CN107841538A - For detecting the primer and detection method of CEBPA gene mutations - Google Patents
For detecting the primer and detection method of CEBPA gene mutations Download PDFInfo
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- CN107841538A CN107841538A CN201711185646.1A CN201711185646A CN107841538A CN 107841538 A CN107841538 A CN 107841538A CN 201711185646 A CN201711185646 A CN 201711185646A CN 107841538 A CN107841538 A CN 107841538A
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Abstract
The invention discloses the primer and detection method for detecting CEBPA gene mutations, and the sense primer of the primer is as shown in SEQ ID NO.1, and anti-sense primer is as shown in SEQ ID NO.2.Method for detecting CEBPA gene mutations, is comprised the steps of：Step 1, testing sample DNA is taken；Step 2, using the kit for including primer described in claim 1, using the DNA described in step 1 as template, pcr amplification reaction is carried out；Step 3, sequencing reaction is carried out using pcr amplification product；Step 4, sequencing reaction product it is purified, denaturation after, upper sequencer；Step 5, sequencing peak figure is analyzed, and be identified with CEBPA gene mutation types.It can be used for the effective detection of CEBPA gene mutations using primer provided by the invention and detection method, be further used for judging the prognosis situation of normal karyotype AML patient, it is higher for the degree of accuracy of detection, specificity and sensitivity.
Description
Technical field
The present invention relates to technical field of biological, and in particular to for detecting primer and the detection of CEBPA gene mutations
Method.
Background technology
Cell propagation, differentiation and the change of apoptotic pathways are acute myeloid leukaemias as caused by gene mutation
The pathogenesis basis of (acutemyeloidleukemia, AML).Recently identified in normal karyotype AML it is a variety of have it is important pre-
The molecular labeling being worth afterwards, wherein medullary system transcription factor CCAAT enhancer binding protein-A (CCAAT-
Enhancerbindingprotein-alpha, CEBPA) myeloid cell is only expressed in hemopoietic system, it is Meloid progenitor
Propagation and an important transcription factor in atomization, have the function that the differentiation and the Inhibiting proliferation that induce grain system.Repaiied newly
In the WHO leukaemia criteria for classifications ordered, the detection of CEBPA mutation has turned into the important diagnosis of AML, layering, prognosis reference index,
It is likely to become new therapy target.The assignment of genes gene mapping is in 19q13.1, total length 3318bp, intronless, cDNA total lengths 2385bp, interior
Containing multiple translation initiation sites.Its structure includes:The leucine in the transcriptional activity area of N-terminal, DNA lands and C-terminal it is abundant two
Dimerization functional areas.Under normal circumstances based on total length 42kD albumen, while a small amount of 30kD albumen be present, ratio changes therebetween
The change of its function will be caused by becoming.In AML patient, CEBPA mutation rates are about 5%-14%, are mainly seen in M1, M2 hypotype,
Wherein about 70% mutation sees normal karyotype patient.In good caryogram group, such as t (15；17), inv (16), t (8；21), still
It there are no CEBPA mutation.
In recent years, a variety of body cell gain mutations have been detected in the normal AML patient of karyotype, including
Mll Gene Partials tandem sequence repeats (PTD), flt3 gene internals tandem sequence repeats (ITD) or tyrosine kinase domain (TKD) are prominent
Change, NPM1, CEBPA, NRAS and WT1 etc., which part mutator have turned into the molecular labeling for assessing relationship with prognosis
Thing.Genetic marker independent as one CEBPA, prognosis bona is prompted, as pre- in NCCNAML treatment guidelines in 2010
Index afterwards, for judging the prognosis situation of normal karyotype AML patient.
The content of the invention
Present invention aims at the primer and detection method provided for detecting CEBPA gene mutations, carried using the present invention
The primer and detection method of confession can be used for the effective detection of CEBPA gene mutations, be further used for judging normal karyotype AML patient
Prognosis situation, it is higher for the degree of accuracy of detection, specificity and sensitivity.
The present invention is achieved through the following technical solutions：
For detecting the primer of CEBPA gene mutations, the sense primer of the primer is as shown in SEQ ID NO.1, downstream
Primer is as shown in SEQ ID NO.2.
Preferably, the kit includes above-mentioned primer.
Method for detecting CEBPA gene mutations, is comprised the steps of：
Step 1, testing sample DNA is taken；
Step 2, using the kit for including primer described in claim 1, using the DNA described in step 1 as template, carry out
Pcr amplification reaction；
Step 3, sequencing reaction is carried out using pcr amplification product；
Step 4, sequencing reaction product it is purified, denaturation after, upper sequencer；
Step 5, sequencing peak figure is analyzed, and be identified with CEBPA gene mutation types.
