CN101701253B - Double fluorescent quantitative PCR kit used for detecting gene expression - Google Patents
Double fluorescent quantitative PCR kit used for detecting gene expression Download PDFInfo
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Abstract
The invention relates to a double fluorescent quantitative PCR kit used for detecting gene expression. The kit comprises PCR reaction liquid, DNA polymerase, a proof sample, a primer, a fluorescent probe, a primer and a fluorescent probe, wherein the primers and the fluorescent probes are used for detecting a target gene and an internal reference gene. The kit is characterized in that the sequence of an upstream primer of the internal reference gene which is an abl for detecting the gene is 5'-GGG CGG CCT GAA TGA AG-3', the sequence of a downstream primer is 5'-GCG CTG AAC AAGTTG GTC TTT-3', and the sequence of the fluorescent probe is 5'-TGA GCG CCT TCT CCC CAA AGA-3'; the 5' end of the fluorescent probe is marked with a fluorescent base group, and the 3' end is marked with a quenching base group. The kit can reach the detection accuracy and sensitivity of the prior single fluorescent quantitative PCR method, but the required sample quantity and the required reagent quantity are saved by nearly half.
Description
Technical field
The present invention relates to be used for biochemical field, be specifically related to the quantitative detecting method of genetic expression.
Background technology
The real-time fluorescence quantitative PCR technology was released by U.S. Applied Biosystems company in 1996; Because this technology has not only realized the leap of PCR from qualitative to quantitative; And compare with conventional PCR; It has that specificity is stronger, level of automation is higher, effectively solve characteristics such as PCR pollution problem, is used widely in medical science and food inspection field at present.
The reaction system of quantitative fluorescent PCR is formed (like Fig. 1) by a pair of primer (upstream primer P1 and downstream primer P2) and a probe T, and the probe marked has a fluorophor R and a quenching group Q.Before the reaction; Fluorescent signal on the probe is because the existence of quenching group and not luminous, after the reaction beginning, and Taq enzymic hydrolysis probe and make quenching group come off from probe; Fluorescent signal can occur when receiving laser radiation, the power of detected fluorescent signal is directly proportional with the PCR reaction times.
The pattern of " primer+probe " has reduced wrong generation, can improve susceptibility and specificity simultaneously, becomes the gold standard that PCR detects, and can be applied to many aspects such as lesion detection, virus detection, transgenic animal screening.The anticancer association in Europe thinks that the RQ-PCR technology is the gold standard with specific fusion gene white blood disease minimal residual leukemia clinical detection.
Quantitative fluorescent PCR has been widely used in the detection of changes in gene expression.Yet, detect changes in gene expression, need the expression of while testing goal gene and internal control gene, this just needs more a large amount of samples.For instance, the bcr-abl of the current international practice
P210Detect actual following 3 parts that comprise: 1. bcr-abl in the sample
P210The absolute quantitation of gene copy number, the 2. absolute quantitation of abl gene copy number, 3. bcr-abl
P210Ratio with the abl gene copy number.Owing to 2. can be used as the comprehensive quality control index of each link of RT-RQ-PCR, eliminate each sample treating processes and test the error between criticizing, therefore,, also be convenient to compare between the laboratory 3. than the variation that 1. can react patient's body endomixis gene masculine cell content more reliably.But it is in different reaction tubess, to carry out with internal control gene (needing another to primer and another probe) that existing RQ-PCR detects fusion gene (needing an a pair of primer and a corresponding probe), promptly is substance RQ-PCR; Simultaneously, in order to improve the accuracy of experiment, every batch of experiment need be provided with multiple hole---and calculate to establish 3 multiple holes, therefore, the single of each sample detects just needs a multiple hole, 2 testing gene * 3=6 pipe samples; And in order accurately to compare, often need be with the serial sample duplicate detection of different time points, the sample consumption is more.In order to guarantee to obtain enough samples, need to extract more marrow of patient and blood specimen.Sampling less, high-throughput, intensification detect is the inevitable direction of modern laboratory diagnosis technical development.
