune deficiency syndrome (AIDS).Mycobacterium tuberculosis resistance situation is serious, and resistant rate constitutes severe challenge for prevention lungy and treatment up to 46%.
Antitubercular agent is the chemotherapeutical basis of tuberculosis, and chemotherapy lungy is the main means of human controlling tuberculosis.Streptomycin sulphate is found in the forties in 20th century, it is the effective antitubercular agent that occurs the earliest, also be treatment one of line medicine lungy (1, Li Zhizhong, Gao Quanjin. status [J] the Chinese mistaken diagnosis magazine of Streptomycin sulphate in current tuberculosis, 2002.2 (1): 62-63).Streptomycin sulphate is a kind of aminocyclitol glucosides class microbiotic, mainly act on the rrna of mycobacterium tuberculosis, combine with the rrna 30S subunit of mycobacterium tuberculosis, the mispairing of induction of genetic password, suppress the beginning of mRNA translation, disturb the check and correction in the translation process, thereby arrestin matter is synthetic.Streptomycin sulphate is often individually dosed, so its resistance development is very fast.The main molecules mechanism of the anti-Streptomycin sulphate of mycobacterium tuberculosis is the rrs transgenation of the proteic rpsL gene of S12 and the coding 16S ribosome-RNA(rRNA) of coding small subunit, thereby the ability that suppresses medicine binding ribosomal body, cause to the Streptomycin sulphate resistance (2, Zhang Chenyu, Liang Jianqin. the Chinese mistaken diagnosis magazine of mycobacterium tuberculosis resistance recent advances in research of molecular mechanism [J] .2006,6 (17): 3303-3305).The chemical sproof detection of mycobacterium tuberculosis Streptomycin sulphate has extremely important meaning for the propagation and the controlling tuberculosis of control resistance mycobacterium tuberculosis.
The Drug Resistance Detection method that adopts clinically can be divided into phenotype detection and genotype detection two classes at present, and based on the former (3, Heifets L.Rapid automated methods (BACTEC System) in clinical mycobacteriology[J] Semin Respir Infect, 1986.1 (4): 242-249).The a variety of causes yet phenotype detects as consuming time, apparatus expensive, have radiological hazard or interfering factors many etc., is difficult to form examination means rapidly and efficiently.Comparatively speaking, gene test is more direct, present already used genetic analysis method has single-strand conformation polymorphism analysis method (SSCP), restriction fragment length polymorphism analytical method (RFLP), the dna sequencing method, linear probe hybrid method (4, Mokrousov I.Multicenter evaluation of reverse line blot assay for detection of drug resistance in Mycobacterium tuberculosis clinical isolates[J] .J Microbiol Methods, 2004.57 (3): 323-335), RNA/RNA mispairing analytical method, and DNA array (chip) detects etc. (5, Wu X.Detection of streptomycin resistance in Mycobacterium tuberculosis clinical isolates using four molecular methods in China[J] .Yi Chuan Xue Bao, 2006.33 (7): 655-663).Though these method recall rates are different with the flux height, common drawback is to carry out the PCR aftertreatment, has not only influenced detection efficiency, causes that easily amplified production pollutes, and has increased operation easier, has improved demand of laboratory.Real time pcr will increase and detect the unification of two steps, have advantages such as quick, special, sensitive, quantitative, be used widely in pathogenic micro-organism detection and sudden change detection range, application on the tubercule bacillus Drug Resistance Detection in recent years also cause the very big interest of people (6, El-Hajj H.Detection of rifampin resistance in Mycobacterium tuberculosis in a single tube with molecular beacons[J] .J Clin Microbiol, 2001.39 (11): 4131-4137).
The fluorescent PCR technology then is on the basis of regular-PCR technology, adds a specific fluorescent probe again in a pair of Auele Specific Primer of adding in amplification reaction system, uses the fluorescent PCR detector of monitoring in real time to detect the technology of target nucleotide sequences.Except the advantage with regular-PCR, it also has the following advantages: (1) specificity is stronger, and sensitivity is higher.Since used more one can with the fluorescent probe of template complementary pairing, improved specificity, and collected fluorescent signal by self-reacting device, avoided the subjectivity of artificial judgment, can further improve sensitivity again.(2) totally-enclosed reaction, online real-time monitoring fluorescence need not the aftertreatment of PCR product, avoids polluting, and has guaranteed result's reliability.(3) can realize the single tube double check, reduce cost.(4) do not contact toxic reagent, operational safety.(5) according to the specificity of probe and target sequence hybridization and the T of hybridizing with incomplete coupling target sequence
mValue difference is different, can utilize melting curve to distinguish wild and sudden change, the easy interpretation of result.
Summary of the invention
One of purpose of the present invention is to provide the detection method of a kind of effective raising sensitivity and specificity, easy and simple to handle, mycobacterium tuberculosis Streptomycin sulphate medicament-resistant mutation that the cycle is short, 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 Streptomycin sulphate medicament-resistant mutation.
