CN102776282B - Kit for detecting relevant expression quantity of AML-EVI1 (acute myelocytic leukemia-ecotropic virus integration site 1) fusion gene - Google Patents
Kit for detecting relevant expression quantity of AML-EVI1 (acute myelocytic leukemia-ecotropic virus integration site 1) fusion gene Download PDFInfo
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Abstract
The invention discloses a kit for detecting relevant expression quantity of an AML-EVI1 (acute myelocytic leukemia-ecotropic virus integration site-1) fusion gene. The kit comprises red blood cell lysis buffer, TRIzol, chloroform, absolute ethyl alcohol, RevertraAceqPCRRTKit, detecting system PCR (polymerase chain reaction) liquid, a positive reference substance and a negative reference substance. The kit is characterized in that the detecting system PCR reaction liquid comprises THUNDERBIRDqPCRMIX; a target gene is detected via upstream and downstream primers AML1-EVI1-F and AML1-EVI1-R and a probe AML1-EVI1-Probe; and an internal control gene Abl is detected via primers abl-F and abl-R and a probe abl-Probe. The kit disclosed by the invention can detect the expression level of the AML1-EVI1 fusion engine in a patient body subjected to CML (chronicmyelognousleukemia), thus, the detecting time can be effectively salved, and the detecting precision can be increased.
Description
Technical field
The invention belongs to life science and biological technical field, a kind of gene detecting kit particularly, adopt the probe for real-time fluorescence quantitative PCR technique, can be to human chronic granulocytic leukemia (chronic myelognous leukemia, CML) AML1-EVI1 fusion gene expression level detects in patient's body, can effectively save detection time, improve accuracy of detection.
Background technology
Leukemia is the unusual clone's property malignant disease of a class hemopoietic stem cell.The a large amount of hyperplasia of leukemia cell are gathered and are soaked into other organs and tissue in marrow and other hemopoietic tissues, and normal hematopoiesis is suppressed, clinical manifestation be anaemia, hemorrhage, infect and each organ soaks into symptom.According to leukemia cell's maturity and natural history, leukemia can be divided into acute and chronic two big classes.The acute leukemia disease progression is rapid, and natural history only has several weeks to the several months.Generally can be divided into acute myeloid leukemia (AML) and acute lymphoblastic leukemia (ALL) two big classes according to leukemia cell's series ownership.And chronic leukemia common chronic myelocytic leukemia (CML), lymphocytic leukemia (CLL) arranged.Because the type of leukemia difference, treatment plan and prognosis also are not quite similar.
Chronic granulocytic leukemia, (chronic myelognous leukemia CML), is the leukemia relatively slowly of a kind of onset and development clinically to be called for short slow grain.The course of disease did not wait from the several months to the several years, should disease can be divided into for 3 phases clinically: chronic phase, acceleration period and acute transformation phase (CML-BC), most of patient is chronic phase during first visit, generally in making a definite diagnosis 2-5, proceed to acceleration period by chronic phase, develop into acute transformation phase (CML-BC) at last, chemotherapy is efficient after the sudden turn of events is no more than 30%, and median survival interval 1-13 month, the prognosis extreme difference.The CML-BC phase can relate to all hematopoietic cells that are, and the prognosis of different sudden turn of events types and principle of reatment are not quite similar, this moment, morphocytology and histological chemistry were difficult to determine fully sudden turn of events type, need be aided with immunophenotyping is differentiated, in conjunction with cytogenetics and molecular biology inspection, help the judgement of diagnosis, treatment and the prognosis of sudden turn of events type.
The t (3 of report such as Nueifora; 21) in, the AML1 on No. 21 karyomit(e) ruptures between runt district and transcriptional activation domain and has formed AML1/MDS1 and AML1/EVI1 fusion gene with MDS1 and EVI1 respectively.In the AML1/EVI1 transcript, the 5th exon of AML1 is connected to the 2nd the untranslated exon of EVI1, caused the generation of a long complex polypeptide, and it has comprised the runt district of AML1 and whole EVI1 almost.The fusion rotein of 180 kD of AML1/EVI1 coding contains 3 DNA lands.In recent years, many clinical and experimental observations all show, slow grain acute transformation phase, and except the bcr/abl fusion gene, 60%-80% also presents some new cell or molecular genetics changes.Wherein the AML1/EVI1 fusion gene also is one of important mechanisms of slow grain acute change.The AML1/EVI1 fusion rotein that EVI1 and AML1 gene fusion produce has dual function, blocking-up differentiation on the one hand; Then stimulate proliferation on the other hand, this depends on structural domain on the AML1 and the 2nd Zinc finger domain of EVI1 respectively.Numerous reports show that in the slow grain sudden turn of events EVI1 negative patients, possible part patient EVI1 gene has produced fusion, uses the specific primer of EVI1 then can not detect.If use more responsive method to detect the predictability that the AMLI/EVI1 fusion gene may improve slow grain acute change simultaneously.