Double fluorescent quantitative PCR detects and is meant that carrying out two PCR in the same reaction tubes reacts (like Fig. 2), comprises 2 groups of primers (P1, P2; P1 ', P2 ') and probe (T and T '), every group contains the different probe of fluorescent signal, and upstream primer P1 and downstream primer P2 guide the position of No. 1 reaction, the process of No. 1 reaction of fluorescence indication on the probe T; Same No. 2 reactions are also carried out separately.Do not influence each other to each other, fluorescent signal can the phase mutual interference when result detected, and can detect 2 signals with different wavelengths simultaneously, carry out reaction result and reacting phase comparison separately separately.Detect changes in gene expression with double fluorescent quantitative PCR, with comparing of substance can be brief reagent and sample consumption about half.Yet double fluorescent quantitative PCR is to the requirement of the reaction system height than substance; Not only the enzyme of the concentration of 6 sequences (two pairs of primers and two kinds of probes) and PCR system, dNTP etc. should reach optimization configuration, and should reduce the interference between two pairs of primers and the two kinds of probes as far as possible.Detect changes in gene expression with double fluorescent quantitative PCR at present, gene of normally every detection all needs again selected internal control gene and designs the primer and the probe of internal control gene, very trouble.
Summary of the invention
The technical problem that the present invention will solve provides a kind of double fluorescent quantitative PCR kit with general internal control gene and probe and primer.
The technical scheme that the present invention addresses the above problem is:
A kind ofly be used to detect the genetic expression double fluorescent quantitative PCR kit.This test kit comprises: the primer of PCR reaction solution, archaeal dna polymerase, standard substance, testing goal gene and the primer and the fluorescent probe of fluorescent probe and internal control gene, it is characterized in that,
Described internal control gene is the abl gene;
The sequence of the upstream primer of said detection internal control gene is 5 '-GGG CGG CCT GAA TGA AG-3 ' (SEQ NO.1); The sequence of downstream primer is 5 '-GCG CTG AAC AAG TTG GTC TTT-3 ' (SEQ NO.2), and the sequence of fluorescent probe is 5 '-TGA GCG CCT TCT CCC CAAAGA-3 ' (SEQ NO.3); 5 ' end of described fluorescent probe is marked with fluorophor, and like HEX, 3 ' end is marked with quenching group, like Elipse.
The described goal gene of test kit of the present invention promptly is a gene to be detected, can be mammiferous gene, to detecting the different gene corresponding primer of should arranging in pairs or groups.
The method of use of test kit of the present invention is:
1. the preparation of typical curve
If 5 standard substance concentration gradients are respectively 1 * 10
7Copies, 1 * 10
6Copies, 1 * 10
5Copies, 1 * 10
4Copies and 1 * 10
3Copies, each gradient is established 3 parallel pipes, totally 15 standard substance reaction tubess;
Add goal gene standard substance and internal control gene abl standard substance, fluorescence quantitative PCR reaction solution, archaeal dna polymerase, the primer of testing goal gene and primer and the fluorescent probe of fluorescent probe and internal control gene abl in each standard substance reaction tubes by above-mentioned being set in, add water then and supply 50 μ L.At last by following program reaction: 50 ℃ of reaction 3min, 95 ℃ of reaction 10min, then at 94 ℃ of following 15s, 60 ℃ of following 1min, 50 circulations of coreaction.Reaction carries the Ct value that software is read goal gene and internal reference abl gene through machine after finishing, and is dependent variable with the Ct value, and the copy number of standard substance is an independent variable(s) drawing standard curve.
2. the detection of sample
(1) preparation of template cDNA: extract the RNA of testing sample, reverse transcription is cDNA; Each sample is established 3 parallel pipes.
(2) pcr amplification: in the sample reaction tubes, add cDNA template, fluorescence quantitative PCR reaction solution, archaeal dna polymerase, the primer of testing goal gene and primer and the fluorescent probe of fluorescent probe and internal control gene abl, add water then and supply 50 μ L.At last by following program reaction: 50 ℃ of reaction 3min, 95 ℃ of reaction 10min, then at 94 ℃ of following 15s, 60 ℃ of following 1min, 50 circulations of coreaction.Reaction carries the Ct value that software is read goal gene and internal reference abl gene through machine after finishing, and substitution gained typical curve draws the copy number of goal gene and internal reference abl gene, and calculates the two ratio.
Test kit of the present invention has been realized in same reaction tubes; Simultaneously goal gene and internal control gene are carried out the PCR detection; Can find out from its method of use, detect a sample and only need 15 standard reaction pipes and 3 sample reaction tubess, compared with prior art save half the reagent and sample consumption.