The detection method of described mycobacterium tuberculosis Streptomycin sulphate medicament-resistant mutation may further comprise the steps:
1), utilizes primer-design software Primer Premier 5 design primer and probes according to mycobacterium tuberculosis complete sequence and mycobacterium tuberculosis genome rpsL and rrs gene order;
2) DNA of extraction mycobacterium tuberculosis sample;
3) make up the PCR reaction system;
4) carrying out pcr amplification and melting curve analysis: with step 2) DNA that extracts adds in the PCR reaction system of step 3), if negative control, blank and positive control, carry out pcr amplification and melting curve analysis, with the T of sample to be tested reaction tubes gained melting curve and positive control reaction tubes gained melting curve
mValue compares, and judge in the sample to be tested on the rpsL and rrs gene the resistance relevant mutational site and whether undergo mutation, thus judgement sample resistance situation.
In step 1), the concrete grammar of described design primer and probe 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 select FAM, ROX, HEX, CY5, TET, CAL-Fluor etc. any one, 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:
Primer rpsL43F:5 '-CAGCCCGCAGCGTCGTGGTGT-3 '
Primer rpsL43R:5 '-GTGGCCCTCGCCGGGAA-3 '
Primer rpsL88F:5 '-GCACTCGATGGTGCTGGTGC-3 '
Primer rpsL88R:5 '-CGTGCCTGTTTGCGGTTCTT-3 '
Primer rrs512F:5 '-ACCTCTTTCACCATCGACGAAGGTCC-3 '
Primer rrs512R:5 '-GTTAAGCCGTGAGATTTCACGAACAA-3 '
Primer rrs904F:5 '-GGGTTTCCTTCCTTGGGATCCGTG-3 '
Primer rrs904R:5 '-GCATGTCAAACCCAGGTAAGGTTC-3 '
Probe rpsL43P:5 '-FAM-CCGCGCCACTCCGAAGAAGCCGAACTCCGCGG-Dabcyl-3 '
Probe rpsL88P:5 '-TET-CCGTCCGGGTGAAGGACCTGCCTGACGG-Dabcyl-3 '
Probe rrs512P:5 '-FAM-CGCACCCAGCAGCCGCGGTAATACGTGCG-Dabcyl-3 '
Probe rrs904P:5 '-TET-CCGCGGGCTAAAACTCAAAGGAATTGACGGCGCGG-Dabcyl-3 '
In step 2) in, described PCR reaction system can be: a kind of rpsL of being system, be used for detecting rpsL gene-correlation medicament-resistant mutation (rpsL codon 43 and rpsL codon 88), another kind is the rrs system, be used for detecting rrs gene-correlation medicament-resistant mutation (rrs512 ring district and rrs904 ring district), reaction solution is 20 μ l for every part, contains following component in every part of reaction solution:
RpsL system: 1 * PCR Buffer[10mM Tris-HCl, 50mM KCl, 50% (v/v) glycerine], dATP, dCTP, dGTP each 200 μ M, dUTP 400 μ M, MgCl
21mM, each 0.6 μ M of primer rpsL43R and rpsL88R, each 0.06 μ M of primer rpsL43F and rpsL88F, each 0.06 μ M of probe rpsL43P and rpsL88P, Taq archaeal dna polymerase 2U, UNG enzyme 0.01U;
Rrs system: 1 * PCR Buffer[10mmol/L Tris, 50mmol/L KCl, 50% (v/v) glycerine], dATP, dCTP, dGTP each 200 μ M, dUTP 400 μ M, MgCl
23mM, each 0.6 μ M of primer rrs512R and rrs904R, primer rrs512F and rrs904F each 0.06 μ M, probe rrs512P and each 0.06 μ M of rrs904P, Taq archaeal dna polymerase 2U, UNG enzyme 0.01U.
The stager also selects the buffer of other type, perhaps finds more suitable buffer type by optimization.
In step 4), the condition of described pcr amplification is: 50 ℃ of 2min, 95 ℃ of 10min; 95 ℃ of 10s, 70 ℃ of 20s (each circulation reduces by 1 ℃), 75 ℃ of 20s constitute a touchdown circulation (carrying out 10 circulations altogether); 95 ℃ of 10s, 60 ℃ of 20s (fluorescence data collection), 75 ℃ of 20s constitute a circulation (carrying out 30 circulations altogether); Be the liquation step afterwards, 95 ℃ of 2min, 40 ℃ of 2min, 40 ℃～80 ℃ per 0.5 ℃ of collection fluorescence.
The test kit of the detection method of described mycobacterium tuberculosis Streptomycin sulphate medicament-resistant mutation is provided with box body, amplifing reagent (comprising STRPCR Mix A, STR PCR Mix B and TB enzyme mixed solution), TB DNA extraction liquid, TB negative control and STR positive control; Described amplifing reagent (comprising STR PCR Mix A, STR PCR Mix B and TB enzyme mixed solution), TB DNA extraction liquid, TB negative control and STR positive control are placed in the box body.
Described STR PCR Mix A comprises 1 * PCR Buffer (10mM Tris-HCl, 50mM KCl, 50% (v/v) glycerine), dATP, dCTP, dGTP each 200 μ M, dUTP 400 μ M, MgCl
21mM, each 0.6 μ M of primer rpsL43R and rpsL88R, each 0.06 μ M of primer rpsL43F and rpsL88F, each 0.06 μ M of probe rpsL43P and rpsL88P.
