CN102229990A - Method and kit for detecting quinolone resistance mutation of Mycobacterium tuberculosis - Google Patents
Method and kit for detecting quinolone resistance mutation of Mycobacterium tuberculosis Download PDFInfo
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- CN102229990A CN102229990A CN2011101378324A CN201110137832A CN102229990A CN 102229990 A CN102229990 A CN 102229990A CN 2011101378324 A CN2011101378324 A CN 2011101378324A CN 201110137832 A CN201110137832 A CN 201110137832A CN 102229990 A CN102229990 A CN 102229990A
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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 quinolone resistance mutation of Mycobacterium tuberculosis, which are rapid, sensitive and specific. The method comprises the following steps of: extracting DNA of a Mycobacterium tuberculosis sample; designing primers and probes according to the quinolone resistance-determining region, constructing a single-tube single-color real-time PCR (polymerase chain reaction) system, performing PCR to detect the drug resistance mutation of Mycobacterium tuberculosis, comparing the difference in melting point of melting curve between the detected sample and a positive control, and judging whether the mutation occurs in the sample. When the melting point of the sample is equal to that of the positive control, the detected strain is of wild type and sensitive to quinolone; and when the melting point of the sample is 2 DEG C less than that of the positive control, the detected strain is of mutation type and resistant to quinolone. The sensitivity and specificity of detecting drug resistance mutation of Mycobacterium tuberculosis are improved and the time required for detection is shortened.
Description
Technical field
The present invention relates to the detection technique of mycobacterium tuberculosis 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 fluoroquinolone medicament-resistant mutation.
Background technology
Since the eighties in 20th century, the tuberculosis epidemic situation rises again, becomes the global harm the most serious transmissible disease arranged side by side with acquired immune deficiency syndrome (AIDS).According to World Health Organization's statistics, the population in the whole world 1/3rd infects mycobacterium tuberculosis at present, annual newly-increased 8,000,000 patients, and dead 200～3,000,000 people are the highest communicable diseases of present mortality ratio.According to the assessment of the World Health Organization, China is that 22 tuberculosis height are born one of country in the world, and patient's number is only second to India, occupies second in the whole world.In the conference of hold this year second whole world containment tuberculosis partner forum, China is listed in first of the countries and regions that need cause caution especially.
Quinolones is the artificial chemosynthesis antimicrobial drug of a class, because such medicine all has the basic structure of 4-quinolone parent nucleus, so name thus.Fluorine promise quinoline ketone is introduced fluorine on the 66 of quinolone parent nucleus, the 7th derivative that go up to introduce piperazinyl or pyrrolinyl, be the 3rd generation the quinolone product.The fluoroquinolones that has been used for clinical anti-mycobacterium has Ciprofloxacin (Ciprofloxacin, CIP), and Ofloxacine USP 23 (Ofloxacin, OFL), levofloxacin (Levofloxacin, LEV), and sparfloxacin (Sparfloxacin, SPX), Gatifloxacin (Gatifloxacin, GAT), and Moxifloxacin (Moxifloxacin, MOX) etc.Along with increasing that carbostyril family antibacterial drugs uses in mycobacterium tuberculosis (MTB) infects, mycobacterium tuberculosis rises gradually to the resistant rate of such antibacterials.Therefore, the detection to the fluoroquinolone medicament-resistant mutation necessitates.