With the retrieval of in genebank (http://www.ncbi.nlm.nih.gov/), comparing of the amplification of DNA fragments of the primer that detects internal control gene abl in the test kit of the present invention; The matching rate overwhelming majority who finds animal abl genes such as this fragment and man like ape, monkey, dog, horse, mouse reaches more than 97.1%; Minimumly also reach 87.9%, and with other genes zone of coupling fully not almost.It is thus clear that the applied range of test kit of the present invention can be used for detecting expression level, the especially Disease-causing gene of range gene in the Mammals, like tumour fusion gene BCR-ABL; PML-RARa, AML1-ETO, SIL-TAL1, CBFB-MYH11; E2A-PBX1, MLL-AF4, TEL-AML1 etc.Therefore, when using test kit of the present invention to detect genetic expression, only need the primer and the probe of purpose of design gene to get final product, need not to design again again the primer and the probe of internal control gene.
Description of drawings
Fig. 1 is the reaction principle of quantitative fluorescent PCR, and wherein P1 and P2 are primer, and T is a probe, and R is a fluorescence generation group, and Q is the fluorescent quenching group.
Fig. 2 is the reaction synoptic diagram of double fluorescent quantitative PCR, and wherein P1, P2, P1 ' and P2 ' are primer, and T and T ' are probe, and R is a fluorescence generation group, and Q is the fluorescent quenching group.
Fig. 3 is that standard substance plasmid concentration is 1 * 10
5Bcr-abl during copies/2 μ L
P210The amplification curve diagram of fusion gene and abl gene, wherein 1～3 be respectively 4 parallel pipes bcr-abl
P210The fusion gene amplification curve, 4～6 are respectively the abl gene amplification curve of 3 parallel pipes.
Fig. 4 is different concns standard substance plasmid Bcr-ab
LP210The amplification curve logarithmic curve chart of T-A, wherein 1～7 is respectively that concentration is 1 * 10
7Copies/2 μ L, 1 * 10
6Copies/2 μ L, 1 * 10
5Copies/2 μ L, 1 * 10
4Copies/2 μ L, 1 * 10
3Copies/2 μ L, 1 * 10
2Copies/2 μ L and 1 * 10
1The amplification curve of the standard substance plasmid of copies/2 μ L, the 8th, negative control, empty horizontal line are threshold value (threshold).
Fig. 5 is standard substance plasmid Bcr-ab
LP210TThe canonical plotting of-A.
Embodiment
Example 1
One, the composition of test kit of the present invention
1.2 the general reaction solution of * TaqMan PCR (2 * TaqMan universal PCR Mastermix is available from American AB I company): contain essential other composition of polysaccharase and PCR reaction, except template, primer and the probe;
2. standard substance are Bcr-ablP210 T-A carrier, promptly are loaded with bcr-ablP210 and internal control gene abl segmental control plasmid carrier to be measured simultaneously, and its preparation method is following:
The following dna solution of preparation in Eppendorf tube, full dose is 5 μ l (table 1);
Table 1
2) the Solution I of adding 5 μ l (equivalent);
3) 16 ℃ were reacted 30 minutes.
Annotate: 1. room temperature (25 ℃) also can normally be carried out ligation, but reaction efficiency reduces a little.
2. also can normally carry out ligation in 5 minutes, but reaction efficiency reduces a little.
4) full dose (10 μ l) is added in the 100 μ l JM109 competent cells, places 30 minutes in the ice;
5) after 42 ℃ of 45 seconds of heating, in ice, placed 1 minute again;
6) add 890 μ l SOC substratum, 37 ℃ of shaking culture 60 minutes;
7) cultivate containing on the L-Agar Plating of X-Gal, IPTG, Amp, form single bacterium colony.Counting white, blue colonies;
8) select white colony, use the PCR method to confirm to insert in the carrier segmental length scale.
3. the primer of internal control gene abl and fluorescent probe are respectively:
The sequence of upstream primer is SEQ NO.1,5 '-GGG CGG CCT GAA TGA AG-3 '
The sequence of downstream primer is SEQ NO.2,5 '-GCG CTG AAC AAG TTG GTC TTT-3 '
The sequence of fluorescent probe is SEQ NO.3,5 '-TGA GCG CCT TCT CCC CAA AGA-3 ', and 5 ' end of this fluorescent probe is marked with fluorophor HEX, and 3 ' end is marked with cancellation reporter group Elipse.