Described STR PCR Mix B comprises 1 * PCR Buffer (10mM Tris, 50mM KCl, 50% (v/v) glycerine), dATP, dCTP, dGTP each 200 μ M, dUTP 400 μ M, MgCl
23mM, each 0.6 μ M of primer rrs512R and rrs904R, each 0.06 μ M of primer rrs512F and rrs904F, each 0.06 μ M of probe rrs512P and rrs904P.
Described TB enzyme mixed solution comprises 2U Taq, 0.01U UNG.
Described STR positive control is a STR wild-type standard plasmid.
Described TB negative control can be TB DNA extraction liquid, also can be 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 Streptomycin sulphate medicament-resistant mutation 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 LSTR PCR Mix A and n * 0.4 μ L 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 STR PCR Mix B and n * 0.4 μ L 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 10s, 70 ℃ of 20s (each circulation reduces by 1 ℃), 75 ℃ of 20s, 10 circulations;
The 3rd step: 95 ℃ of 10s, 60 ℃ of 20s, 75 ℃ of 20s, 30 circulations, fluorescent signal is collected at 60 ℃ of 20s places;
The 4th step: 95 ℃ of 2min, 40 ℃ of 2min;
The 5th step: 40～80 ℃, 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 57.0 ℃ ± 1 ℃ among the reaction system A; The Tm value at TET passage wild-type contrast peak is 65.0 ℃ ± 1 ℃.
The Tm value at FAM passage wild-type contrast peak is 63.5 ℃ ± 1 ℃ among the reaction system B; The Tm value at TET passage wild-type contrast peak is 60.0 ℃ ± 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 Streptomycin sulphate sensitivity; The fusing point of sample is lower than 2 ℃ of positive controls and (Δ T when above
m〉=2 ℃) be judged to be mutant, test strain is to the Streptomycin sulphate 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 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 Streptomycin sulphate 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.
Concrete principle of the present invention is to utilize Auele Specific Primer and 4 specificity fluorescent probes of 4 pairs of target nucleotide sequences, respectively at testing in two pipes, adopt hot resistant DNA polymerase (Taq enzyme), four kinds of nucleotide monomer compositions such as (dNTP), and use real time pcr and realize the accounting fragment amplification of target nucleotide sequences and melting curve analysis afterwards.Employed probe is the oligonucleotide chain of two ends difference mark fluorescent reporter group and fluorescent quenching group.When probe is free state, the reporter group fluorescent signal emitted is absorbed by quenching group, and in system, exist in a large number can be with probe bonded strand amplicon the time, probe and amplicon are in conjunction with making the fluorescence report group separate with the fluorescent quenching group, the fluorescence report group sends fluorescence.Like this in the process of liquation, along with the bonding state of variation of temperature probe and amplicon is reflected in the change of fluorescent signal, whether undergo mutation at resistant mutational site and can judge sample to be tested by the difference of target sequence on fluorescent signal changes of relatively mating fully and not exclusively mating with probe, thus judgement sample resistance situation.Whether the present invention directly increases a liquation step and can suddenly change to the resistance related locus and judge after the PCR EP (end of program).This method need not be carried out the PCR aftertreatment, has reduced the probability that pollution and false positive occur.Fluorescent PCR melting curve method specificity is good, can detect mycobacterium tuberculosis Streptomycin sulphate resistance common mutations quickly and accurately, is expected to be directly used in clinical sample Streptomycin sulphate resistance and detects.
Outstanding advantage of the present invention is as follows:
(1) because the present invention adopts fluorescent PCR melting curve method as detection method, entire reaction is all carried out in the reaction tubes of sealing, has avoided other nucleic acid detection method such as PCR-electrophoresis etc. to be easy to form aerosol and has polluted and cause false positive results.
(2) easy and simple to handle, and the PCR product monitored in real time, detection time saved greatly.
(3) because each reagent dosage is all less, greatly reduce cost.
Description of drawings
Fig. 1 is rpsL43 site typical case's melting curve figure as a result.In Fig. 1, X-coordinate is a temperature, and ordinate zou is that (dF/dT), curve 1 and 2 is rpsL43 site mutation peak, and curve 3 is wild peak, rpsL43 site, and curve 4 is a blank for the negative derivative of fluorescence intensity and temperature.
Fig. 2 is rpsL88 site typical case's melting curve figure as a result.In Fig. 2, X-coordinate is a temperature, and ordinate zou is that (dF/dT), curve 1,2 and 3 is rpsL88 site mutation peak, and curve 4 is wild peak, rpsL88 site, and curve 5 is a blank for the negative derivative of fluorescence intensity and temperature.
Fig. 3 is rrs513 site typical case's melting curve figure as a result.In Fig. 3, X-coordinate is a temperature, and ordinate zou is that (dF/dT), curve 1 and 2 is rrs513 site mutation peak, and curve 3 is wild peak, rrs513 site, and curve 4 is a blank for the negative derivative of fluorescence intensity and temperature.
