CN105838793A - Primers, kit and method for qualitatively detecting leukaemia fusion genes - Google Patents
Primers, kit and method for qualitatively detecting leukaemia fusion genes Download PDFInfo
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Abstract
The invention belongs to the technical field of gene engineering, and discloses a primer combination for detecting leukaemia fusion genes, a kit containing the primer combination, and a multiplex nested RT-PCR (reverse transcription-polymerase chain reaction) method for performing leukaemia fusion gene detection by using the primer combination or kit. The method is based on a multiplex nested RT-PCR technique, and thus, is simple and quick and has high sensitivity. Besides, the reasonable primer combination is utilized to effectively avoid interactions among multiple primer pairs, thereby reducing the detection errors. The detection method can be utilized to comprehensively perform qualitative detection on 43 leukaemia fusion genes, thereby saving the reagent consumption and lowering the detection cost. The detection method has wide detection range, and is suitable for detecting mass samples in clinic.
Description
Technical field
The present invention relates to gene engineering technology field, be particularly used for qualitative detection leukemia and merge base
The primer of cause, test kit and method.
Background technology
Leukemia is modal a kind of malignant clone disease in child and youth.Multiple studies have shown that
Most leukaemic exists certain chromosome structure distortion (lack, repeat, inversion,
Transposition etc.), these are to cause leukemia to occur and the major reason of development.The distortion of chromosome structure,
The structure variation of proto-oncogene and antioncogene can be caused so that oncogene, Oncogene Mutation activate,
Antioncogene disappearance or inactivation, produce new fusion gene, encoding fusion protein.Thus different melting
Close gene and have become as dissimilar leukemic molecular biology specificity marker.Such as chronic myelocytic
BCR-ABL1 fusion gene in leukemia (CML), acute promyelocytic leukemia (APL)
In PML-RARA fusion gene become respective molecular marker and become the target for the treatment of further
Mark.Within 2000, exception common for some of them is returned especially by WHO about in the standard of leukemia classification
One of standard received as leukemia basic diagnosis.In view of the detection of leukemia correlation fusion gene is in vain
Disorders of blood diagnosis, typing, clinical treatment selection, prognosis therapeutic evaluation and minimal residual disease (MRD)
Clinical value in detection, the exploitation of fusion gene detection technique has great clinical meaning and market
It is worth.
At present, the detection method of fusion gene mainly includes chromosome karyotype analysis, fluorescence in situ hybridization
Technology and polymerase chain reaction (PCR).With chromosome karyotype analysis and fluorescence in situ hybridization technique
Comparing, PCR detection method has the most effectively, sensitivity advantages of higher, has wider clinically
Application.Multiple PCR technique refers to add multipair primer in same PCR system, it is possible to simultaneously
Amplify multiple DNA fragmentation.As the deriving technology that PCR is important, multiplex PCR can expand simultaneously
Increase genes of interest, have time-consuming, reduce cost, put forward high efficiency advantage.Nest-type PRC
Being the round pcr of a kind of variation, it uses two to the complete fragment of primer amplification.Pair of primers expands
Increase fragment similar with regular-PCR.Second pair of primer is referred to as nested primer, and it is combined in PCR for the first time
The inside of product so that the second amplified fragments is shorter than amplification for the first time.The advantage of nest-type PRC is,
If amplification creates false segments for the first time, then second time can carry out primer pairing in false segments
And the probability that expands is extremely low.Therefore, nest-type PRC can improve the sensitivity of PCR and reduce non-
Specific amplified.
But, multiplex-nested PCR technology needs to add multipair drawing in same PCR reaction system
Thing, which adds multipair primer and interacts in reaction system and form the several of primer dimer
Rate, thus reduce the specificity and efficiency of PCR reaction, even can not amplify specific targets and produce
Thing.Additionally, along with leukemia research is deepened continuously by people, increasing leukemia merges base
Because having been observed that, the method for current detection leukemia fusion gene can not meet the most far away people for
The demand of the fusion gene of the detection overwhelming majority simultaneously, needs badly a kind of quick, effective and is prone to standardized
The method of energy qualitative detection overwhelming majority leukemia fusion gene simultaneously.