4. detect the primer and the fluorescent probe of bcr-ablP210 fusion gene, its sequence is respectively:
The primer of bcr-ablP210 fusion gene and fluorescent probe can adopt the sequence of recommending like European anticancer association (EAC), are respectively:
Upstream primer is SEQ NO.4:5 '-TCC GCT GAC CAT CAA TAA GGA-3 '
Downstream primer is SEQ NO.5:5 '-CAC TCA GAC CCT GAG GCT CAA-3 '
The sequence of fluorescent probe is SEQ NO.6:5 '-CCC TTC AGC GGC CAG TAG CAT CTG A-3 ', and 5 ' end of this fluorescent probe is marked with fluorophor FAM, and 3 ' end is marked with cancellation reporter group, TAMRA.
Precious biotech firm of Ying Jun biotech company and Dalian synthesizes in Shanghai for above-mentioned primer and probe.
Two, testing sample
Year December in May, 2004 to 2006 is in Nanfang Hospital's outpatient service or CML chronic phase (CP) patient 46 examples of being in hospital, male 23 examples, and women 23 examples, at 16～65 years old age, The median age 38.1 years old is got patient's peripheral blood or marrow.
Three, the processing of sample
1. isolation of RNA: get leukaemic's marrow or peripheral blood and separate through the Ficoll lymphocyte separation medium and obtain mononuclearcell; Concentration with 5 * 106～1 * 107 cell/pipes adds 1mL Trizol Reagent; Use immediately or-80 ℃ of preservations, separate total RNA with single stage method.RNA measures the A260/A280 value through ultraviolet spectrophotometer, measures RNA concentration and purity.
2. synthetic template cDNA: in 25 μ l systems, contain the total RNA of 1.8 μ g, 0.5 μ g random primer, 40U RNA enzyme inhibitors, 1mmol/LdNTPs, 200U M-MLV reversed transcriptive enzyme, 50mM Tris-HCl (pH8.3), 75mM KCl, 3mM MgCl2,10mM DTT.More than be the Promega Company products.Total RNA is mixed with random primer, placed 5 minutes, add RNA enzyme inhibitors, dNTPs, reversed transcriptive enzyme and Buffer then, placed 45 minutes at 37 ℃ at 65 ℃.
Four, the detection of sample
1. detect with test kit of the present invention
Instrument: Biorad company fluorescent quantitative PCR appearance and analytical system
(1) preparation of typical curve:
Get Bcr-ablP210 T-A carrier; Be diluted with water to following concentration: 1 * 107copies/2 μ L, 1 * 106copies/2 μ L, 1 * 105copies/2 μ L, 1 * 104copies/2 μ L, 1 * 103copies/2 μ L, 1 * 102copies/2 μ L and 1 * 101copies/2 μ L;
Add Bcr-ablP210 T-A carrier 2 μ L, the general reaction solution 25 μ L of 2 * TaqMan PCR in each standard substance reaction tubes by above-mentioned being set in; Adding the primer that detects the bcr-abl fusion gene and primer to each primer concentration that detects internal control gene abl is 0.3 μ moL/L; The probe that detects the bcr-abl fusion gene is 0.2 μ moL/L with probe to each concentration and probe concentration that detects internal control gene abl, and adding adds water then and supplies 50 μ L.At last by following program reaction: 50 ℃ of reaction 3min, 95 ℃ of reaction 10min, then at 94 ℃ of following 15s, 50 circulations of 60 ℃ of following 1min coreactions.Reaction carries the Ct value that software is read bcr-abl gene and internal reference abl gene through machine after finishing, and is dependent variable with the Ct value, and the copy number of standard substance is an independent variable(s) drawing standard curve; Each Bcr-ablP210 T-A carrier concn is established 3 parallel pipes, totally 15 standard substance reaction tubess.
(2) detection of sample
In the sample detection pipe, add template cDNA2 μ L; The primer of bcr-ablP210 gene and abl internal control gene (being SEQ NO.4, SEQ NO.5, SEQNO.1 and SEQNO.2) is respectively to 0.3 μ moL/L; The probe (SEQNO.3) of probe of bcr-ablP210 gene (SEQNO.6) and abl gene is respectively to 0.2 μ moL/L; The general reaction solution 25 μ L of 2 * TaqMan PCR, the total system 50 μ L of the reaction of every pipe, insufficient section replenishes with aseptic double-distilled water.Response procedures: 50 ℃ * 3min+95 ℃ * 10min, 94 ℃ * 15s, 60 ℃ * 1min totally 50 circulations then.Reaction carries the copy number that software is read bcr-ablP210 goal gene and abl internal reference through machine after finishing, and calculates the ratio of the two.Each sample is all made 3 parallel pipes, to guarantee result's accuracy.
