CN104593505A - Kit for detecting activity degree of telomerase in whole blood or serum sample - Google Patents
Kit for detecting activity degree of telomerase in whole blood or serum sample Download PDFInfo
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Abstract
The invention provides a kit for detecting activity degree of telomerase in a whole blood or serum sample, belonging to the field of molecular biology detection. The kit comprises the following primers: (1) primer sequences of telomerase hTERT genes: hTERT-F: 5'-GGCCGATTGTGAACATGGA-3'; hTERT-R: 5'-CCTCTTTTCTCTGCGGAACGT-3'; and (2) probe sequences of telomerase hTERT genes: hTERT-P: 5'-TACGTCGTGGGAGCCA-3', the 5' end is connected with fluorescence reporter genes, and the 3' end is connected with fluorescence quenching genes. The kit for detecting activity degree of telomerase in the whole blood or serum sample has the advantages of high detection sensitivity, short analysis time and high stability.
Description
Technical field
The invention belongs to technical field of molecular biology, be specifically related to a kind of test kit detecting the level of activity of whole blood or serum sample telomerase.
Background technology
Telomerase is a kind of ribonucleoprotein complex be made up of RNA and protein, has the activity of reversed transcriptive enzyme.Its component comprises telomerase RNA template (human telomerase RNA, hTR), telomerase associated proteins 1 (humantelomerase associated protein1, and reverse transcriptase of telomere (human telomerase reversetranscriptase, hTERT) three parts hTEP1).Research shows, hTERT is the core of Telomerase, is only expressed in telomerase-positive cells, and is the rate-limiting factor of Telomerase, and its mrna expression level directly determines the level of activity of Telomerase.
The general no telomerase activity of mankind's normal somatic cell, and the malignant tumour of 85% ~ 90% can detect telomerase activation.Human cell is early stage in fetal development, and Telomerase is active state, suppressed subsequently and be in inactivated state always, thus the usual no telomerase activity of somatocyte or activity very low.The Telomerase reactivation when somatocyte generation canceration, to escape normal cellular senescence process, obtains the potential of infinite multiplication.Thus, Telomerase reactivate is malignant tumour significant biological property.The level of activity of Telomerase can be determined by the expression level detecting hTERT mRNA, thus diagnose person to be checked whether to suffer from malignant tumour.
Quantitative fluorescent PCR is a real-time detection passing through to each circulation products fluorescent signal in pcr amplification reaction, thus realizes the detection technique of starting template being carried out to quantitative analysis.
But this detection method also imperfection, exists the shortcoming that detection sensitivity is low in prior art, often cause a part of patient to fail to pinpoint a disease in diagnosis, or occur " false positive ", make troubles to patient.Therefore, need to set up that a kind of susceptibility is high, specificity good, widely applicable detection method.
Summary of the invention
The technical issues that need to address of the present invention are, provide a kind of test kit detecting the level of activity of whole blood or serum sample telomerase, accurately can detect the level of activity of testing sample telomerase, thus overcome the deficiencies in the prior art.
For solving the problem, the present invention adopts following technical scheme:
Detect the primer of telomerase hTERT mrna expression level, comprise following primer sequence:
(1) primer sequence of telomerase hTERT gene:
SEQ ID NO:1(hTERT-F)：5’-GGCCGATTGTGAACATGGA-3’；
SEQ ID NO:2(hTERT-R)：5’-CCTCTTTTCTCTGCGGAACGT-3’；
(2) probe sequence of telomerase hTERT gene:
SEQ ID NO:3 (hTERT-P): 5 '-TACGTCGTGGGAGCCA-3 ', and its 5 ' end is connected with fluorescent reporter gene, 3 ' end is connected with fluorescent quenching gene.
Wherein, the probe sequence 5 ' of telomerase hTERT gene holds the fluorescent reporter group connected to be FAM, and the fluorescent quenching group that 3 ' end connects is MGB.