At present clinically with the AML1-EVI1 fusion gene as one of means of dynamic evaluation conditions of patients and prognosis.Detection technique commonly used has fluorescence in situ hybridization technique (FISH), methods such as RT-PCR, RQ-PCR.Although FISH method result is comparatively directly perceived, process of the test is loaded down with trivial details, and it is various to relate to reagent type, waste time and energy, and the result needs the seasoned professional to come interpretation, and there is bigger subjectivity in interpretation as a result.And simple RT-PCR rule is that terminal point is quantitative, can't estimate accurately the starting template amount.In addition, because the application of arsenical and all-trans-retinoic acid, the APL result for the treatment of is greatly improved, minimal residual disease is the major cause of its recurrence, thereby is badly in need of a kind of hypersensitivity, high specific, high automation degree, pollutes the method for controlling well and come to detect the AML1-EVI1 fusion gene mRNA is residual.
RQ-PCR adopts Taqman fluorescence probe quantitative technology, comprehensive organism, zymetology and fluorescence chemical are in one, all under PCR reaction tubes closed state, carry out to interpretation of result from amplification, solve the PCR product pollution and caused false-positive problem, also improved simultaneously susceptibility, its result represents with copy number, realized to the PCR product accurately quantitatively, be easy to unified standard, compare with the qualitative PCR technology, it is good to have specific degree, highly sensitive, linear relationship is good, and is simple to operate, level of automation height, anti-pollution has bigger advantages such as linearity range.Can satisfy the detection of AML1-EVI1 fusion gene mRNA minimal residue, be considered to present first-selected detection method, be used for estimating result for the treatment of, prediction prognosis.Common method has SYBR GreenI dye method in the real-time fluorescence quantitative PCR, two probe hybridization methods and Taqman technology etc.Wherein SYBR GreenI is owing to be non-saturable dye, and specificity must be judged its specificity by observing solubility curve not as two probe hybridization methods and Taqman method; And two probe method hybrid method cost is comparatively expensive.Therefore this research adopts the real-time fluorescence PCR technology to be applied to the gene test of AML1-EVI1 in conjunction with the Taqman probe method.
Summary of the invention
In view of the deficiency that detects AML1-EVI1 fusion gene in the prior art, the present invention has designed detection confidential reference items/goal gene with primer, probe sequence, detects leukemia AML1-EVI1 fusion gene relative expression quantity with fluorescent quantitative PCR technique.By adjusting primer concentration and probe concentration and the ratio of two genes, optimize reaction system and the reaction conditions of PCR, developed a kind of test kit for detection of leukemia AML1-EVI1 fusion gene relative expression quantity.
Test kit for detection of AML1-EVI1 fusion gene relative expression quantity, comprise erythrocyte cracked liquid, TRIzol, chloroform, dehydrated alcohol, ReverTra Ace qPCR RT Kit, detection architecture PCR reaction solution, positive reference substance and negative control product, it is characterized in that:
Detection architecture PCR reaction solution comprises that THUNDERBIRD qPCR MIX, testing goal gene use downstream primer AML1-EVI1-F, AML1-EVI1-R, and probe AML1-EVI1-Probe detects internal control gene Abl primer abl-F, abl-R, probe abl-Probe; Wherein,
AML1-EVI1-F: GACCATCACTGTCTTCA
AML1-EVI1-R: AGTCTTCGCAGCGATAT
AML1-EVI1-Probe: FAM-AATCACAGTGGATGGGCCCCGAG-TAMRA
abl-F: GCCGTGAAGACCTTGAAGGAG
abl-R: ATGATATAGAACGGGGGCTC
abl-Probe: FAM-ACCTGGTGCAGCTCCTTGGG-TAMRA。
Further, detecting the ratio of using downstream primer and probe is preferably: the mol ratio of AML1-EVI1-F ︰ AML1-EVI1-R ︰ AML1-EVI1-Probe is 2 ︰, 2 ︰ 1.