2. the contrast experiment 1---and (FISH) detects with fluorescence in situ hybridization
The interphase fluorescence in situ hybridization operation instructions that provides by test kit is carried out operation such as wash-out after sample disposal, sex change, hybridization, the hybridization.
3. the contrast experiment 2---use the substance fluorescence quantitative PCR detection
(1) reagent: the standard substance plasmid is the plasmid Bcr-ablP210 plasmid and the T-A carrier that contains the abl internal control gene that contains the bcr-ablP210 gene, and all the other reagent are all identical with test kit of the present invention.
(2) method:
1. the drafting of Bcr-ablP210 gene typical curve
Getting the Bcr-ablP210 plasmid vector carries out 10 times of gradient dilutions and contains 1 * 106copies, 1 * 105copies, 1 * 104copies respectively to per 2 μ L; 1 * 103copies, 1 * 102copies, 1 * 101copies plasmid; Each gradient is all made 3 parallel pipes, to guarantee result's accuracy.Reaction system: add 2 μ L positive templates in every pipe, the upstream and downstream primer of bcr-ablP210 gene (being SEQ NO.4 and SEQ NO.5) is respectively to 0.3 μ moL/L, and the TaqMan probe of bcr-ablP210 gene FAM mark (being SEQ NO.6) is to 0.2 μ moL/L; The general common component of 2 * TaqMan PCR (2 * TaqMan universal PCR Mastermix, American AB I company) 25 μ L, the total system 50 μ L of the reaction of every pipe, insufficient section replenishes with aseptic double-distilled water.
2. the drafting of abl gene typical curve
Get and have internal control gene abl segmental T-A carrier to be measured and carry out 10 times of gradient dilutions and contain 1 * 106copies respectively to per 2 μ L; 1 * 105copies, 1 * 104copies, 1 * 103copies; 1 * 102copies; 1 * 101copies plasmid, each gradient are all made 3 parallel pipes, to guarantee result's accuracy.Reaction system: add 2 μ L positive templates in every pipe, the upstream and downstream primer of abl internal control gene (being SEQ NO.1 and SEQ NO.2) is respectively to 0.3 μ moL/L, and the TaqMan probe of abl gene HEX mark (being SEQ NO.3) is to 0.2 μ moL/L; The general common component of 2 * TaqManPCR (2 * TaqMan universal PCR Mastermix, American AB I company) 25 μ L, the total system 50 μ L of the reaction of every pipe, insufficient section replenishes with aseptic double-distilled water.
Response procedures: 50 ℃ * 3min+95 ℃ * 10min, 94 ℃ * 15s, 60 ℃ * 1min totally 50 circulations then.Reaction carries the copy number that software is read bcr-ablP210 goal gene and abl internal reference through machine after finishing, and calculates the ratio of the two, draws typical curve separately.
Five, result
1. the drafting of test kit typical curve of the present invention
Fig. 3 shows when the initial copy number when bcr-abl fusion gene and abl gene is identical; The Ct value of using primer separately to increase is identical, explains that it is feasible that typical curve with standard substance plasmid of the present invention replaces the typical curve of bcr-abl fusion gene and abl gene.Fig. 4 is that concentration is 1 * 107copies/2 μ L, 1 * 106copies/2 μ L, 1 * 105copies/2 μ L, 1 * 104copies/2 μ L and the standard substance plasmid of 1 * 103copies/2 μ L and the amplification curve of negative control.Typical curve according to Fig. 4 result draws is as shown in Figure 5, and the typical curve equation is y=-3.381X+16.40, r=0.995.The sample CV value (n=5) of 7 gradients of 1 * 107 to 10 copy number is respectively 4.89%, 3.88%, 4.34%, 1.90%, 3.93%, 4.26% and 3.45%, and the good stability of the inventive method is described.