Summary of the invention
The present invention is directed to drawbacks described above present in prior art, based on up-to-date leukemia fusion gene
Data base, has redesigned the detection primer for detecting leukemia fusion gene and multiplex nested
RT-PCR method.The detection method of the present invention is quick, effective and is prone to 43 kinds of leukemia are merged bases
Because of the qualitative detection being standardized.
To this end, one aspect of the present invention provides a kind of primer combination for detecting leukemia fusion gene,
It is made up of the primer shown in SEQ ID NO.1-138.
In a preferred embodiment of the present invention, drawing shown in SEQ ID NO.1-69 in the combination of this primer
Thing composition first organizes greatly detection primer, the primer second largest group of inspection of composition shown in SEQ ID NO.70-138
Survey primer；First group detection primer greatly is further by first shown in SEQ ID NO.1-8,68,69
Group's detection primer, second group's detection primer shown in SEQ ID NO.9-15,68,69, SEQ ID
The 3rd group's detection primer shown in NO.16-21,68,69, SEQ ID NO.22-26,68,69
The 4th shown group's detection primer, the 5th group inspection shown in SEQ ID NO.27-33,68,69
Survey primer, the 6th group's detection primer shown in SEQ ID NO.34-38,68,69, SEQ ID
The 7th group's detection primer shown in NO.39-46,68,69, SEQ ID NO.47-50,68,69
The 8th shown group's detection primer, the 9th group inspection shown in SEQ ID NO.51-54,68,69
Survey primer, the tenth group's detection primer shown in SEQ ID NO.55-59,68,69, SEQ ID
The tenth a small group detection primer composition shown in NO.60-67,68,69；Second largest group of detection primer is entered
One step is by first group's detection primer shown in SEQ ID NO.70-77,137,138, SEQ ID
Second group's detection primer shown in NO.78-84,137,138, SEQ ID NO.85-90,137,
The 3rd group's detection primer shown in 138, the 4th shown in SEQ ID NO.91-95,137,138 is little
Group detection primer, the 5th group's detection primer shown in SEQ ID NO.96-102,137,138, SEQ
The 6th group's detection primer shown in ID NO.103-107,137,138, SEQ ID NO.108-115,
137, the 7th group's detection primer shown in 138, shown in SEQ ID NO.116-119,137,138
The 8th group's detection primer, shown in SEQ ID NO.120-123,137,138 the 9th group inspection
Survey primer, the tenth group's detection primer shown in SEQ ID NO.124-128,137,138, SEQ ID
The tenth a small group detection primer composition shown in NO.129-136,137,138.
The present invention, by selecting related gene broken site upstream sequence and downstream sequence, carries out specificity
The design of multiple PCR primer.Designed primer Tm, all near 60 DEG C, is integrated and is arranged in pairs or groups each
Primer, thus avoid primer dimer as far as possible and produce, improve the specificity and efficiency of PCR amplification.
For 43 kinds of leukemia fusion genes, the concrete primer situation of present invention design is as shown in table 1.
Table 1, the primer information slip of the present invention
Another aspect of the present invention provides a kind of test kit for detecting leukemia fusion gene, and it comprises
Primer of the present invention combines.
In a preferred embodiment of the present invention, in test kit, the primer shown in SEQ ID NO.1-67 is dense
Degree is 3.5pmol/ μ l for the primer concentration shown in 2.5pmol/ μ l, SEQ ID NO.70-136, SEQ
Primer concentration shown in ID NO.68,69,137 and 138 is 2.5pmol/ μ l.
In further preferred embodiment of the present invention, test kit comprises the DMSO of 5% further.