At present, the Drug Resistance Detection method of Cai Yonging can be divided into phenotype detection and gene test two classes clinically, and based on the phenotype detection.The biological TRAP of conventional drug sensitive test method during phenotype detects, BACTEC TB-460 liquid nutrient medium drug sensitive test method and phage has been applied to clinical, yet these methods all have tangible limitation.Conventional drug sensitive test method is slowly influenced by growth of bacillus tubercle, needs 6～8 weeks even longer time just can obtain the result.Though BACTEC TB-460 liquid nutrient medium drug sensitive test method is sensitive, speed is very fast, needs expensive device, and easily is bacterial contamination, and radiological hazard is arranged.The biological amplification of phage rule is subjected to interfering factors many, and complicated operation.Because present phenotype detects the detection means that is difficult to form efficiently and accurately, usually cause the state of an illness to incur loss through delay, more be difficult to realize following the trail of treatment.Existing genotype detection method has dna sequencing method (1, Dauendorffer, JN, Guillemin I, Aubry A et al, Identification of mycobacterial species by PCR sequencing of quinolone resistance-determining regions of DNA gyrAse genes.J Clin Microbiol.2003,41:1311-1315.), linear hybrid method (2, Giannoni F, Iona E, Sementilli F et al.Evaluation of a new line probe assay for rapid identification of gyrA mutations in Mycobacterium tuberculosis.Antimicrob Agents Chemother.2005,49:2928-2933.), PCR-SSCP method (3, Cheng AF, Yew WW, Chan EW et al.Multiplex PCR amplimer conformation analysis for rapid detection of gyrA mutations in fluoroquinolone-resistant Mycobacterium tuberculosis clinical isolates.Antimicrob Agents Chemother.2004,48:596-601), DHPLC method (4, Shi R, Otomo K, Yamada H et al.Temperature-mediated heteroduplex analysis for the detection of drug-resistant gene mutations in clinical isolates of Mycobacterium tuberculosis by denaturing HPLC, SURVEYOR nuclease.Microbes Infect.2006,8:128-135), biochip method (5, Antonova OV, Gryadunov DA, Lapa SA et al.Detection of mutations in Mycobacterium tuberculosis genome determining resistance to fluoroquinolones by hybridization on biological microchips.Bull Exp Biol Med.2008,145:108-113), real-time PCR method (6, van Doorn H R, An DD, de Jong MD et al.Fluoroquinolone resistance detection in Mycobacterium tuberculosis with locked nucleic acid probe real-time PCR.Int.J.Tuberc.Lung Dis.2008,12:736-742) etc., the existing GenoType MTBDRsl test kit that can detect the gyrA transgenation of German HAIN company.
Summary of the invention
One of purpose of the present invention be to provide a kind of fast, the detection method of sensitive, special mycobacterium tuberculosis fluoroquinolone medicament-resistant mutation, 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 fast, the test kit of the detection method of sensitive, special mycobacterium tuberculosis fluoroquinolone medicament-resistant mutation.
The detection method of described mycobacterium tuberculosis fluoroquinolone medicament-resistant mutation may further comprise the steps:
1) DNA of extraction mycobacterium tuberculosis sample;
2), utilize primer-design software Primer 5 design primer and probes according to mycobacterium tuberculosis fluoroquinolone resistance determining area (QRDR);
In step 2) in, 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 any one among FAM, ROX, HEX, CY5, TET, the CAL-Fluor etc., and quenching group can be selected BHQ or Dabcyl etc., and probe can carry out various types of modifications (as the LNA mark) etc.
The concrete sequence of described primer and probe is:
Primer-F：5’-CCAAGTCGGCCCGGTCGGTTG-3’
Primer-R：5’-GACCAGGGCTGGGCCATG-3’
Probe-A：5′-FAM-GGCGACGCGTCGATCTACGACCGCC-BHQ-3′
3) make up the monochromatic PCR in real time system of single tube, the PCR that carries out mycobacterium tuberculosis fluoroquinolone medicament-resistant mutation detects, relatively fusing point (the T of melting curve between institute's test sample and the positive control
mValue) difference, whether judgement sample undergos mutation, and is 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 ℃), and test strain is to the fluoroquinolone sensitivity; The fusing point of sample is judged to be mutant when being lower than at least 2 ℃ of positive controls, and test strain is to the fluoroquinolone resistance.