2. respectively organize the sample detection result
As shown in table 2, the detected result of test kit of the present invention and substance fluorescence quantitative PCR detection be basically identical as a result, explain that the accuracy of test kit of the present invention is suitable with the substance quantitative fluorescent PCR, but the consumption of sample and reagent has been saved one times.
Table 2
| Divide into groups | Sample number (example) | Test kit detected result of the present invention | The FISH detected result | Substance quantitative fluorescent PCR detected result |
| 1 | 8 | 0.492±0.263 | 63.9％±16.1％ | 0.579±0.271 |
| 2 | 6 | 0.023±0.033 | 3.8％±1.8％ | 0.031±0.013 |
| 3 | 9 | (4.33±3.84)×10 -4 | Negative | (3.99±1.32)×10 -4 |
Six, model case is for example:
Just send out the patient of treatment, it is 95% that FISH detects bcr-abl gene masculine rate.
Through 3 months patients of imatinib mesylate treatment, it is 5% that FISH detects bcr-abl gene masculine rate.
Through recessive allele HSCT patient, it is negative that FISH detects bcr-abl gene masculine rate.
The detected result of The above results explanation FISH and the result of clinical diagnosis are consistent, conform to test kit detected result of the present invention.
Example 2
One, the composition of test kit of the present invention:
The general reaction solution of 2 * TaqMan PCR (2 * TaqMan universal PCR Mastermix is available from American AB I company): contain essential other composition of polysaccharase and PCR reaction, except template, primer and the probe;
Standard substance are Bcr-abl
P190The T-A carrier promptly is loaded with bcr-abl simultaneously
P190With the control plasmid carrier of internal control gene abl, its preparation method is following:
1) press table 2 and in Eppendorf tube, prepare following dna solution, full dose is 5 μ l.
Table 3
2) the Solution I of adding 5 μ l (equivalent).
3) 16 ℃ were reacted 30 minutes.
Notes) 1. room temperature (25 ℃) also can normally be carried out ligation, but reaction efficiency reduces a little.
2. also can normally carry out ligation in 5 minutes, but reaction efficiency reduces a little.
4) full dose (10 μ l) is added in the 100 μ l JM109 competent cells, places 30 minutes in the ice.
5) after 42 ℃ of 45 seconds of heating, in ice, placed 1 minute again.
6) add 890 μ l SOC substratum, 37 ℃ of shaking culture 60 minutes.
7) cultivate containing on the L-Agar Plating of X-Gal, IPTG, Amp, form single bacterium colony.Counting white, blue colonies.
8) select white colony, use the PCR method to confirm to insert in the carrier segmental length scale.
Primer and the fluorescent probe of internal control gene abl are respectively
The sequence of upstream primer is SEQ NO.1:5 '-GGG CGG CCT GAA TGA AG-3 '
The sequence of downstream primer is SEQ NO.2:5 '-GCG CTG AAC AAG TTG GTC TTT-3 '
The sequence of fluorescent probe is SEQ NO.3:5 '-TGA GCG CCT TCT CCC CAA AGA-3 ', and 5 ' end of this fluorescent probe is marked with fluorophor HEX, and 3 ' end is marked with cancellation reporter group Elipse.
Bcr-abl
P190The primer of fusion gene and fluorescent probe can adopt the sequence of recommending like European anticancer association (EAC), are respectively
Upstream primer is SEQ NO.7:5 '-CTG GCC CAA CGA TGG CGA-3 ';
Downstream primer is SEQ NO.5:5 '-CAC TCA GAC CCT GAG GCT CAA-3 ';
The sequence of fluorescent probe is SEQ NO.6:5 '-CCC TTC AGC GGC CAG TAG CAT CTG A-3 ', and 5 ' end of this fluorescent probe is marked with fluorophor FAM, and 3 ' end is marked with cancellation reporter group TAMRA.
Precious biotech firm of handsome biotech company and Dalian synthesizes in Shanghai for above-mentioned primer and probe.
Two, the processing of sample
1, isolation of RNA: get leukaemic's marrow or peripheral blood and obtain mononuclearcell, with 5 * 10 through the separation of Ficoll lymphocyte separation medium
6～1 * 10
7The concentration of individual cell/pipe adds 1mL Trizol Reagent, uses immediately or-80 ℃ of preservations, separates total RNA with single stage method.RNA measures A through ultraviolet spectrophotometer
260/ A
280Value is measured RNA concentration and purity.