Another aspect of the present invention provides primer sets of the present invention and is combined in preparation detection leukemia fusion
Application in kit gene.
Further aspect of the present invention provides primer of the present invention combination, or reagent of the present invention
Box application in detection leukemia fusion gene.
Last aspect of the present invention provides a kind of multiplex nested RT-PCR detecting leukemia fusion gene
Method, it comprises the steps of
1, the cDNA template of testing sample is obtained.
2, use primer SEQ ID NO.1-69 of the present invention, or use reagent of the present invention
Box, the cDNA obtained in step 1, as template, carries out the amplification of first round PCR.
The primer of first round PCR amplification is Outside primer, is divided into 11 pipes, is often multi-primers in pipe,
Use the reaction system of 20 μ l: deionized water 8.18 μ l, 10 × Stanard Taq Reaction Buffer 2
μl、2.5mM dNTPs 1.6μl、25mM MgCl2 0.12μl、2.5pmol/μl Primes 2μl、
DMSO 1 μ l, cDNA Template 5 μ l, Hot Start Taq archaeal dna polymerase 0.1 μ l, instead
The condition is answered to be: first 95 DEG C 30s；Secondly 95 DEG C of 30s, 58 DEG C of 30s, 68 DEG C of 50s, totally 25
Circulation.
3, use primer SEQ ID NO.70-138 of the present invention, or use examination of the present invention
Agent box, the amplified production obtained in step 2, as template, carries out second and takes turns PCR amplification.
Second primer taking turns PCR amplification is inner primer, and the most corresponding 11 pipes that are divided into, often in pipe
Also multi-primers it is, the same reaction system using 20 μ l: deionized water 12.3 μ l, 10 × Stanard
Taq Reaction Buffer 2μl、2.5mM dNTPs 1.6μl、3.5pmol/μl Primes 2μl、
DMSO 1 μ l, the 1st take turns PCR primer 1 μ l, Hot Start Taq archaeal dna polymerase 0.1 μ l,
Reaction condition is: first 95 DEG C 30s；Secondly 95 DEG C of 30s, 58 DEG C of 30s, 68 DEG C of 50s, totally 20
Individual circulation.
4, second take turns pcr amplification product after electrophoresis, carry out result interpretation.
The present invention in the detection of multiplex nested RT-PCR, internal reference comparison primer for people's E2A gene,
And the first round and second take turns PCR amplification time, every Guan Zhongjun adds reference gene E2A primer, feminine gender
Then without any cDNA template in comparison (water).Therefore, when using electrophoresis to detect,
Only amplify the purpose band of 690bp when reference gene, and wherein 1 pipe can amplify corresponding
Fusion gene purpose band, when no template control product are without amplified signal, the testing result of sample to be tested
Just effectively and be positive.
In a preferred embodiment of the present invention, the primer concentration shown in SEQ ID NO.1-67 is
Primer concentration shown in 2.5pmol/ μ l, SEQ ID NO.70-136 is 3.5pmol/ μ l, SEQ ID
Primer concentration shown in NO.68,69,137 and 138 is 2.5pmol/ μ l.
In further preferred embodiment of the present invention, when the first round and second take turns PCR amplification,
The DMSO that final volume is 5% is all added, to improve PCR amplification efficiency and specificity in system.
Seen from the above description, compared with prior art, the present invention possesses following advantage.
1, compared with existing chromosome karyotype analysis and fluorescence in situ hybridization technique, the detection of the present invention
Method is based on multiplex nested RT-PCR technology, because the method is simple, quick, highly sensitive.
2, the detection method of the present invention is arranged in pairs or groups by rational primer, can be prevented effectively from multipair primer it
Between interaction, thus decrease detection error.
3, the detection method of the present invention can carry out qualitative detection to 43 kinds of leukemia fusion genes all sidedly,
It is possible not only to save reagent dosage, reduces testing cost, and detection range is wide, is suitable to clinically
Sample detects in high volume.