In step 3), the monochromatic PCR in real time system of described single tube can be:
Contain 1 * PCR buffer (10mM Tris-HCl, pH8.6,50mM KCl, 5% glycerine), 2.0mM MgCl
2, dATP, dCTP, each 0.2mM of dGTP, dUTP 0.4mM, 2U Taq, 0.01U UNG, 0.8 μ M Primer-R, 0.08 μ M Primer-F, 0.08 μ M Probe-A.
The stager also can select the buffer of other type, perhaps finds more suitable buffer type by optimization.
Get mycobacterium tuberculosis sample 5 μ l DNA as template, and to get concentration be 10
4The positive plasmid of copies/ μ l is as positive control, and distilled water is as negative control;
The response procedures of described PCR is:
The first step: 50 ℃ of 2min, 95 ℃ of 10min;
Second step: 95 ℃ of 10s, 65 ℃ of 20s (each circulation reduces by 1 ℃), 78 ℃ of 25s, 10 circulations;
The 3rd step: 95 ℃ of 10s, 55 ℃ of 20s, 78 ℃ of 25s, 40 circulations, fluorescent signal is collected at 65 ℃ of 20s places;
The 4th step: 95 ℃ of 2min;
The 5th step: 40 ℃ of 2min;
The 6th step: 45～85 ℃, collect fluorescent signal, select the FAM fluorescence channel for use for per 1 ℃.
The present invention has detected 279 clinical samples, comprises 270 parts of sensitive strains and 9 parts of Resistant strain.About heterozygosis result's interpretation, divide 3 kinds of situations: 1. occur being the heterozygosis sample when bimodal when sample; 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 fluoroquinolone resistance, when the heterozygosis sample appears in suggestion with the sample duplicate detection, to determine the sample resistance.
The test kit of the detection method of described mycobacterium tuberculosis fluoroquinolone medicament-resistant mutation is provided with box body, amplifing reagent, TB enzyme mixed solution, FQ positive control, TB negative control and TB DNA extraction liquid; Amplifing reagent, TB enzyme mixed solution, FQ positive control, TB negative control and TB DNA extraction liquid are placed in the box body;
Described amplifing reagent is FQ PCR Mix, and its composition is 1 * PCR buffer (10mM Tris-HCl, pH8.6,50mM KCl, 5% glycerine), 2.0mM MgCl
2, dATP, dCTP, each 0.2mM of dGTP, dUTP 0.4mM, 0.8 μ M Primer-R, 0.08 μ M Primer-F, 0.08 μ M Probe-A.
Described TB enzyme mixed solution comprises 2U Taq, 0.01U UNG.
Described FQ positive control is a FQ wild-type standard plasmid, and the TB negative control can be selected from 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.
Adopt the test kit of the detection method of described mycobacterium tuberculosis fluoroquinolone medicament-resistant mutation that the method for inspection of sample be 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 LFQ PCR Mix and n * 0.4 μ L TB enzyme mixed solution a and join in the 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 is sub-packed in PCR thin-walled reaction tubes with the PCR reaction solution with every pipe 20 μ L.
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, 65 ℃ of 20s (each circulation reduces by 1 ℃), 78 ℃ of 25s, 10 circulations;
The 3rd step: 95 ℃ of 10s, 55 ℃ of 20s, 78 ℃ of 25s, 40 circulations;
The 4th step: 95 ℃ of 2min;
The 5th step: 40 ℃ of 2min;
The 6th step: 45～85 ℃, collect fluorescent signal, select the FAM fluorescence channel for use for per 1 ℃.
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
mThe value scope: the Tm value at FAM passage wild-type contrast peak is 71 ℃ ± 1 ℃ in the reaction system.
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.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 fluoroquinolone 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 fluoroquinolone resistance.