The primer of above-mentioned detection telomerase hTERT mrna expression level detects application in the reagent of the level of activity of whole blood or serum sample telomerase also within protection scope of the present invention in preparation.
Detect a test kit for the level of activity of whole blood or serum sample telomerase, this test kit comprises the primer (hTERT-F and hTERT-R) of above-mentioned telomerase hTERT gene and the probe (hTERT-P) of telomerase hTERT gene.
Wherein, this test kit also comprises following reagent: total RNA extraction reagent, Reverse Transcription, quantitative fluorescent PCR reaction reagent, quality control product.
Wherein, described total RNA extraction reagent comprise following reagent:
(1) cell pyrolysis liquid Trizol: volume fraction is the heavily steaming phenol of 56%, the guanidinium isothiocyanate of 28.2g/L, 14.9mmol/L sodium citrate solution, 3g/L sarcosyl, sodium-acetate 112.7mmol/L, and solvent is distilled water;
(2) extraction liquid: chloroform;
(3) precipitation agent: Virahol;
(4) rinsing liquid: volume fraction be 75% ethanol-;
(5) RNA solvent: without the water of RNA enzyme.
Wherein, described Reverse Transcription comprises following component:
(1) reverse transcription reaction damping fluid: 250mmol/L Tris-cl, 375mmol/L KCl, 15mmol/L MgCl
2, 50mmol/L DTT, pH8.3;
(2) dNTP mixture: 10mmol/L dATP, 10mmol/L dTTP, 10mmol/L dCTP, 10mmol/LdGTP;
(3) reversed transcriptive enzyme liquid: 20mmol/L Tris-cl, 200mmol/L NaCl, 0.1mmol/L EDTA, 1.0mmol/L DTT, volume fraction is the glycerine of 50%, 200U/ μ L reversed transcriptive enzyme, 40U/ μ L RNA enzyme inhibitors, pH7.5;
(4) primer mixture: oligodeoxythymidylic acid primer, random 6 nucleotide primers;
(5) ultrapure water: resistance 18.2 megaohm.
Wherein, described quantitative fluorescent PCR reaction reagent comprise following component:
(1) RT-PCR reaction solution: 10mmol/L dATP, 10mmol/L dTTP, 10mmol/L dCTP, 10mmol/L dGTP, 25mmol/L MgCl
2, 5U/ μ L Taq DNA polymerase, 100mmol/L Tris-cl, 500mmol/LKCl, 15mmol/L MgCl
2, pH8.5;
(2) ultrapure water: resistance 18.2 megaohm.
Wherein, described quality control product comprises following component:
(1) positive quality control product: containing the recombinant plasmid pGM-hTERT of telomerase hTERT gene, extraction purification concentration is 250mg/mL, and copy number is 2.6x10
4copies/ μ L;
(2) negative quality control product: ultrapure water.
The test kit of the level of activity of above-mentioned detection whole blood or serum sample telomerase is preparing the application in the telomerase activity degree reagent of detecting end also within protection scope of the present invention.
Beneficial effect:
Test kit of the present invention compared with prior art has following advantage:
1, test kit of the present invention is applicable to fluorescence quantitative PCR method and detects whole blood or serum Telomerase Activity degree.
2, the present invention has highly sensitive, sample telomerase hTERT mrna expression level>=10
0during copies/ μ L, all can detect.
3, the present invention is by production standard curve, realizes the absolute quantitation to sample Telomerase Activity degree.
4, the present invention detects only needs 250 μ L samples, not only saves sample, also has analysis time short, without the need to software analysis after detection, directly can read result.
Accompanying drawing explanation
Fig. 1 typical curve amplification figure.
Fig. 2 canonical plotting.
Fig. 3 sample telomerase hTERT gene amplification figure.
Embodiment
According to following embodiment, the present invention may be better understood.But those skilled in the art will readily understand, the content described by embodiment only for illustration of the present invention, and should can not limit the present invention described in detail in claims yet.