Fig. 4 is rrs905 site typical case's melting curve figure as a result.In Fig. 4, X-coordinate is a temperature, and ordinate zou is that (dF/dT), curve 1 is rrs905 site mutation peak, and curve 2 is wild peak, rrs905 site, and curve 3 is a blank for the negative derivative of fluorescence intensity and temperature.
Fig. 5 is the test kit example structure synoptic diagram of the detection method of mycobacterium tuberculosis Streptomycin sulphate medicament-resistant mutation of the present invention.
Embodiment
Respectively design in four sites according to mycobacterium tuberculosis wild type gene group sequence many to primer and probe, the primer 3 ' no complementary sequence of end.Primer, probe sequence combination can be:
Primer rpsL43F:
5′-CAGCCCGCAGCGTCGTGGTGT-3′
Primer rpsL43R:
5′-GTGGCCCTCGCCGGGAA-3′
Primer rpsL88F:
5′-GCACTCGATGGTGCTGGTGC-3′
Primer rpsL88R:
5′-CGTGCCTGTTTGCGGTTCTT-3′
Primer rrs512F:
5′-ACCTCTTTCACCATCGACGAAGGTCC-3′
Primer rrs512R:
5′-GTTAAGCCGTGAGATTTCACGAACAA-3′
Primer rrs904F:
5′-GGGTTTCCTTCCTTGGGATCCGTG-3′
Primer rrs904R:
5′-GCATGTCAAACCCAGGTAAGGTTC-3′
Probe rpsL43P:
5′-FAM-CCGCGCCACTCCGAAGAAGCCGAACTCCGCGG-Dabcyl-3′
Probe rpsL88P:
5′-TET-CCGTCCGGGTGAAGGACCTGCCTGACGg-Dabcyl-3′
Probe rrs512P:
5′-FAM-CGCACCCAGCAGCCGCGGTAATACGTGCG-Dabcyl-3′
Probe rrs904P:
5′-TET-CCGCGGGCTAAAACTCAAAGGAATTGACGGCGCGG-Dabcyl-3′
Primer and probe are given birth to the biological company limited of worker by Shanghai and are synthesized.
Extract the mycobacterium tuberculosis genomic dna with pyrolysis method, detailed process is:
The mycobacterium tuberculosis of growing on A, 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.
B, 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.
C. sample is stored in-20 ℃, and finishes test in 1 month.Note not multigelation sample.
The foundation and the optimization of embodiment 3 reaction systems
Utilize wild-type mycobacterium tuberculosis H37Rv genomic dna as sample to be checked, be stored in after the packing-20 ℃ standby.
2.1 the optimization of primer ratio: under the situation that other condition is identical in reaction system, adjusting the ratio of upstream and downstream primer, the primer ratio was transferred to respectively 1: 5,1: 10,1: 15 and 1: 20, was the ratio of upstream and downstream primer in selected 1: 10 through repeated experiments.
2.2MgCl
2The optimization of concentration: under the situation that other condition is identical in reaction system, with MgCl
2Concentration increase progressively with 1mmol/L with 1mmol/L to 5mmol/L, be the magnesium ion concentration in the test kit reaction system through selected 1mmol/L (rpsL system) of repeated experiments and 3mmol/L (rrs system).
2.3Taq the optimization of enzyme dosage: by comparing the optimization experiment result of Taq enzyme dosage (in the Unit of unit), selected 2U is as the consumption of Taq enzyme in the test kit reaction system.
Utilize above-mentioned primer and probe to carry out the foundation of reaction system, the reaction system of the fluorescent PCR melting curve method rapid screening mycobacterium tuberculosis Streptomycin sulphate medicament-resistant mutation that determine to adopt at last is 25 μ l systems, and required each component and respective concentration see Table 1 and table 2.Each component situation in the reaction of table 1 fluorescent PCR melting curve method rapid screening mycobacterium tuberculosis Streptomycin sulphate rpsL gene medicament-resistant mutation, each the component situation in the reaction of table 2 fluorescent PCR melting curve method rapid screening mycobacterium tuberculosis Streptomycin sulphate rrs gene medicament-resistant mutation
Table 1
Table 2
Annotate: the instrument difference that a. uses, reaction parameter should be done suitable adjustment.
B. different according to detecting the sample source, should suitably adjust the template dosage.
Embodiment 4PCR detects
When carrying out the fluorescent PCR reaction, the collection of tackling reaction tubes fluorescent signal in the used instrument is provided with, and the fluorescence detection channel of selection is consistent with the fluorescence report group of probe institute mark.Concrete method to set up is different because of instrument, should be with reference to the instrument working instructions.(among the present invention with Bio-Rad CFX 96
TMReal-Time system is an example)
The PCR condition is selected as follows:
50℃2min，95℃10min；
95 ℃ of 10s, 70 ℃ of 20s (each circulation reduces by 1 ℃), 75 ℃ of 20s constitute touchdown circulation, 10 circulations;
95 ℃ of 10s, 60 ℃ of 20s (fluorescence data collection), 75 ℃ of 20s, 30 circulations;
The liquation step, 95 ℃ of 2min, 40 ℃ of 2min, 40 ℃～80 ℃ per 0.5 ℃ of collection fluorescence.