Interpretation about the heterozygosis sample result: when melting curve occurs bimodal or merge the peak, be the heterozygosis sample, divide 3 kinds of situations: 1. occur being the heterozygosis sample when bimodal when sample; 2. sample is one and merges the peak, and is consistent with the fusing point of positive control, but the peak type has than big-difference with the peak type of positive control, small peak or projection occur in the position at the peak that might occur suddenling change, and is the heterozygosis sample; 3. sample is a fusion peak, is the sudden change fusing point, but small peak or projection occurs in the position at wild peak, is the heterozygosis sample.The heterozygosis sample is to the fluoroquinolone 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.
Compare with existing mycobacterium tuberculosis fluoroquinolone Drug Resistance Detection method, the present invention has following outstanding advantage:
1) primer is to design at the two ends of gyrA gene fluoroquinolone resistance determining area, has the specificity of height.
2) design of probe has taken into full account the emphasis detection in main mutational site and effective covering of other mutation type, thereby has reached the purpose that improves the sudden change recall rate.In addition, optional fluorophor and the quenching group of selecting broad variety of probe, and can carry out various modifications.
3) divide wild-type and mutation type by analysis area to fusing point.Complete and the probe sequence coupling of wild-type, so fusing point is higher; Mutant can not be fully and the probe sequence coupling, so fusing point is low than wild-type, and the sudden change that single base causes can cause the decline of about 2～6 ℃ of fusing points.
The present invention has improved sensitivity, specificity that mycobacterium tuberculosis fluoroquinolone medicament-resistant mutation detects greatly, has shortened the needed time of detecting greatly.
Description of drawings
Fig. 1 is the typical consequence figure of probe in detecting mycobacterium tuberculosis single base mutation.In Fig. 1, X-coordinate is a temperature of reaction, ordinate zou be fluorescence intensity and temperature negative derivative (dF/ dT).Curve 1,2,3,4,5 expressions respectively: sudden change 94GAC＞GCC, sudden change 94GAC＞GGC, sudden change 90GCG＞GTG, wild-type, negative control.
Fig. 2 is the test kit example structure synoptic diagram of the detection method of mycobacterium tuberculosis fluoroquinolone medicament-resistant mutation of the present invention.
Embodiment
Following examples will the present invention will be further described in conjunction with the accompanying drawings:
The plant and instrument that adopts is referring to table 1
Table 1
| Instrument title (specification) | Production firm |
| Quantitative real time PCR Instrument (CFX96) | U.S. Bio-Rad company |
| Small desk supercentrifuge (centrifuge 5415D) | Germany Eppendorf company |
| Regular-PCR instrument (T3) | Germany Biometra company |
| Ultramicrospectrophotometer (Nanodrop ND-1000) | U.S. Nanodrop company |
The extraction of embodiment 1 mycobacterium tuberculosis DNA
1) strain culturing
Mycobacterium tuberculosis adopts acid Russell medium to cultivate.
2) DNA extraction
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.
Embodiment 2 mycobacterium tuberculosis detect the design of primer and probe
The design of primer and probe:
According to mycobacterium tuberculosis fluoroquinolone drug resistance related gene gyrA sequence, softwares such as application Primer Premier 5, Tm Utilityv1.3 and mfold are assisted and are finished.Primer and probe sequence are as follows:
Primer-F：5’-CCAAGTCGGCCCGGTCGGTTG-3’
Primer-R：5’-GACCAGGGCTGGGCCATG-3’
Probe-A：5′-FAM-GGCGACGCGTCGATCTACGACCGCC-BHQ-3′
Primer and probe are synthetic by Shanghai Sangon Biological Engineering Technology And Service Co., Ltd.Keep in Dark Place behind the probe quantitative.
Carrying out the monochromatic fluoroquinolone medicament-resistant mutation of single tube with single reaction system detects:
FQ PCR MIX is 19.6 μ l for every part, contains 1 * PCR buffer (10mM Tris-HCl, pH8.6,50mMKCl, 5% glycerine) in every part of reaction solution, 2.0mM MgCl
2, dATP, dCTP, each 0.2mM of dGTP, dUTP 0.4mM, 0.8 μ M Primer-R, 0.08 μ M Primer-F, 0.08 μ M Probe-A.Add 2U enzyme mixed solution (0.4 μ l) before detecting, template add-on to be measured is 5 μ l.