Preferably, the sense primer of the primer and the concentration of anti-sense primer are 2.5 μ L.
Preferably, the pcr amplification reaction step includes successively：
Step (1) pre-degeneration；Step (2) is denatured；Step (3) is annealed；Step (4) extends；Step (5) extends；Step (6)
Preserve.
Preferably, step (1) denaturation temperature is 95 DEG C, time 10min.
Preferably, step (2) denaturation temperature is 95 DEG C, time 30s；Step (3) annealing temperature is 60 DEG C, the time
For 30s；Step (4) elongating temperature is 72 DEG C, time 1min.
Preferably, step (5) elongating temperature is 72 DEG C, time 7min.
Preferably, the step 3 and step 4 are using the detection of Sanger PCR sequencing PCRs.
Application based on above-mentioned primer in CEBPA gene mutations are detected.
The present invention compared with prior art, has the following advantages and advantages：
The invention provides a kind of primer and detection method for being used to detect CEBPA gene mutations, provided using the present invention
Primer and detection method can be used for CEBPA gene mutations effective detection, be further used for judging normal karyotype AML patient's
Prognosis situation is higher for the degree of accuracy of detection, specificity and sensitivity.Specifically, EDTA anti-freezings are derived from available for detection
The DNA sample of peripheral blood or marrow blood, by extracting blood sample DNA, examined after expanding CEBPA genes using Sanger PCR sequencing PCRs
Survey, sequencing result is analyzed using Sequencher softwares.
Brief description of the drawings
Accompanying drawing described herein is used for providing further understanding the embodiment of the present invention, forms one of the application
Point, do not form the restriction to the embodiment of the present invention.In the accompanying drawings：
Fig. 1 is CEBPA-FR2 provided by the invention (BZIP)-M13F＆CEBPA-FR2 (BZIP)-M13R amplified fragments.
Embodiment
For the object, technical solutions and advantages of the present invention are more clearly understood, with reference to embodiment and accompanying drawing, to this
Invention is described in further detail, and exemplary embodiment of the invention and its explanation are only used for explaining the present invention, do not make
For limitation of the invention.
Embodiment 1：Primer
The invention provides a kind of primer of detection CEBPA gene mutations, according to GenBank announcements and AML prognosis phases
The sequence of each mutator closed, design of primers is carried out using Primer Premier5.0 primer-design softwares, it is designed
Primer synthesizes by Sangon Biotech (Shanghai) Co., Ltd..General survey is added at the sense primer 5' ends of each extron
Sequence primer M13F, anti-sense primer 5' ends add universal sequencing primer thing M13R, the specific primer situation such as institute of table 1 that the present invention designs
Show：
The primer situation table of the present invention detection CEBPA gene mutations of table 1
| Primer | Primer sequence |
| CEBPA_FR2(BZIP)_M13F | M13F-CCGCTGGTGATCAAGCAGGA |
| CEBPA_FR2(BZIP)_M13R | M13R-CACGGTCTGGGCAAGCCTCGAGAT |
Sequencing primer provided by the invention is detected primarily directed to BZIP saltation zones, and above-mentioned sequencing primer and PCR
Amplimer is consistent, and in order to carry out two-way sequencing, reduces the possibility of missing inspection.
Embodiment 2：CEBPA detection in Gene Mutation
The key instrument that detection process uses is as shown in table 2：
The detection in Gene Mutation of table 2 uses instrument
| Sequence number | Instrument title | Purposes | Brand | Remarks |
| 1 | BioRad S1000 | PCR is expanded | BioRad | Numbering：FB86 |
| 2 | ABI 9700 | PCR is expanded | Life | Numbering：FB148 |
| 3 | ABI 3500xl | Sanger is sequenced | Life | Numbering：FB110 |
Concretely comprise the following steps：
Step 1, the DNA sample of EDTA anticoagulation cirumferential bloods is taken, extracting method is with reference to Qiagen company's T IANamp Blood
DNA kit (article No. DP318) specification, DNA sample is diluted to 100ng/ μ L, it is standby.
Step 2, using the kit for including primer described in claim 1, using the DNA described in step 1 as template, carry out
Pcr amplification reaction；Kit is purchased from ABI Products AmpliTaq Gold 360Master Mix【KY】, article No. 4398881.
The pcr amplification reaction system is as shown in table 3：
Table 3PCR amplification reaction systems
Pcr amplification reaction condition is as shown in table 4：
Table 4PCR amplification reaction conditions
Step 3, detected using pcr amplification product using Sanger PCR sequencing PCRs, sequencing result uses Sequencher softwares
Analyzed.