Carrying out the result after the PCR reaction judges:
1, the FAM passage has a peak in the pipe 1, detects rpsL gene 43 bit codons, and wild-type contrast peak Tm value is respectively 57.0 ± 1 ℃.The TET passage has a peak, detects rpsL gene 88 bit codons, and wild-type contrast peak Tm value is respectively 65.0 ± 1 ℃.The FAM passage has a peak in the pipe 2, detects rrs gene 513～517 zones, and wild-type contrast peak Tm value is respectively 63.5 ± 1 ℃.The TET passage has a peak, detects rrs gene 905～908 zones, and wild-type contrast peak Tm value is respectively 60.0 ± 1 ℃.
2, pipe 1 and manage in 2, 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.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.Be judged to be wild-type during the fusing point of sample consistent with the fusing point of positive control (error is no more than 1 ℃), test strain is to the Streptomycin sulphate sensitivity; The fusing point of sample is lower than 2 ℃ of positive controls and (Δ T when above
m〉=2 ℃) be judged to be mutant, test strain is to the Streptomycin sulphate resistance.
3, about heterozygosis result's interpretation, divide 3 kinds of situations: 1. arbitrary passage occurs being the heterozygosis sample when bimodal when sample in four passages; 2. sample is a fusion 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 Streptomycin sulphate resistance, when the heterozygosis sample appears in suggestion with the sample duplicate detection, to determine the sample resistance.
The sensitivity of embodiment 5 reaction systems and specificity are investigated
1, the sensitivity of reaction system is investigated: get Streptomycin sulphate wild-type sample and typical mutant sample (each site one strain), every kind of sample is from 2 * 10
8The concentration of bacterium/mL is with 10 times of gradient serial dilution to 2 * 10
3Bacterium/mL, each sample is made three parts of replications, and three parts all is that the minimum cause of disease scale of construction of male is the detection limit of test kit.Display sensitivity all can reach 1000 copy/reaction tubess as a result.
May have mixed bacterium in the actual sample, therefore need to investigate the ability that detects mixed bacterium, be 2 * 10 with concentration
7The wild-type sample of bacterium/mL and concentration are 2 * 10
7(rpsL43AAG → AGG) be mixed in proportion, wherein the ratio of mutant sample is respectively the mutant sample of bacterium/mL: 100%, 80%, 70%, 60%, 50%, 40%, 30%, 0%.Equally, concentration is 2 * 10
6Bacterium/mL and concentration are 2 * 10
5The sudden change of bacterium/mL and wild reference material are mixed in proportion as above mutant proportion.Detected result shows, can detect in the mixed bacterium 40% sudden change exactly.
2, the analysis specificity of reaction system is investigated: get 10 strain wild-type samples and 8 plant mutant type samples, concentration 2 * 10
6Bacterium/mL, each reaction system adds 5 μ L, and wild-type pattern detection result is a wild-type, and mutant pattern detection result is a mutant, no false negative and false positive phenomenon, no omission phenomenon, and the T of wild-type sample
mT with positive control
mFluctuation range is in ± 1.0 ℃, and the T of all mutant samples
mThan positive control T
mLow more than 2.0 ℃.
5.1 the preparation of template to be measured: extract 92 parts of Mycobacterium tuberculosis genomic dnas with pyrolysis method.
5.1.1 collect the mycobacterium tuberculosis bacterium colony of an amount of 2～3 all cell ages and be suspended in the 300 μ l pure water with transfering loop;
Seal 99 ℃ of heating 20min 5.1.2 seal film;
5.1.314000rpm centrifugal 10min shifts supernatant to new 1.5ml centrifuge tube, supernatant is the pcr amplification template;
51.4 sample is stored in-20 ℃, and finishes experiment in 1 month, notes not multigelation sample.
5.2 the amplification and the fusion of the reaction of fluorescent PCR melting curve method rapid screening mycobacterium tuberculosis Streptomycin sulphate medicament-resistant mutation
According to table 1 and table 2 application of sample, will add excellent PCR pipe and be placed on Bio-Rad CFX 96
TMAmong the Real-Time system, design and increase after the corresponding phosphor collection condition and fuse, program is as follows:
5.2.150℃2min，95℃10min；
5.2.295 ℃ 10s, 70 ℃ of 20s (each circulation reduces by 1 ℃), 75 ℃ of 20s constitute touchdown circulation, 10 circulations;
5.2.395 ℃ 10s, 60 ℃ of 20s (collection fluorescence), 75 ℃ of 20s, 30 circulations;
5.2.4 the liquation step, 95 ℃ of pre-sex change 2min, 40 ℃ of insulation 2min, 40～80 ℃ of per 1 ℃ of collection fluorescence.
5.3 the detected result of fluorescent PCR melting curve method rapid screening mycobacterium tuberculosis Streptomycin sulphate medicament-resistant mutation reaction
After testing, if sample to be checked is then melting curve demonstration wild-type T of wild-type
mValue is if sample to be checked is then its T of mutant
mThan positive control T
mLow more than 2.0 ℃, prompting present method can be distinguished wild-type and mutant sample.Concrete outcome is referring to Fig. 1～4.