Following examples key instrument agent producer used is as shown in table 1.
Table 1 laboratory apparatus and producer
| Instrument | Producer |
| Quantitative real time PCR Instrument (Lightcycler480) | Roche company of Switzerland |
| Medical high speed freezing centrifuge | Beijing Bai Yang Medical Devices Co., Ltd. |
| Ultrapure water instrument | The limited public affairs of the excellent general ultrapure science and technology in Chengdu |
| Constant-temperature metal bath (CHB-100) | Hangzhou BIOER company |
| Adjustable pipette (0.5-10 μ L, 10-100 μ L, 100-1000 μ L) | Eppendorf company of Germany |
| Spectrophotometer (NanoDrop2000) | Thermo Fisher company of the U.S. |
Embodiment 1: the preparation method detecting the test kit of the level of activity of whole blood or serum sample telomerase.
1, total RNA extraction reagent:
(1) cell pyrolysis liquid Trizol: measure 500mL and heavily steam phenol, 250g guanidinium isothiocyanate, 17.6mL 0.75mol/L sodium citrate solution (pH >=7), 26.4mL 100g/L sarcosyl solution, 50mL 2mol/L sodium acetate soln (pH >=4), 293mL distilled water, in 4 DEG C of preservations after mixing;
(2) extraction liquid: chloroform;
(3) precipitation agent: Virahol;
(4) rinsing liquid: measure 75mL dehydrated alcohol, be settled to 100mL;
(5) RNA solvent: the distilled water removing RNA enzyme ,-20 DEG C of preservations.
2, Reverse Transcription:
(1) reverse transcription reaction damping fluid: take 302.95mg Tris, 279.60mg KCl, 30.50mg MgCl
2, 77.13mg DTT, adds 9mL ddH
2o, regulates pH to 8.3 with 1mol/L HCl, is settled to 10mL, is distributed into 1mL/ pipe ,-20 DEG C of preservations;
(2) dNTP Mix: take 11.784mg dATP, 10.22mg dCTP, 11.024mg dGTP, 11.400mgdTTP, is settled to 2mL, and be distributed into 100 μ L/ and manage, final concentration is 10mmol/L ,-20 DEG C of preservations;
(3) reversed transcriptive enzyme liquid: take 242.28mg Tris, 1.17g NaCl, 3.72mg EDTA, 15.43mg DTT, 5mg reversed transcriptive enzyme, 1.25mg RNA enzyme inhibitors, adds 40mL ddH
2o, 50mL glycerine, 1mol/L HCl regulates pH to 7.5, is settled to 100mL, is distributed into 100 μ L/ and manages, and the final concentration of reversed transcriptive enzyme is 200U/ μ L, and the final concentration of RNA enzyme inhibitors is 40U/ μ L ,-20 DEG C of preservations;
(4) Primer Mix: take oligodeoxythymidylic acid primer and random 6 nucleotide primer dry powder, be dissolved into the primer mixed solution that final concentration is 100 μm of ol/L, dilution is distributed into 100 μ L/ and props up, and final concentration is 10 μm of ol/L ,-20 DEG C of preservations;
(5) ultrapure water: resistance 18.2 megaohm ,-20 DEG C of preservations.