Embodiment 3：Primer specificity
The sequencing primer that embodiment 1 is provided carries out primer Blasting, as a result as follows, CEBPA-FR2 in UCSC
(BZI P)-M13F＆CEBPA-FR2 (BZIP)-M13R amplified fragments are located at chr19:Between 33301244-33301964, length is
703b p.There is no other homologous genes, coincide with CEBPA gene reference sequences, extension increasing sequence is as shown in Figure 1.
Embodiment 4：The degree of accuracy
This detection uses the contrast scheme sequencing primer in sequencing primer and table 5 in table 1 respectively to 20 blood samples
DNA is detected, as a result as shown in table 6.
The sequencing primer of contrast scheme is：
Table 5 contrasts the sequencing primer of scheme
As shown in table 6,20 samples detect through three sets of different primers, and sequencing primer testing result provided by the invention is with giving
The examination criteria result of random sample product is consistent, the degree of accuracy 100%.
The present invention of table 6 and contrast scheme testing result degree of accuracy comparative result
Embodiment 5：Detection specificity and detection sensitivity
The detection specificity of this detection is defined as negative match-rate, and detection sensitivity is defined as positive coincidence rate.
This detection is examined using the detection primer in table 1 and contrast scheme sequencing primer to 20 blood sample DNA
Survey, as a result as shown in table 7-1, table 7-2, table 7-3.
Two pairs of primer detection results are the sample of negative (being not detected by mutation), completely the same with given sample, this detection
Detection specificity is 100%.
Two pairs of primer detection results are the sample of positive (detecting mutation), mutational site completely the same with given sample
And type is also completely the same, the detection sensitivity of this detection is 100%.
Table 7-1 sequencing primer detection specificity of the present invention and detection sensitivity test result
Table 7-2 contrast scheme 1 primer detection specificity and detection sensitivity test knot
Table 7-3 contrast scheme 2 primer detection specificity and detection sensitivity test knot
Embodiment 6：Detect precision
The precision of this detection is defined as that sample is carried out repeating to detect to obtain the ability of same result.This detection is carried out not
Contrast experiment between same personnel, different time, different instruments, same sample difference hole：Specifically include：
(1) withinrun precision
This detection has carried out the detection of the multiple holes of CEBPA genes 3 to 1 positive sample and 1 negative sample.Same sample is different
Amplification between hole is successful, and Sanger sequencing results are as shown in table 8.As a result show, the testing result between same sample difference hole
Unanimously.The withinrun precision of this detection is 100%.
The withinrun precision test result of table 8
(2) betweenrun precision
This detection has carried out the detection three times of CEBPA gene mutations to 10 samples.The amplification of same sample different batches
Successful, Sanger sequencing results are as shown in table 9.As a result show, the testing result of same sample different batches is consistent.This detection
Withinrun precision is 100%.
The betweenrun precision test result of table 9
Embodiment 7：Monitoring lower-cut
(1) this detection is detected using Sanger PCR sequencing PCRs, and this laboratory is carried out when establishing Sanger microarray datasets
Associated verification, the result show that the lowest detection lower limit (LOD) of Sanger PCR sequencing PCRs is 20%, when mutant cell and normally
When the ratio of cell is less than 40%, however not excluded that the possibility of false negative.
(2) shown according to the testing result of DNA input quantities, between 12.5~200ng/reaction of amount of DNA input range,
It can ensure that sudden change sample testing result is consistent (as shown in the testing result of embodiment 4).
The result shows that this detection method can be used for CEBPA gene mutations to carry out effective detection.
Above-described embodiment, the purpose of the present invention, technical scheme and beneficial effect are carried out further
Describe in detail, should be understood that the embodiment that the foregoing is only the present invention, be not intended to limit the present invention
Protection domain, within the spirit and principles of the invention, any modification, equivalent substitution and improvements done etc., all should include
Within protection scope of the present invention.
Sequence table
<110>Hefei gold domain Co., Ltd of medical test institute
<120>For detecting the primer and detection method of CEBPA gene mutations
<160> 2
<170> SIPOSequenceListing 1.0
<210> 1
<211> 38
<212> DNA
<213>Sequencing primer artificial sequence (1)
<400> 1
tgtaaaacga cggccagtcc gctggtgatc aagcagga 38
<210> 2
<211> 42
<212> DNA
<213>Sequencing primer artificial sequence (2)
<400> 2
caggaaacag ctatgaccca cggtctgggc aagcctcgag at 42
Claims (10)
1. the primer for detecting CEBPA gene mutations, it is characterised in that the sense primer of the primer such as SEQ ID NO.1
Shown, anti-sense primer is as shown in SEQ ID NO.2.