Accompanying drawing explanation
The operation principle schematic diagram of Fig. 1: 43 kinds of leukemia fusion gene RT-Nested PCR.
Fig. 2: embodiment 1 uses leukemia fusion gene positive cell line and the detection of negative cells system
Detection system of the present invention specific PCR product result figure.
Fig. 3: embodiment 2 uses leukemia fusion gene positive cell line and the detection of negative cells system
The PCR product result figure of detection system susceptiveness of the present invention.
Fig. 4: embodiment 3 use detection system of the present invention carry out the result figure of clinical sample detection.
Detailed description of the invention
Below by embodiment, the present invention is described in further detail, it is intended to is used for the present invention is described
And the non-limiting present invention.It should be pointed out that, to those skilled in the art, without departing from the present invention
On the premise of principle, it is also possible to the present invention is carried out some improvement and modification, these improve and modify also
Fall under the scope of the present invention equally.
Embodiment 1: utilize fusion gene positive cell line and negative cells system to detect detection bodies of the present invention
The specificity of system
The present embodiment with determine K562 positive cell line containing BCR-ABL1 fusion gene,
The Kasumi cell line of AML1-ETO fusion gene and the HL60 without fusion gene are negative thin
Born of the same parents system verifies feasibility and the specificity of detection method.Wherein K562 cell line, Kasumi
Cell line, HL60 cell line are purchased from Beijing DingGuo ChangSheng Biology Technology Co., Ltd.
The concrete detection method of the present embodiment is as follows:
1, cell is cultivated
K562 cell line, Kasumi cell line and HL60 are cultivated in the standard operation cultivated according to cell
Cell line, is placed in 37 DEG C, 5%CO2Cultivate in incubator.
2, nucleic acid extraction
Suggestion uses TIANGEN RNAprep Pure Blood Kit test kit, illustrates according to test kit
The RNA in cell line is extracted in book operation, carries out reverse transcription the most immediately.
3, reverse transcription
Suggestion uses Promega GoScriptTMReverse Transcriptase test kit, according to reagent
The operation of box description obtains cDNA.The cDNA Nuclease-Free Water of reverse transcription is carried out
Correspondingly dilution (calculating according to total rna concentration), the cDNA after dilution is used for carrying out nido
RT-PCR detects.
4, multiplex nested RT-PCR detection
(1) nest-type PRC the 1st takes turns reaction
The reaction system of 20ul is sequentially added into deionized water 8.18ul, 10 × Stanard Taq Reaction
Buffer 2ul、2.5mM dNTPs 1.6ul、25mM MgCl2 0.12ul、2.5pmol/ul Primes 2ul、
DMSO 1ul, cDNA Template 5ul, Hot Start Taq archaeal dna polymerase 0.1ul.
PCR reaction condition is: 95 DEG C of denaturations 30s；95 DEG C of degeneration 30s, 58 DEG C of annealing 30s, 68 DEG C
Extend 50s, totally 25 circulations.The product that PCR terminates is as the 2nd template taking turns reaction.
(2) nest-type PRC the 2nd takes turns reaction
The reaction system of 20ul is sequentially added into deionized water 12.3ul, 10 × Stanard Taq Reaction
Buffer 2ul, 2.5mM dNTPs 1.6ul, 3.5pmol/ul Primes 2ul, DMSO 1ul, the 1st
Wheel PCR primer 1ul, Hot Start Taq archaeal dna polymerase 0.1ul.
PCR reaction condition is: 95 DEG C of denaturations 30s；95 DEG C of degeneration 30s, 58 DEG C of annealing 30s, 68 DEG C
Extend 50s, totally 20 circulations；Last 68 DEG C of abundant extension 5min.