3, quantitative fluorescent PCR reaction reagent:
(1) RT-PCR reaction buffer: measure 8.0mL 10 × Buffer (100mmol/L Tris-cl, 500mmol/LKCl, 15mmol/L MgCl
2, pH8.5), 500 μ l 10mmol/L dNTP Mixture, 1.5mL 25mmol/LMgCl
2, 5mg Taq DNA polymerase, mixing is distributed into 1mL/ and props up, and the final concentration of Taq DNA polymerase is 5U/ μ L ,-20 DEG C of preservations;
(2) Primer-Probe: take the primer after synthesis and probe dry powder respectively, is dissolved into the solution that final concentration is 100 μm of ol/L, and dilution is distributed into 100 μ L/ and props up, and final concentration is 10 μm of ol/L ,-20 DEG C of preservations;
The primer sequence of telomerase hTERT gene
hTERT-F：5’-GGCCGATTGTGAACATGGA-3’，
hTERT-R：5’-CCTCTTTTCTCTGCGGAACGT-3’；
The probe sequence of telomerase hTERT gene
hTERT-P：5’-FAM-TACGTCGTGGGAGCCA-MGB-3‘；
(3) ultrapure water: resistance 18.2 megaohm;
4, quality control product:
(1) positive quality control product: build the recombinant plasmid pGM-hTERT containing telomerase hTERT gene, be transformed into intestinal bacteria TOP10, screening positive clone is also cultivated extraction purification and is become 250mg powder, 1mL elutriant (10mmol/LTris-cl, 1mmol/L EDTA, pH8.0) dissolve, forming concentration is the solution of 250mg/mL, and 1 μ L is containing 2.6x10 after measured
4copies.
(2) negative quality control product: the ultrapure water of resistance 18.2 megaohm.
Embodiment 2: the extraction of whole blood/serum total serum IgE.
(1) homogenization effect: draw 0.25mL whole blood/serum and add 0.75mL Trizol, with sample loading gun piping and druming liquid sample several times to help cell in lysate sample.
(2) separation phase: homogenised sample is hatched under 15-30 DEG C of condition and decomposes completely to make ribosome for 5 minutes.Every 0.75mL Trizol adds 0.2mL extraction liquid.Cover tightly sample hose lid, to exert oneself shaking test tube 15s it is hatched 15min at 30 DEG C with hand.To be no more than the centrifugal force high speed frozen centrifugation 15min of 12,000xg at 2-8 DEG C.Centrifugal rear mixture is divided into three layers: lower floor's phenol chloroform layer, middle layer, the water sample layer that upper strata is colourless.RNA is present in the middle of water sample layer bar none.The capacity of water sample layer is approximately 70% of added Trizol capacity.
(3) RNA precipitation: water sample layer is transferred in a clean EP pipe, by water sample layer and precipitant mix are carried out precipitated rna.The corresponding 0.5mL precipitation agent of every 0.75mL Trizol during initial homogenizing.The sample of mixing is hatched 10min under 15-30 DEG C of condition and to be no more than the centrifugal force high speed frozen centrifugation 10min of 12,000xg at 2-8 DEG C.RNA precipitation is usually invisible before centrifugation, forms a gluey flaky precipitate and is attached at the bottom of pipe.
(4) rinsing: remove upper strata suspension, precipitate once with rinsing liquid washing RNA, the Trizol of every 0.75mL at least adds the rinsing liquid of 1mL.Vortex oscillation biased sample to be no more than the centrifugal force high speed frozen centrifugation 5min of 7,500xg at 2-8 DEG C.
(5) the dissolving again of RNA: simple dry RNA precipitation, draw 30 μ LRNA solvents with liquid-transfering gun and dissolve RNA, and hatch 10min at 55-60 DEG C, the RNA after dissolving is kept at-70 DEG C.
Embodiment 3: reverse transcription reaction.
Reaction solution preparation is please carried out on ice.In order to ensure the accuracy that reaction solution is prepared, when carrying out every reaction, first should press the amount preparation Master Mix of stoichiometric number+2, and then packing 10 μ L is in each reaction tubes.Various composition in reaction solution and practical amount, in table 1, carry out reverse transcription reaction after soft mixing immediately.
Reagent in the reaction solution of table 1 reverse transcription and consumption
Hatch 60min for 42 DEG C, hatch 15min for 70 DEG C, 4 DEG C of placements.
Embodiment 4: real-time fluorescence quantitative PCR reacts.
(1) real-time fluorescence quantitative PCR reaction system
Reaction solution preparation is please carried out on ice, by the component preparation real-time fluorescence quantitative PCR reaction solution shown in table 2.