Be preferably with reference to the ratio of using downstream primer and probe: the mol ratio of abl-F ︰ abl-R ︰ abl-Probe is 2 ︰, 2 ︰ 1.
Described positive reference substance is the solution that contains the AML1-EVI1 gene; Described negative control product are the solution of no AML1-EVI1 gene.
The qPCR that ReverTra Ace qPCR RT Kit wherein produces for TOYOBO company cDNA test kit product.
The quantitative PCR reagent that THUNDERBIRD qPCR MIX produces for TOYOBO company, product specification is QPS-101.
Use test kit of the present invention, the real-time fluorescence PCR technology in conjunction with adopting the Tapman probe, can be detected the expression level of AML1-EVI1 fusion gene, the accuracy of detection height, and also simple to operate, can reduce the detection cost, save detection time.Adopt two calibration curve methods, by making up the standard quantitative curve of AML1-EVI1 and internal control gene abl, the internal control gene copy number of accurate quantification sample and AML1-EVI1 copy number, than in the past immunohistochemical methods method and △ △ CT method now, this test kit has the precision height, and the result is convenient to advantages such as interpretation.This test kit primer, probe that reaction system is required carries out rational proportion and optimization in addition, make experiment conditions such as amplification efficiency and speed reach best, thereby having saved loaded down with trivial details condition gropes link, has promoted conventional efficient greatly.This test kit specificity after tested is good, highly sensitive, easy and simple to handle.Help chronic granulocytic leukemia (chronic myelognous leukemia clinically, CML) minimal residue of AML1-EVI1 fusion gene detection in patient's body is avoided hematology recurrence, adjustment treatment plan, evaluation result for the treatment of, prediction prognosis, is prevented clinical recurrence all significant for timely therapeutic intervention.
Description of drawings
Fig. 1: AML1-EVI1 type positive findings synoptic diagram.
Fig. 2: AML1-EVI1 type negative findings synoptic diagram.
Embodiment
Test kit for detection of AML1-EVI1 fusion gene relative expression quantity of the present invention comprises:
Erythrocyte cracked liquid;
TRIzol；
Chloroform;
Dehydrated alcohol;
Detection architecture PCR reaction solution: ReverTra Ace qPCR RT Kit(TOYOBO company); THNDERBIRD Probe qPCR Mix(2 *), AML1-EVI1 upstream and downstream each 0.8uM of primer, AML1-EVI1 probe 0.4 uM; Abl upstream and downstream each 0.8uM of primer, abl-probe(probe) 0.4uM;
Wherein: AML1-EVI1-F:GACCATCACTGTCTTCA;
AML1-EVI1-R: AGTCTTCGCAGCGATAT；
AML1-EVI1-Probe: FAM-AATCACAGTGGATGGGCCCCGAG-TAMRA；
abl-F: GCCGTGAAGACCTTGAAGGAG；
abl-R: ATGATATAGAACGGGGGCTC；
abl-Probe: FAM-ACCTGGTGCAGCTCCTTGGG-TAMRA；
Positive reference substance: contain AML1-EVI1 genome solution; Negative control product: do not contain AML1-EVI1 genome solution.
The using method of test kit of the present invention:
(1) organizes RNA in the extracting blood: in the centrifuge tube of the 1.5ml of cleaning, add the 1ml erythrocyte cracked liquid, get anticoagulation 0.5ml mixing.Room temperature leaves standstill 10min; The centrifugal 5min of 5000rpm abandons supernatant, collects the cell of bottom; Add the 0.5ml erythrocyte cracked liquid again, the centrifugal 5min of 5000rpm abandons supernatant, collects the cell of bottom; In cell, add 1ml TRIzol, blow and beat repeatedly until precipitation dissolving fully, the static 5min of room temperature; Add the 0.2ml chloroform, concussion evenly; 4 ℃ of centrifugal 10min of 14000rpm draw the supernatant layer and are transferred in another new centrifuge tube; Add isopyknic Virahol, abundant mixing up and down, room temperature leaves standstill 10min; 4 ℃ of centrifugal 10min of 14000rpm abandon supernatant, add 75% ethanol 1ml, and washing tube wall gently turns upside down; 4 ℃ of centrifugal 5min of 14000rpm abandon ethanol; Drying at room temperature 10-15min adds 20ulRNase-free water dissolution precipitation.