Detect and divide two reaction systems to carry out dual double-colored detection:
STR PCR MIX A is 19.6 μ l for every part, and every part of reaction solution comprises 1 * PCR Buffer (10mM Tris-HCl, 50mMKCl, 50% (v/v) glycerine), dATP, dCTP, dGTP each 200 μ M, dUTP 400 μ M, MgCl
21mM, each 0.6 μ M of primer rpsL43R and rpsL88R, each 0.06 μ M of primer rpsL43F and rpsL88F, each 0.06 μ M of probe rpsL43P and rpsL88P.Add 2U enzyme mixed solution (0.4 μ l) before detecting, template add-on to be measured is 5 μ l.
STR PCR Mix B is 19.6 μ l for every part, and every part of reaction solution comprises 1 * PCR Buffer (10mM Tris, 50mM KCl, 50% (v/v) glycerine), dATP, dCTP, dGTP each 200 μ M, dUTP 400 μ M, MgCl
23mM, each 0.6 μ M of primer rrs512R and rrs904R, each 0.06 μ M of primer rrs512F and rrs904F, each 0.06 μ M of probe rrs512P and rrs904P.Add 2U enzyme mixed solution (0.4 μ l) before detecting, template add-on to be measured is 5 μ l.
Each detects sample and gets 5 μ lDNA as template, and STR wild-type standard plasmid is as positive control, and TB DNA extraction liquid is as negative control.The PCR response procedures:
The first step: 50 ℃ of 2min, 95 ℃ of 10min;
Second step: 95 ℃ of 10s, 70 ℃ of 20s (each circulation reduces by 1 ℃), 75 ℃ of 20s, 10 circulations;
The 3rd step: 95 ℃ of 10s, 60 ℃ of 20s, 75 ℃ of 20s, 30 circulations, fluorescent signal is collected at 60 ℃ of 20s places;
The 4th step: 95 ℃ of 2min, 40 ℃ of 2min;
The 5th step: 40～80 ℃, collect fluorescent signal for per 0.5 ℃, fluorescence channel is selected FAM and TET for use.
Interpretation as a result:
FAM passage wild-type contrast peak Tm value is 57.0 ℃ ± 1 ℃ among the reaction system A; TET passage wild-type contrast peak Tm value is 65.0 ℃ ± 1 ℃.
FAM passage wild-type contrast peak Tm value is 63.5 ℃ ± 1 ℃ among the reaction system B; TET passage wild-type contrast peak Tm value is 60.0 ℃ ± 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 Streptomycin sulphate sensitivity; The fusing point of sample is lower than 2 ℃ of positive controls and (Δ T when above
m〉=2 ℃) be judged to be mutant, test strain is to the Streptomycin sulphate resistance.
Fig. 5 provides the structure of test kit embodiment of the detection method of described mycobacterium tuberculosis Streptomycin sulphate medicament-resistant mutation and forms, and is provided with box body 1, amplifing reagent (comprising ST RPCR Mix A2, STR PCR Mix B3 and TB enzyme mixed solution 4), TB DNA extraction liquid 5, TB negative control 6 and STR positive control 7; Described amplifing reagent (comprising STR PCR Mix A2, STR PCRMix B3 and TB enzyme mixed solution 4), TB DNA extraction liquid 5, TB negative control 6 and STR positive control 7 are placed in the box body 1.
Described STR PCR Mix A comprises 1 * PCR Buffer (10mM Tris-HCl, 50mM KCl, 50% (v/v) glycerine), dATP, dCTP, dGTP each 200 μ M, dUTP 400 μ M, MgCl
21mM, each 0.6 μ M of primer rpsL43R and rpsL88R, each 0.06 μ M of primer rpsL43F and rpsL88F, each 0.06 μ M of probe rpsL43P and rpsL88P.
Described STR PCR Mix B comprises 1 * PCR Buffer (10mM Tris, 50mM KCl, 50% (v/v) glycerine), dATP, dCTP, dGTP each 200 μ M, dUTP 400 μ M, MgCl
23mM, each 0.6 μ M of primer rrs512R and rrs904R, each 0.06 μ M of primer rrs512F and rrs904F, each 0.06 μ M of probe rrs512P and rrs904P.
Described TB enzyme mixed solution comprises 2U Taq, 0.01U UNG.
Described STR positive control is a STR wild-type standard plasmid.
Described TB negative control can be TB DNA extraction liquid, also can be 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 Streptomycin sulphate medicament-resistant mutation 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 LSTR PCR Mix A and n * 0.4 μ L 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 STR PCR Mix B and n * 0.4 μ L 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 10s, 70 ℃ of 20s (each circulation reduces by 1 ℃), 75 ℃ of 20s, 10 circulations;
The 3rd step: 95 ℃ of 10s, 60 ℃ of 20s, 75 ℃ of 20s, 30 circulations, fluorescent signal is collected at 60 ℃ of 20s places;
The 4th step: 95 ℃ of 2min, 40 ℃ of 2min;
The 5th step: 40～80 ℃, 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 57.0 ℃ ± 1 ℃ among the reaction system A; The Tm value at TET passage wild-type contrast peak is 65.0 ℃ ± 1 ℃.