Reagent in table 2 real-time fluorescence quantitative PCR reaction solution and consumption
(2) real-time fluorescence quantitative PCR reaction conditions
Stage1: denaturation
95℃30s 1Cycle
Stage2:PCR increases
95℃5s
60℃20s 10Cycle
Stage3: phosphor collection
95℃5s
60 DEG C of 20s 40Cycle collect fluorescence
Stage4: place
37℃10s
Embodiment 5: production standard curve.
Draw positive quality control product (2.6x10
4copies/ μ L) 50 μ L, add 80 μ L ddH
2o mixes.Again as stoste 10
4copies/ μ L, is diluted to 10
3copies/ μ L, 10
2copies/ μ L, 10
1copies/ μ L, 10
0copies/ μ L, carries out quantitative fluorescent PCR reaction according to the reaction conditions in embodiment 4, then production standard curve.As shown in Figure 1, typical curve as shown in Figure 2 for typical curve amplification figure.
Embodiment 6: betweenrun precision and withinrun precision experiment.
Betweenrun precision (low value and high level): detect the high and low value sample in available sample, with the expression level of typical curve quantitative assay telomerase hTERT mRNA, detect 4 every day, continuous detecting 5 days, whole blood sample interassay coefficient of variation (CV%) is respectively 2.89% and 1.41%, the results are shown in Table 3; Serum sample interassay coefficient of variation (CV%) is respectively 4.90% and 2.68%, the results are shown in Table 5; The interassay coefficient of variation (CV%) that laboratory allows is 10%.This experiment meets laboratory allowed band, and comparatively prior art sensitivity is higher, and repeatability is better.
Withinrun precision (low value and high level): with the high and low value sample in a collection of experiment detection 20 available samples, with the expression level of typical curve quantitative assay telomerase hTERT mRNA, whole blood sample variation within batch coefficient (CV%) is respectively 1.54% and 0.87%, the results are shown in Table 4; Serum sample variation within batch coefficient (CV%) is respectively 1.43% and 1.18%, the results are shown in Table 6; The variation within batch coefficient (CV%) that laboratory allows is 7.5%.This experiment meets laboratory allowed band, and comparatively prior art sensitivity is higher, and repeatability is better.
Table 3 whole blood sample betweenrun precision experimental result
Table 4 whole blood sample withinrun precision experimental result
Table 5 serum sample betweenrun precision experimental result
Table 6 serum sample withinrun precision experimental result
Embodiment 7: the linearity range of positive quality control product.
Draw positive quality control product (2.6x10
4copies/ μ L), by its gradient dilution to 10
4copies/ μ L, 10
3copies/ μ L, 10
2copies/ μ L, 10
1copies/ μ L, 10
0copies/ μ L.Each gradient detects 5 times, and changes into LOG value calculating linearly dependent coefficient, and linearly dependent coefficient R is 0.9999, the results are shown in Table 7, Laboratory Request R > 0.97, for linearity range performance verification can accept.This experiment meets Laboratory Request, and comparatively prior art, the absolute quantitation to sample telomerase activation degree can be realized, sample telomerase hTERT mrna expression level>=10
0during copies/ μ L, all can detect.
Table 7 positive quality control product linearity range the result
Embodiment 8: reference interval is verified.
Choose 50 routine Whole Blood of Healthies respectively and 50 routine Healthy Human Serum samples carry out quantitative fluorescent PCR reaction, to determine reference interval ("-" represents feminine gender).
In Whole Blood of Healthy pattern detection, 50 routine sample results display hTERT mrna expression level≤10copies/ μ L, the results are shown in Table 8, interval consistent with theoretical reference.Comparatively prior art, has good consistence.Fig. 3 is one of them sample telomerase hTERT gene amplification figure
In Healthy Human Serum pattern detection, 47 routine sample results display hTERT mrna expression levels are 0copies/ μ L, namely negative, the results are shown in Table 9, interval consistent with theoretical reference.Comparatively prior art, has good consistence.