2. the kit for detecting CEBPA gene mutations, it is characterised in that the kit is included described in claim 1
Primer.
3. the method for detecting CEBPA gene mutations, it is characterised in that comprise the steps of：
Step 1, testing sample DNA is taken；
Step 2, using the kit for including primer described in claim 1, using the DNA described in step 1 as template, performing PCR expansion is entered
Increase reaction；
Step 3, sequencing reaction is carried out using pcr amplification product；
Step 4, sequencing reaction product it is purified, denaturation after, upper sequencer；
Step 5, sequencing peak figure is analyzed, and be identified with CEBPA gene mutation types.
4. the method according to claim 3 for detecting CEBPA gene mutations, it is characterised in that the primer it is upper
The concentration for swimming primer and anti-sense primer is 2.5 μ L.
5. the method according to claim 3 for detecting CEBPA gene mutations, it is characterised in that the PCR amplifications are anti-
Step is answered to include successively：
Step (1) pre-degeneration；Step (2) is denatured；Step (3) is annealed；Step (4) extends；Step (5) extends；Step (6) is protected
Deposit.
6. the method according to claim 5 for detecting CEBPA gene mutations, it is characterised in that the step (1) is pre-
Denaturation temperature is 95 DEG C, time 10min.
7. the method according to claim 5 for detecting CEBPA gene mutations, it is characterised in that the step (2) becomes
Warm-natured degree is 95 DEG C, time 30s；Step (3) annealing temperature is 60 DEG C, time 30s；Step (4) elongating temperature is 72 DEG C,
Time is 1min.
8. the method according to claim 5 for detecting CEBPA gene mutations, it is characterised in that the step (5) is prolonged
It is 72 DEG C to stretch temperature, time 7min.
9. the method according to claim 3 for detecting CEBPA gene mutations, it is characterised in that the step 3 and step
Rapid 4 using the detection of Sanger PCR sequencing PCRs.
10. the application based on the primer described in claim 1 in CEBPA gene mutations are detected.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109753939A (en) * | 2019-01-11 | 2019-05-14 | 银丰基因科技有限公司 | A kind of HLA sequencing peak figure recognition methods |
| CN110057799A (en) * | 2019-04-26 | 2019-07-26 | 南京市妇幼保健院 | A kind of kit, detection method and its application of amido modified gold nano carrier sense cell C/EBP α |
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| WO2006048262A2 (en) * | 2004-11-04 | 2006-05-11 | Roche Diagnostics Gmbh | Classification of acute myeloid leukemia |
| CN103484552A (en) * | 2013-10-09 | 2014-01-01 | 武汉康录生物技术有限公司 | Simplified Sanger gene sequencing method |
| US20150283270A1 (en) * | 2014-04-04 | 2015-10-08 | Crown Bioscience Inc. | Modeling anti-leukemic therapy by patient derived aml xenografts with distinct phenotypes/genotypes |
| CN105861674A (en) * | 2016-04-27 | 2016-08-17 | 上海荻硕贝肯生物科技有限公司 | Primers, kit and method for detecting gene mutation related to AML (acute myeloid leukemia) prognosis |
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2017
- 2017-11-23 CN CN201711185646.1A patent/CN107841538A/en active Pending
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2006048262A2 (en) * | 2004-11-04 | 2006-05-11 | Roche Diagnostics Gmbh | Classification of acute myeloid leukemia |
| CN103484552A (en) * | 2013-10-09 | 2014-01-01 | 武汉康录生物技术有限公司 | Simplified Sanger gene sequencing method |
| US20150283270A1 (en) * | 2014-04-04 | 2015-10-08 | Crown Bioscience Inc. | Modeling anti-leukemic therapy by patient derived aml xenografts with distinct phenotypes/genotypes |
| CN105861674A (en) * | 2016-04-27 | 2016-08-17 | 上海荻硕贝肯生物科技有限公司 | Primers, kit and method for detecting gene mutation related to AML (acute myeloid leukemia) prognosis |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109753939A (en) * | 2019-01-11 | 2019-05-14 | 银丰基因科技有限公司 | A kind of HLA sequencing peak figure recognition methods |
| CN109753939B (en) * | 2019-01-11 | 2021-04-20 | 银丰基因科技有限公司 | HLA sequencing peak graph identification method |
| CN110057799A (en) * | 2019-04-26 | 2019-07-26 | 南京市妇幼保健院 | A kind of kit, detection method and its application of amido modified gold nano carrier sense cell C/EBP α |
| CN110057799B (en) * | 2019-04-26 | 2021-12-24 | 南京市妇幼保健院 | Kit for detecting cell C/EBP alpha by using amino modified gold nano-carrier, detection method and application thereof |
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