Getting concentration is 10
4The FQ wild-type standard plasmid of copies/ μ l 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, 65 ℃ of 20s (each circulation reduces by 1 ℃), 78 ℃ of 25s, 10 circulations;
The 3rd step: 95 ℃ of 10s, 55 ℃ of 20s, 78 ℃ of 25s, 40 circulations, fluorescent signal is collected at 65 ℃ of 20s places;
The 4th step: 95 ℃ of 2min;
The 5th step: 40 ℃ of 2min;
The 6th step: 45～85 ℃, collect fluorescent signal, select the FAM fluorescence channel for use for per 1 ℃.
1. the somatotype principle of probe
Molecular beacon keeps stable hair clip shape secondary structure under the situation that does not have target sequence to exist, fluorophor and quenching group are comparatively close, and the fluorescence of fluorophor is by cancellation, and detect less than fluorescence this moment.When target sequence exists, probe can combine with complementary base on the target sequence, promptly hybridizes, and fluorophor and quenching group are because the extended position of probe is no longer close, the fluorescence of fluorophor can't be by the quenching group cancellation, and just can detect fluorescent signal this moment.In the testing process of melting curve, along with the rising of temperature, probe and target sequence present by being attached to dissociated process fully, therefore can be by the difference identification probe of fusing point and the matching degree of target sequence.
2. interpretation as a result
FAM passage wild-type contrast peak Tm value is 71 ℃ ± 1 ℃ in the reaction system.
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 fluoroquinolone 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 fluoroquinolone resistance.
3. adopt the test kit (fluorescent PCR melting curve method) of the detection method of mycobacterium tuberculosis fluoroquinolone medicament-resistant mutation to detect 279 strain bacterial strains
Referring to Fig. 2, the test kit embodiment of the detection method of described mycobacterium tuberculosis fluoroquinolone medicament-resistant mutation is provided with box body 1, amplifing reagent 2, TB enzyme mixed solution 3, FQ positive control 4, TB negative control 5 and TB DNA extraction liquid 6; Amplifing reagent 2, TB enzyme mixed solution 3, FQ positive control 4, TB negative control 5 and TB DNA extraction liquid 6 are placed in the box body;
Described amplifing reagent 2 is FQ PCR Mix, and its composition is 1 * PCR buffer (10mM Tris-HCl, pH8.6,50mMKCl, 5% glycerine), 2.0mM MgCl
2, dATP, dCTP, each 0.2mM of dGTP, dUTP 0.4mM, 0.8 μ M Primer-R, 0.08 μ M Primer-F, 0.08 μ M Probe-A.
Described TB enzyme mixed solution 3 comprises 2U Taq, 0.01U UNG.
Described FQ positive control 4 is a FQ wild-type standard plasmid, and TB negative control 5 can be selected from TB DNA extraction liquid, H
2O, Tris or physiological saline etc.
The composition of described TB DNA extraction liquid 6 is disodium ethylene diamine tetraacetate, Tris and TritonX-100.
Utilize mycobacterium tuberculosis fluoroquinolone medicament-resistant mutation detection kit (fluorescent PCR melting curve method) to detect 279 strain bacterial strains, the result shows, wherein 270 parts is the fluoroquinolone sensitive strain, 9 parts is the fluoroquinolone Resistant strain, compare with clinical drug sensitivity tests, total rate of accuracy reached 99.28% of this test kit, sensitivity 77.78%, specificity 100%.