2, synthetic template cDNA: in 25 μ l systems, contain the total RNA of 1.8 μ g, 0.5 μ g random primer, 40U RNA enzyme inhibitors, 1mmol/LdNTPs, 200U M-MLV reversed transcriptive enzyme, 50mM Tris-HCl (pH8.3), 75mM KCl, 3mM MgCl
2, 10mM DTT.More than be the Promega Company products.Total RNA is mixed with random primer, placed 5 minutes, add RNA enzyme inhibitors, dNTPs, reversed transcriptive enzyme and Buffer then, placed 45 minutes at 37 ℃ at 65 ℃.
Three, the detection of sample
Instrument: Biorad company fluorescent quantitative PCR appearance and analytical system
(1) preparation of typical curve:
Get Bcr-abl
P190The T-A carrier is diluted with water to every microlitre and contains Bcr-abl
P190T-A carrier 1 * 10
6Copies, 1 * 10
5Copies, 1 * 10
4Copies, 1 * 10
3Copies, 1 * 10
2Copies, 1 * 10
1Copies;
Add Bcr-ab by above-mentioned being set in each standard substance reaction tubes
LP190 T-A carrier 2 μ L, the general reaction solution 25 μ L of 2 * TaqMan PCR; Adding the primer that detects the bcr-abl fusion gene and primer to each primer concentration that detects internal control gene abl is 0.3 μ moL/L; The probe that detects the bcr-abl fusion gene is 0.2 μ moL/L with probe to each concentration and probe concentration that detects internal control gene abl; Add and to add water then and supply 50 μ L, at last by following program reaction: 50 ℃ * 3min+95 ℃ * 10min, 94 ℃ * 15s, 60 ℃ * 1min totally 50 circulations then.Reaction carries the Ct value that software is read bcr-abl gene and internal reference abl gene through machine after finishing, and is dependent variable with the Ct value, and the copy number of standard substance is an independent variable(s) drawing standard curve; Each Bcr-ab
LP190The T-A carrier concn is established 3 parallel pipes, totally 15 standard substance reaction tubess.
(2) detection of sample
In the sample detection pipe, add template cDNA2 μ L, bcr-abl
P190The primer of gene and abl internal control gene (being SEQ NO.7, SEQ NO.5, SEQ NO.1 and SEQ NO.2) is respectively to 0.3 μ moL/L, bcr-abl
P190The probe (SEQ NO.3) of probe of gene (SEQ NO.6) and abl gene each to 0.2 μ moL/L, the general reaction solution 25 μ L of 2 * TaqMan PCR, the total system 50 μ L of the reaction of every pipe, insufficient section replenishes with aseptic double-distilled water.Response procedures: 50 ℃ * 3min+95 ℃ * 10min, 94 ℃ * 15s, 60 ℃ * 1min totally 50 circulations then.Reaction carries software through machine and reads bcr-abl after finishing
P190The copy number of goal gene and abl internal reference calculates the two ratio.Each sample is all made 3 parallel pipes, to guarantee result's accuracy.
Sequence table
< 110>Nanfang Medical Univ
< 120>a kind ofly be used to detect the genetic expression double fluorescent quantitative PCR kit
<160>7
<170>PatentIn?version?3.3
<210>1
<211>17
<212>DNA
< 213>artificial sequence
<400>1
gggcggcctg?aatgaag 17
<210>2
<211>21
<212>DNA
< 213>artificial sequence
<400>2
gcgctgaaca?agttggtctt?t 21
<210>3
<211>21
<212>DNA
< 213>artificial sequence
<400>3
tgagcgcctt?ctccccaaag?a 21
<210>4
<211>21
<212>DNA
< 213>artificial sequence
<400>4
tccgctgacc?atcaataagg?a 21
<210>5
<211>21
<212>DNA
< 213>artificial sequence
<400>5
cactcagacc?ctgaggctca?a 21
<210>6
<211>25
<212>DNA
< 213>artificial sequence
<400>6
cccttcagcg?gccagtagca?tctga 25
<210>7
<211>18
<212>DNA
< 213>artificial sequence
<400>7
ctggcccaac?gatggcga 18
Claims (1)
1. one kind is used to detect the genetic expression double fluorescent quantitative PCR kit, and this test kit comprises: the primer of PCR reaction solution, archaeal dna polymerase, standard substance, testing goal gene and the primer and the fluorescent probe of fluorescent probe and internal control gene, it is characterized in that,