(2) with reference to the Rever Tra Ace qPCR RT Kit test kit specification sheets of TOYOBO company, RNA is reversed to cDNA.
(3) reagent configuration: by detecting each X ul of people's umber configuration detection system PCR reaction solution, everyone part 23ul packing:
X=23ul reaction solution * (8 parts of confidential reference items (typical curve)+8 part goal gene (typical curve)+n part sample+1 part of positive control+1 part negative control+1 part of blank);
(4) application of sample: add 2ulcDNA in the detection architecture PCR reaction solution; Positive control and negative control directly add 2ul positive reference substance and negative control product; Blank adds 2ul physiological saline or does not add any material.
(5) detect: detect and carry out at the real-time fluorescence PCR instrument, available instrument comprises ABI7300,7500(U.S. Applied Biosystems company) etc.Reaction conditions: 95 ℃ of pre-sex change 1min; 95 ℃ of 15s, 40 circulations of 58 ℃ of 35sec, fluorescent signal is gathered when 58 ℃ of 35 sec.
(6) result judges: threshold line is adjusted to background signal and negative the amplification more than the line, and system calculates copy number automatically according to typical curve and CT value.
When 1) confidential reference items were positive, it is effective that detected result is just thought;
2) positive judging criterion:, Ct＜36, positive; 35≤Ct≤38 are the doubtful positive, need checking again; Ct＞38, negative.
Adopt kit for detecting nucleic acid of the present invention to detect clinical samples
Chronic granulocytic leukemia (CML) the patient anticoagulation sample of fetching and delivering inspection is totally 80 examples, presses embodiment 2 described methods and extracts geneome RNAs, reagent preparation and detect.
Every part of sample adds 2ul in the detection architecture PCR reaction solution.Do the positive simultaneously, feminine gender, blank, each portion of the typical curve of internal control gene/goal gene.The fluorescent PCR instrument in 96 holes can detect 38 duplicate samples simultaneously, and each sample repeats a positive control, a negative control and a blank 2 times.Only be 100 minutes detection time.
Experimental result is compared with special reporting the result of laboratory of inspection, determines the accuracy rate of sample detection.Part positive findings such as following table:
| Sample number | Special inspection laboratory detection result (%) | Test kit detected result (%) |
| 5348 | 44.55 | 44.39 |
| 5303 | 1.46 | 1.47 |
| 5327 | 21.70 | 21.63 |
| 5314 | 49.44 | 49.57 |
| 5321 | 31.34 | 31.40 |
| 5330 | 5.50 | 5.55 |
| 5329 | 66.33 | 66.22 |
| 5303 | 34.24 | 34.30 |
| 5310 | 18.71 | 18.78 |
| 5348 | 66.63 | 66.10 |
| 5348 | 45.04 | 45.36 |
| 5303 | 25.41 | 25.58 |
| 5330 | 74.50 | 74.85 |
| 5320 | 76.03 | 76.32 |
| 5316 | 11.61 | 11.54 |
| 5339 | 20.81 | 20.65 |
| 5343 | 56.89 | 56.64 |
| 5337 | 1.55 | 1.56 |
| 5325 | 39.93 | 39.61 |
| 5334 | 24.35 | 24.16 |
| 5333 | 74.07 | 74.04 |
| 5303 | 60.33 | 60.36 |
| 5338 | 56.23 | 56.38 |
Table 1 is this experimental result and PCR Lab result's contrast, as can be seen from the above table, 30 routine samples all conform to the detected result in special inspection laboratory, especially the minimal residue detected result, the result who examines the laboratory with the spy is identical splendid, and whole coincidence rate reaches 100%.Illustrating that detection kit of the present invention and spy examine utilizes conventional fluorescent quantitation method to compare, detected result accuracy height not only, and shortened detection time, improved detection efficiency.