Adopt the test kit of the detection method of described mycobacterium tuberculosis fluoroquinolone medicament-resistant mutation that the method for inspection of sample be 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 LFQ PCR Mix and n * 0.4 μ L TB enzyme mixed solution a and join in the 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 is sub-packed in PCR thin-walled reaction tubes with the PCR reaction solution with every pipe 20 μ L.
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, 65 ℃ of 20s (each circulation reduces by 1 ℃), 78 ℃ of 25s, 10 circulations;
The 3rd step: 95 ℃ of 10s, 55 ℃ of 20s, 78 ℃ of 25s, 40 circulations;
The 4th step: 95 ℃ of 2min;
The 5th step: 40 ℃ of 2min;
The 6th step: 45～85 ℃, collect fluorescent signal, select the FAM fluorescence channel for use for per 1 ℃.
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
mThe value scope: the Tm value at FAM passage wild-type contrast peak is 71 ℃ ± 1 ℃ in the reaction system.
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.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 fluoroquinolone 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 fluoroquinolone resistance.
Interpretation about the heterozygosis sample result: when melting curve occurs bimodal or merge the peak, be the heterozygosis sample, divide 3 kinds of situations: 1. occur being the heterozygosis sample when bimodal when sample; 2. sample is one and merges the peak, and is consistent with the fusing point of positive control, but the peak type has than big-difference with the peak type of positive control, small peak or projection occur in the position at the peak that might occur suddenling change, and is the heterozygosis sample; 3. sample is a fusion peak, is the sudden change fusing point, but small peak or projection occurs in the position at wild peak, is the heterozygosis sample.The heterozygosis sample is to the fluoroquinolone 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.
By the The above results analysis as seen, this test kit is according to mycobacterium tuberculosis fluoroquinolone drug resistance related gene gyrA sequence, this fragment of design primer amplification, and design molecular beacon probe covering fluoroquinolone resistance determining area, realized the detection that this zone is suddenlyd change by the monochromatic real-time fluorescence PCR 7 melting curve methods of single tube.
Claims (7)
1. the detection method of a mycobacterium tuberculosis fluoroquinolone medicament-resistant mutation is characterized in that may further comprise the steps:
1) DNA of extraction mycobacterium tuberculosis sample;
2), utilize primer-design software Primer 5 design primer and probes according to mycobacterium tuberculosis fluoroquinolone resistance determining area;
3) make up the monochromatic PCR in real time system of single tube, the PCR that carries out mycobacterium tuberculosis fluoroquinolone medicament-resistant mutation detects, and compares the difference of the fusing point of melting curve between institute's test sample and the positive control, and whether judgement sample undergos mutation.The fusing point of sample is judged to be wild-type when consistent with the fusing point of positive control, and test strain is to the fluoroquinolone sensitivity; The fusing point of sample is lower than positive control and is judged to be mutant more than 2 ℃ or 2 ℃ the time, and test strain is to the fluoroquinolone resistance.
2. the detection method of a kind of mycobacterium tuberculosis fluoroquinolone medicament-resistant mutation as claimed in claim 1, it is characterized in that in step 2) in, the concrete grammar of described design primer and probe is as follows: a, and 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 any one, quenching group can be selected BHQ, Dabcyl.
3. the detection method of a kind of mycobacterium tuberculosis fluoroquinolone medicament-resistant mutation as claimed in claim 1 is characterized in that in step 2) in, the concrete sequence of described primer and probe is:
Primer-F：5’-CCAAGTCGGCCCGGTCGGTTG-3’
Primer-R：5’-GACCAGGGCTGGGCCATG-3’
Probe-A：5′-FAM-GGCGACGCGTCGATCTACGACCGCC-BHQ-3′。
4. the detection method of a kind of mycobacterium tuberculosis fluoroquinolone medicament-resistant mutation as claimed in claim 1 is characterized in that in step 3), and the monochromatic PCR in real time system of described single tube is:
Contain 1 * PCR buffer, 2.0mM MgCl
2, dATP, dCTP, dGTP 0.2mM, dUTP 0.4mM, 2U Taq, 0.01UUNG, 0.8 μ M of Primer-R, 0.08 μ M of Primer-F, 0.08 μ M of Probe-A;
Get mycobacterium tuberculosis sample 5 μ l DNA as template, and to get concentration be 10
4The positive plasmid of copies/ μ l is as positive control, and distilled water is as negative control.