Table 8 whole blood sample reference interval confirmatory experiment result
| 4.83E+00 | 2.95E+00 | 7.54E+00 | 8.12E+00 | 7.12E+00 |
| 5.12E+00 | 5.36E+00 | 7.62E+00 | 8.17E+00 | 7.58E+00 |
| 8.69E+00 | 4.85E+00 | 8.74E+00 | — | — |
| 9.42E+00 | 6.77E+00 | 1.33E+00 | 8.54E+00 | 8.26E+00 |
| 6.23E+00 | 5.33E+00 | 9.66E+00 | 1.32E+00 | 8.17E+00 |
| 5.59E+00 | — | 4.06E+00 | 5.54E+00 | 9.43E+00 |
| 4.16E+00 | — | 4.05E+00 | 8.76E+00 | 7.49E+00 |
| 4.03E+00 | 1.92E+00 | 1.39E+00 | 8.75E+00 | 2.64E+00 |
| 3.58E+00 | 2.65E+00 | 4.53E+00 | — | 3.78E+00 |
| 6.29E+00 | 5.14E+00 | 7.45E+00 | 7.39E+00 | 5.44E+00 |
Table 9 serum sample reference interval confirmatory experiment result
| — | — | — | — | — |
| — | — | — | — | — |
| — | — | — | 3.25E-01 | — |
| — | — | — | — | — |
| — | — | — | — | — |
| — | — | — | — | — |
| — | — | — | — | — |
| — | — | — | — | — |
| — | 5.90E-01 | — | — | — |
| — | 4.21E-01 | — | — | — |
According to the above results, determine reference interval: whole blood :≤10copies/ μ L, serum: 0copies/ μ L
The present invention adopts Taqman fluorescence probe quantitative PCR technology to detect whole blood/serum telomerase hTERT mRNA, determines the level of activity of human body telomerase.Taqman probe is a kind of and oligonucleotide probe complementary between target sequence upstream and downstream primer, and fluorophor is connected to 5 ' end of probe, and quenching group is connected to 3 ' end of probe.
In PCR process, probe elder generation and target sequence pairing, then extend the stage at PCR, probe is carried out enzyme and cuts by 5 ' 5 prime excision enzyme activity of polysaccharase, fluorophor is separated with quenching group and sends fluorescence.Along with the increase of amplification cycles number, the fluorophor discharged constantly is accumulated, the proportional relation of quantity of fluorescence intensity and amplified production, thus reaches quantitative object.
From the result of different batch processed, there is otherness in various degree in batch and between criticizing, because the stability of primer and probe and starting template amount all can cause test result deviation.In experimentation, use after primer and probe packing, avoid multigelation; Starting template calculates loading volume according to nucleic acid concentration, to ensure the consistence of starting template amount; Choose 50 routine Whole Blood of Healthies respectively and 50 routine Healthy Human Serum samples carry out reference interval checking, consistent with notional result.
Present method is compared with traditional quantitative methods, and reagent dosage is few, highly sensitive, and analysis time is short, good stability, is accurate, quick, stable, reliable analytical procedure.
Claims (10)
1. detect the primer of telomerase hTERT mrna expression level, it is characterized in that, comprise following primer sequence:
(1) primer sequence of telomerase hTERT gene:
hTERT-F：5’-GGCCGATTGTGAACATGGA-3’；
hTERT-R：5’-CCTCTTTTCTCTGCGGAACGT-3’；
(2) probe sequence of telomerase hTERT gene:
HTERT-P:5 '-TACGTCGTGGGAGCCA-3 ', and its 5 ' end is connected with fluorescent reporter group, 3 ' end is connected with fluorescent quenching group.
2. the primer of detection telomerase hTERT mrna expression level according to claim 1, is characterized in that, the probe sequence 5 ' of telomerase hTERT gene holds the fluorescent reporter group connected to be FAM, and the fluorescent quenching group that 3 ' end connects is MGB.