The Tm value at FAM passage wild-type contrast peak is 63.5 ℃ ± 1 ℃ among the reaction system B; The Tm value at TET passage wild-type contrast peak is 60.0 ℃ ± 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 Streptomycin sulphate sensitivity; The fusing point of sample is lower than 2 ℃ of positive controls and (Δ T when above
m〉=2 ℃) be judged to be mutant, test strain is to the Streptomycin sulphate 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 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 Streptomycin sulphate 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 (7)
1. the detection method of a mycobacterium tuberculosis Streptomycin sulphate medicament-resistant mutation is characterized in that may further comprise the steps:
1), utilizes primer-design software Primer Premier 5 design primer and probes according to mycobacterium tuberculosis complete sequence and mycobacterium tuberculosis genome rpsL and rrs gene order;
2) DNA of extraction mycobacterium tuberculosis sample;
3) make up the PCR reaction system;
4) carrying out pcr amplification and melting curve analysis: with step 2) DNA that extracts adds in the PCR reaction system of step 3), if negative control, blank and positive control, carry out pcr amplification and melting curve analysis, with the T of sample to be tested reaction tubes gained melting curve and positive control reaction tubes gained melting curve
mValue compares, and judge in the sample to be tested on the rpsL and rrs gene the resistance relevant mutational site and whether undergo mutation, thus judgement sample resistance situation.
2. the detection method of a kind of mycobacterium tuberculosis Streptomycin sulphate medicament-resistant mutation as claimed in claim 1 is characterized in that in step 1), and the concrete grammar of described design primer and probe 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, and quenching group is selected BHQ, Dabcyl.
3. the detection method of a kind of mycobacterium tuberculosis Streptomycin sulphate medicament-resistant mutation as claimed in claim 1 is characterized in that in step 1), and the concrete sequence of described primer and probe is:
Primer rpsL43F:5 '-CAGCCCGCAGCGTCGTGGTGT-3 '
Primer rpsL43R:5 '-GTGGCCCTCGCCGGGAA-3 '
Primer rpsL88F:5 '-GCACTCGATGGTGCTGGTGC-3 '
Primer rpsL88R:5 '-CGTGCCTGTTTGCGGTTCTT-3 '
Primer rrs512F:5 '-ACCTCTTTCACCATCGACGAAGGTCC-3 '
Primer rrs512R:5 '-GTTAAGCCGTGAGATTTCACGAACAA-3 '
Primer rrs904F:5 '-GGGTTTCCTTCCTTGGGATCCGTG-3 '
Primer rrs904R:5 '-GCATGTCAAACCCAGGTAAGGTTC-3 '
Probe rpsL43P:5 '-FAM-CCGCGCCACTCCGAAGAAGCCGAACTCCGCGG-Dabcyl-3 '
Probe rpsL88P:5 '-TET-CCGTCCGGGTGAAGGACCTGCCTGACGG-Dabcyl-3 '
Probe rrs512P:5 '-FAM-CGCACCCAGCAGCCGCGGTAATACGTGCG-Dabcyl-3 '
Probe rrs904P:5 '-TET-CCGCGGGCTAAAACTCAAAGGAATTGACGGCGCGG-Dabcyl-3 '.
4. the detection method of a kind of mycobacterium tuberculosis Streptomycin sulphate medicament-resistant mutation as claimed in claim 1, it is characterized in that in step 2) in, described PCT reaction system is: a kind of is the rpsL system, be used for detecting rpsL gene-correlation medicament-resistant mutation, another kind is the rrs system, be used for detecting rrs gene-correlation medicament-resistant mutation, reaction solution is 20 μ l for every part, contains following component in every part of reaction solution:
RpsL system: 1 * PCR Buffer[10mmol/L Tris, 50mmol/L KCl, 50% (v/v) glycerine], 200 μ mol/L dATP, 200 μ mol/L dCTP, 200 μ mol/L dGTP, 400 μ mol/L dUTP, 1mmol/L MgCl
2, each 0.6 μ mol/L of primer rpsL43R and primer rpsL88R, each 0.06 μ mol/L of primer rpsL43F, primer rpsL88F, probe rpsL43P and probe rpsL88P, Taq archaeal dna polymerase 2U, UNG enzyme 0.01U;
Rrs system: 1 * PCR Buffer[10mmol/L Tris, 50mmol/L KCl, 50% (v/v) glycerine], 200 μ mol/L dATP, 200 μ mol/L dCTP, 200 μ mol/L dGTP, 400 μ mol/L dUTP, 3mmol/LMgCl
2, each 0.6 μ mol/L of primer rrs512R and primer rrs904R, each 0.06 μ mol/L of primer rrs512F, primer rrs904F, probe rrs512P and probe rrs904P, Taq archaeal dna polymerase 2U, UNG enzyme 0.01U.
5. the detection method of a kind of mycobacterium tuberculosis Streptomycin sulphate medicament-resistant mutation as claimed in claim 1 is characterized in that in step 4), the condition of described pcr amplification is: 50 ℃ of 2min, 95 ℃ of 10min; 95 ℃ of 10s, 70 ℃ of 20s, each circulation reduces by 1 ℃, 75 ℃ 20s and constitutes a touchdown circulation, carries out 10 circulations altogether; 95 ℃ of 10s, 60 ℃ of 20s, 75 ℃ of 20s constitute a circulation, carry out 30 circulations altogether; Be the liquation step afterwards, 95 ℃ of 2min, 40 ℃ of 2min, 40～80 ℃ of per 0.5 ℃ of collection fluorescence.