Described PCR reaction solution and archaeal dna polymerase are the general pcr amplification premix of TaqMan reagent,
Described internal control gene is the abl gene;
The sequence of the upstream primer of said detection internal control gene is 5 '-GGG CGG CCT GAA TGA AG-3 '; The sequence of downstream primer is 5 '-GCG CTG AAC AAG TTG GTC TTT-3 ', and the sequence of fluorescent probe is 5 '-TGA GCG CCT TCT CCC CAA AGA-3 '; 5 ' end of described fluorescent probe is marked with fluorophor HEX, and 3 ' end is marked with cancellation reporter group Elipse;
Described goal gene is bcr-abl
P210Or bcr-abl
P190, wherein,
Said goal gene is bcr-abl
P210Primer and fluorescent probe be respectively: described standard substance are for being loaded with bcr-abl
P210T-A carrier with internal control gene abl; Testing goal gene bcr-abl
P210The sequence of upstream primer be 5 '-TCC GCT GAC CAT CAA TAA GGA-3 '; The sequence of downstream primer is 5 '-CAC TCA GAC CCT GAG GCT CAA-3 ', and the sequence of fluorescent probe is 5 '-CCC TTC AGC GGC CAG TAG CAT CTG A-3 '; 5 ' end of described fluorescent probe is marked with fluorophor FAM, and 3 ' end is marked with cancellation reporter group TAMRA;
Said goal gene is bcr-abl
P190Primer and fluorescent probe be respectively: described standard substance are for being loaded with bcr-abl
P190T-A carrier with internal control gene abl; Testing goal gene bcr-abl
P190The sequence of upstream primer be 5 '-CTG GCC CAA CGA TGG CGA-3 '; The sequence of downstream primer is 5 '-CAC TCA GAC CCT GAG GCT CAA-3 ', and the sequence of fluorescent probe is 5 '-CCC TTC AGC GGC CAG TAG CAT CTG A-3 '; 5 ' end of described fluorescent probe is marked with fluorophor FAM, and 3 ' end is marked with cancellation reporter group TAMRA.
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| CN102925556B (en) * | 2012-09-29 | 2014-09-17 | 童永清 | Kit for detecting mRNA expression quantity of m BCR fusion gene |
| MA36330B1 (en) * | 2013-10-10 | 2016-04-29 | Mascir Morrocan Foundation For Advanced Science Innovation & Res | Kit for the diagnosis of chronic myeloide leukemia in multiplex reaction and its applications in the monitoring of treatment and residual disease |
| CN107674914A (en) * | 2016-08-02 | 2018-02-09 | 熊术道 | One step single tube standard measure RT PCR detect the expression quantity of chronic myelogenous leukemia BCR ABL1 genes |
| CN109971836A (en) * | 2017-12-28 | 2019-07-05 | 上海细胞治疗研究院 | The method and kit of double fluorescent quantitative PCR measurement CAR copy number |
| CN109554472A (en) * | 2018-12-18 | 2019-04-02 | 武汉迪安医学检验实验室有限公司 | Kit for detecting TEL-AML1 fusion gene |
| CN113512592A (en) * | 2021-04-23 | 2021-10-19 | 中国科学院深圳先进技术研究院 | Primer, probe and kit for detecting galanin secretory cells |
| CN113943816A (en) * | 2021-12-03 | 2022-01-18 | 郑州思勤生物科技有限公司 | Nucleic acid compositions, kits and uses thereof |
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| WO2004048608A1 (en) * | 2002-11-28 | 2004-06-10 | Peter Kufer | Novel real-time rt-pcr for the sensitive detection of multiple mage gene transcripts |
| CN101041864A (en) * | 2007-03-06 | 2007-09-26 | 扬子江药业集团北京海燕药业有限公司 | Chlamydi trachomatis and Neisseria gonorrhoeae dual real-time fluorescence PCR detection method |
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| WO2004048608A1 (en) * | 2002-11-28 | 2004-06-10 | Peter Kufer | Novel real-time rt-pcr for the sensitive detection of multiple mage gene transcripts |
| CN101041864A (en) * | 2007-03-06 | 2007-09-26 | 扬子江药业集团北京海燕药业有限公司 | Chlamydi trachomatis and Neisseria gonorrhoeae dual real-time fluorescence PCR detection method |
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