Embodiment 4Clinical sample detects
Get 2 parts of clinical samples to be checked, press embodiment 2 described methods and extract genome, reagent preparation and detection.
Every duplicate samples adds 2ul in the detection architecture PCR reaction solution.Do the positive simultaneously, feminine gender, each portion of blank.Detect with the fluorescent PCR instrument, the time is 100 minutes.
The detected result figure of sample 1 as shown in Figure 1, the amplified signal of internal control gene abl shows that tested sample genome extracts successfully, detected result is effective, amplified signal is arranged before AML1-EVI1 CT＜36 of sample 1, so sample 1 is the AML1-EVI1 positive.
The detected result figure of sample 2 as shown in Figure 2, the amplified signal of internal control gene abl shows that tested sample genome extracts successfully, detected result is effective, the AML1-EVI1 CT of sample 2〉do not have amplified signal after 38, so sample 2 is the AML1-EVI1 feminine gender.
SEQUENCE LISTING
＜110〉the limited company of Wuhan Ai Dikang medical test
＜120〉for detection of the test kit of AML-EVI1 fusion gene relative expression quantity
<130>
<160> 6
<170> PatentIn version 3.3
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<212> DNA
＜213〉artificial sequence
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<212> DNA
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aatcacagtg gatgggcccc gag 23
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<212> DNA
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Claims (4)
1. for detection of the test kit of AML1-EVI1 fusion gene relative expression quantity, comprise erythrocyte cracked liquid, TRIzol, chloroform, dehydrated alcohol, ReverTra Ace qPCR RT Kit, detection architecture PCR reaction solution, positive reference substance and negative control product, it is characterized in that:
Detection architecture PCR reaction solution comprises that THUNDERBIRD qPCR MIX, testing goal gene use downstream primer AML1-EVI1-F, AML1-EVI1-R, and probe AML1-EVI1-Probe detects internal control gene Abl primer abl-F, abl-R, probe abl-Probe; Wherein,
AML1-EVI1-F: GACCATCACTGTCTTCA
AML1-EVI1-R: AGTCTTCGCAGCGATAT
AML1-EVI1-Probe: FAM-AATCACAGTGGATGGGCCCCGAG-TAMRA
abl-F: GCCGTGAAGACCTTGAAGGAG
abl-R: ATGATATAGAACGGGGGCTC
abl-Probe: FAM-ACCTGGTGCAGCTCCTTGGG-TAMRA。
2. test kit as claimed in claim 1, the mol ratio that it is characterized in that AML1-EVI1-F ︰ AML1-EVI1-R ︰ AML1-EVI1-Probe is 2 ︰, 2 ︰ 1.
3. test kit as claimed in claim 1, the mol ratio that it is characterized in that abl-F ︰ abl-R ︰ abl-Probe is 2 ︰, 2 ︰ 1.
4. test kit as claimed in claim 1 is characterized in that described positive reference substance is the solution that contains AML1-EVI1 gene; Described negative control product are the solution of no AML1-EVI1 gene.
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| CN102094074A (en) * | 2009-12-11 | 2011-06-15 | 上海裕隆临床检验中心有限公司 | Fluorescent reverse transcription-polymerase chain reaction (RT-PCR) kit for quantitatively detecting leukemia fusion gene TEL-AML1 |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN102094074A (en) * | 2009-12-11 | 2011-06-15 | 上海裕隆临床检验中心有限公司 | Fluorescent reverse transcription-polymerase chain reaction (RT-PCR) kit for quantitatively detecting leukemia fusion gene TEL-AML1 |
Non-Patent Citations (4)
| Title |
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| Consistent intergenic splicing and production of multiple transcripts between AML1 at 21q22 and unrelated genes at 3q26 in (3;21)(q26;q22)translocations;GIUSEPPINA NUCIFORA等;《PNS》;19940430;第91卷;第4004-4008页 * |
| Minimal residual disease levels assessed by NPM1 mutation-specific RQ-PCR provide important prognostic infomation in AML;Susanne Schnittger等;《Blood》;20090910;第114卷(第11期);第2220-2231页 * |
| 利用多重PCR技术进行急性髓系白血病患者常见12种融合基因筛查及临场意义;高丽等;《中国实验血液学杂志增刊》;20111231;第466页 * |
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