5. the detection method of a kind of mycobacterium tuberculosis fluoroquinolone medicament-resistant mutation as claimed in claim 1 is characterized in that in step 3), and the response procedures of described PCR is:
The first step: 50 ℃ of 2min, 95 ℃ of 10min;
Second step: 95 ℃ of 10s, 65 ℃ of 20s (each circulation reduces by 1 ℃), 78 ℃ of 25s, 10 circulations;
The 3rd step: 95 ℃ of 10s, 55 ℃ of 20s, 78 ℃ of 25s, 40 circulations, fluorescent signal is collected at 65 ℃ of 20s places;
The 4th step: 95 ℃ of 30s;
The 5th step: 40 ℃ of 2min;
The 6th step: 40～85 ℃, collect fluorescent signal, select the FAM fluorescence channel for use for per 1 ℃.
6. the test kit of the detection method of mycobacterium tuberculosis fluoroquinolone medicament-resistant mutation according to claim 1 is characterized in that being provided with box body, amplifing reagent, TB enzyme mixed solution, FQ positive control, TB negative control and TB DNA extraction liquid; Amplifing reagent, TB enzyme mixed solution, FQ positive control, TB negative control and TB DNA extraction liquid are placed in the box body;
Described amplifing reagent is FQ PCR Mix, and its composition is 1 * PCR buffer (10mM Tris-HCl, pH8.6,50mM KCl, 5% glycerine), 2.0mM MgCl
2, dATP, dCTP, each 0.2mM of dGTP, dUTP 0.4mM, 0.8 μ M Primer-R, 0.08 μ M Primer-F, 0.08 μ M Probe-A;
Described TB enzyme mixed solution comprises 2U Taq, 0.01U UNG;
Described FQ positive control is a FQ wild-type standard plasmid, and the TB negative control is selected from 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. as the test kit of the detection method of mycobacterium tuberculosis fluoroquinolone medicament-resistant mutation as described in the claim 6 the method for inspection, it is characterized in that may further comprise the steps to sample:
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 LFQ PCR Mix and n * 0.4 μ L TB enzyme mixed solution a and join in the 1.5mL centrifuge tube, 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 is sub-packed in PCR thin-walled reaction tubes with the PCR reaction solution with every pipe 20 μ L;
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, 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, 65 ℃ of 20s, each circulation reduces by 1 ℃, 78 ℃ of 25s, 10 circulations;
The 3rd step: 95 ℃ of 10s, 55 ℃ of 20s, 78 ℃ of 25s, 40 circulations;
The 4th step: 95 ℃ of 2min;
The 5th step: 40 ℃ of 2min;
The 6th step: 45～85 ℃, collect fluorescent signal, select the FAM fluorescence channel for use for per 1 ℃;
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
Positive control, the i.e. T of wild-type
mThe value scope: the Tm value at FAM passage wild-type contrast peak is 71 ℃ ± 1 ℃ in the reaction system;
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, select effective T with reference to the peak type of positive control and negative control
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;
(4) interpretation as a result
By comparing the fusing point T of melting curve between institute's test sample and the positive control
mWhether the difference judgement sample of value undergos mutation, and the fusing point of sample is consistent with the fusing point of positive control, is judged to be wild-type when promptly error is no more than 1 ℃, and test strain is to the fluoroquinolone sensitivity; When the fusing point of sample is lower than at least 2 ℃ of positive controls, i.e. Δ T
m〉=2 ℃, be judged to be mutant, test strain is to the fluoroquinolone resistance.
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