3. the primer of detection telomerase hTERT mrna expression level according to claim 1 detects the application in the reagent of the level of activity of whole blood or serum sample telomerase in preparation.
4. detect a test kit for the level of activity of whole blood or serum sample telomerase, it is characterized in that, this test kit comprises the primer described in claim 1 ~ 2.
5. the test kit of the level of activity of detection whole blood according to claim 4 or serum sample telomerase, is characterized in that, this test kit also comprises following reagent: total RNA extraction reagent, Reverse Transcription, quantitative fluorescent PCR reaction reagent, quality control product.
6. the test kit of the level of activity of detection whole blood according to claim 4 or serum sample telomerase, is characterized in that, described total RNA extraction reagent comprise following reagent:
(1) cell pyrolysis liquid Trizol: volume fraction is the heavily steaming phenol of 56%, guanidinium isothiocyanate 28.2g/L, sodium citrate solution 14.9mmol/L, sarcosyl 3g/L, sodium-acetate 112.7mmol/L, and solvent is distilled water;
(2) extraction liquid: chloroform;
(3) precipitation agent: Virahol;
(4) rinsing liquid: volume fraction is the ethanol of 75%;
(5) RNA solvent: without the water of RNA enzyme.
7. the test kit of the level of activity of detection whole blood according to claim 4 or serum sample telomerase, is characterized in that, described Reverse Transcription comprises following reagent:
(1) reverse transcription reaction damping fluid: 250mmol/L Tris-cl, 375mmol/L KCl, 15mmol/L MgCl
2, 50mmol/L DTT, pH8.3;
(2) dNTP mixture: 10mmol/L dATP, 10mmol/L dTTP, 10mmol/L dCTP, 10mmol/LdGTP;
(3) reversed transcriptive enzyme liquid: 20mmol/L Tris-cl, 200mmol/L NaCl, 0.1mmol/L EDTA, 1.0mmol/L DTT, volume fraction is the glycerine of 50%, 200U/ μ L reversed transcriptive enzyme, 40U/ μ L RNA enzyme inhibitors, pH7.5;
(4) primer mixture: oligodeoxythymidylic acid primer, random 6 nucleotide primers;
(5) ultrapure water: resistance 18.2 megaohm.
8. the test kit of the level of activity of detection whole blood according to claim 4 or serum sample telomerase, is characterized in that, described quantitative fluorescent PCR reaction reagent comprise following reagent:
(1) RT-PCR reaction solution: 10mmol/L dATP, 10mmol/L dTTP, 10mmol/L dCTP, 10mmol/L dGTP, 25mmol/L MgCl
2, 5U/ μ L Taq DNA polymerase, 100mmol/L Tris-cl, 500mmol/LKCl, 15mmol/L MgCl
2, pH8.5;
(2) ultrapure water: resistance 18.2 megaohm.
9. the test kit of the level of activity of detection whole blood according to claim 4 or serum sample telomerase, is characterized in that, described quality control product comprises following component:
(1) positive quality control product: containing the recombinant plasmid pGM-hTERT of telomerase hTERT gene, extraction purification concentration is 250mg/mL, and copy number is 2.6x10
4copies/ μ L;
(2) negative quality control product: ultrapure water.
10. the application of test kit in the telomerase activity degree reagent of preparation detecting end of the detection whole blood described in claim 4 ~ 9 or the level of activity of serum sample telomerase.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105154563A (en) * | 2015-09-30 | 2015-12-16 | 陕西师范大学 | Triple-amplification-technology-based homogeneous-phase label-free method for detecting activity of telomerase |
| CN116042792A (en) * | 2022-09-05 | 2023-05-02 | 杭州拜赛思生物科技有限责任公司 | Telomerase activity gene detection kit, primer set, probe primer and detection method |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN116042792A (en) * | 2022-09-05 | 2023-05-02 | 杭州拜赛思生物科技有限责任公司 | Telomerase activity gene detection kit, primer set, probe primer and detection method |
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