6. the test kit of the detection method of mycobacterium tuberculosis Streptomycin sulphate medicament-resistant mutation as claimed in claim 1 is characterized in that being provided with box body, amplifing reagent, TB DNA extraction liquid, TB negative control and STR positive control; Described amplifing reagent comprises STR PCR Mix A, STR PCR Mix B and TB enzyme mixed solution, and STR PCR Mix A, STR PCR Mix B, TB enzyme mixed solution, TB DNA extraction liquid, TB negative control and STR positive control are placed in the box body;
Described STR PCR Mix A comprises 1 * PCR Buffer, dATP, dCTP, dGTP each 200 μ M, dUTP 400 μ M, MgCl
21mM, each 0.6 μ M of primer rpsL43R and rpsL88R, each 0.06 μ M of primer rpsL43F and rpsL88F, each 0.06 μ M of probe rpsL43P and rpsL88P; Described Buffer is 10mM Tris-HCl, 50mM KCl, 50% (v/v) glycerine;
Described STR PCR Mix B comprises 1 * PCR Buffer, dATP, dCTP, dGTP each 200 μ M, dUTP 400 μ M, MgCl
23mM, each 0.6 μ M of primer rrs512R and rrs904R, each 0.06 μ M of primer rrs512F and rrs904F, each 0.06 μ M of probe rrs512P and rrs904P, described Buffer is 10mM Tris, 50mM KCl, 50% (v/v) glycerine;
Described TB enzyme mixed solution comprises 2U Taq, 0.01U UNG;
Described STR positive control is a STR 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.
7. the method for the test kit inspection specimen of the detection method of mycobacterium tuberculosis Streptomycin sulphate medicament-resistant mutation as claimed in claim 6 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 LSTR PCR Mix A and n * 0.4 μ L 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 STR PCR Mix B and n * 0.4 μ L 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 10s, 70 ℃ of 20s, each circulation reduces by 1 ℃, 75 ℃ of 20s, 10 circulations;
The 3rd step: 95 ℃ of 10s, 60 ℃ of 20s, 75 ℃ of 20s, 30 circulations, fluorescent signal is collected at 60 ℃ of 20s places;
The 4th step: 95 ℃ of 2min, 40 ℃ of 2min;
The 5th step: 40～80 ℃, 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 57.0 ℃ ± 1 ℃ among the reaction system A; The Tm value at TET passage wild-type contrast peak is 65.0 ℃ ± 1 ℃;
The Tm value at FAM passage wild-type contrast peak is 63.5 ℃ ± 1 ℃ among the reaction system B; The Tm value at TET passage wild-type contrast peak is 60.0 ℃ ± 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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| CN103993065A (en) * | 2014-05-07 | 2014-08-20 | 济宁医学院 | Diphasic quick differential medium of mycobacterium tuberculosis and application of medium |
| CN106367528A (en) * | 2016-08-26 | 2017-02-01 | 厦门大学 | Detection method of hepatitis B virus drug-resistant mutation |
| CN106480183A (en) * | 2016-09-29 | 2017-03-08 | 凯杰（苏州）转化医学研究有限公司 | Pyrosequencing detection mycobacterium tuberculosis kanamycin, amikacin, the method for capreomycin drug resistance |
| CN110438205A (en) * | 2019-07-31 | 2019-11-12 | 天津市泌尿外科研究所 | Joint multiplex PCR nest-type PRC and touchdown PCR are used for the kit of pathogenic mycobacterium tuberculosis detection |
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| CN114032319A (en) * | 2021-11-30 | 2022-02-11 | 广州达安基因股份有限公司 | Detection method and detection kit for streptomycin drug resistance mutation of mycobacterium tuberculosis |
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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 |
| CN106480183A (en) * | 2016-09-29 | 2017-03-08 | 凯杰（苏州）转化医学研究有限公司 | Pyrosequencing detection mycobacterium tuberculosis kanamycin, amikacin, the method for capreomycin drug resistance |
| CN110438205A (en) * | 2019-07-31 | 2019-11-12 | 天津市泌尿外科研究所 | Joint multiplex PCR nest-type PRC and touchdown PCR are used for the kit of pathogenic mycobacterium tuberculosis detection |
| CN110643722A (en) * | 2019-09-10 | 2020-01-03 | 中国医学科学院病原生物学研究所 | Neisseria gonorrhoeae drug-resistant site multiple detection method and kit |
| CN113388690A (en) * | 2021-07-02 | 2021-09-14 | 海南医学院 | Primer, probe and kit for detecting mycobacterium tuberculosis and gene mutation sites related to drug resistance of therapeutic drugs |
| CN114032319A (en) * | 2021-11-30 | 2022-02-11 | 广州达安基因股份有限公司 | Detection method and detection kit for streptomycin drug resistance mutation of mycobacterium tuberculosis |
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Application publication date: 20111102 |