(3) electrophoresis detection
Agarose gel electrophoresis with 2% detects the 2nd and takes turns amplified production, and it is attached that testing result sees description
Fig. 2.Wherein Fig. 2 A is the fusion gene testing result of K562 positive cell line；Fig. 2 B is Kasumi
The fusion gene testing result of positive cell line；Fig. 2 C is the fusion gene inspection of HL60 negative cells system
Survey result；Fig. 2 D is the testing result of negative control (water).Swimming lane 1-11 represents 1-11 respectively
Product in pipe (R1-R11), M is 500bp standard molecular weight.
From accompanying drawing 2 it can be seen that Fig. 2 A, 2B and 2C, each swimming lane of R1-R11 expands
Go out the reference gene E2A of 690bp.K562 positive cell line contains the fusion base of BCR-ABL1
Cause, can amplify the purpose band of BCR-ABL1, as shown in Figure 2 A in R6 swimming lane；Kasumi
Positive cell line contains the fusion gene of AML1-ETO, R4 swimming lane can amplify
The purpose band of AML1-ETO, as shown in Figure 2 B；Without fusion gene in HL60 negative cells system,
R1-R11 swimming lane all can not expand purpose band, as shown in Figure 2 C；Negative control is without template amplification
Do not go out reference gene E2A and fusion gene, as shown in Figure 2 D.
As can be seen here, the testing result of the present embodiment and fusion gene positive cell line and negative cells system
Characteristic consistent, show that the detection system utilizing the present invention can specifically carry out leukemia fusion
The qualitative detection of gene.
Embodiment 2: utilize fusion gene positive cell line and negative cells system to detect detection bodies of the present invention
The sensitivity of system
The present embodiment equally with determine K562 positive cell line containing BCR-ABL1 fusion gene,
The Kasumi cell line of AML1-ETO fusion gene and the HL60 negative cells without fusion gene
System verifies the susceptiveness of detection system of the present invention.Concrete detection method is as follows:
With HL-60 cell line (the negative cells system without fusion gene) respectively to K562 cell line
(containing the positive cell line of BCR-ABL1 fusion gene) and Kasumi cell line (contain
The positive cell line of AML-ETO fusion gene) carry out 100%, 10%, 1%, 1 ‰, 0.5 ‰,
The dilution of 0.25 ‰, 0.125 ‰ (positive cell line and the ratios of negative cells system after dilution), with dilute
The cell mixing system released extracts RNA and reverse transcription becomes cDNA, carries out RT-Nested PCR detection.
Using nest-type PRC reaction system same as in Example 1 and reaction condition, second takes turns PCR
The agarose gel electrophoresis result of amplification afterproduct sees Figure of description 3.In Fig. 3 A, swimming lane 1-7 divides
Do not represent 100%K562,10%K562,1%K562,1 ‰ K562,0.5 ‰ K562,0.25 ‰
K562 and 0.125 ‰ K562；In Fig. 3 B swimming lane 1-7 represent respectively 100%Kasumi, 10%
Kasumi, 1%Kasumi, 1 ‰ Kasumi, 0.5 ‰ Kasumi, 0.25 ‰ Kasumi and 0.125 ‰
Kasumi, M are 500bp standard molecular weight.Fig. 3 A and 3B amplifies 690bp in each swimming lane
The swimming lane 1-6 of reference gene E2A, Fig. 3 A all amplify the BCR-ABL1 of 472bp and merge base
Cause, the swimming lane 1-6 of Fig. 3 B amplifies the AML1-ETO fusion gene of 353bp.
As can be seen here, when reaching 0.125 ‰ K562 or 0.125 ‰ Kasumi cells (8000 HL60
Containing 1 K562 or 1 Kasumi cell in cell) time, the multiplex nested RT-PCR of the present invention
Method can't detect corresponding fusion gene, shows the multiplex nested RT-PCR detection system of the present invention
Detection sensitivity between 1/4000-1/8000 (cancerous cell and normal cell number than).
Embodiment 3: use the detection system of the present invention to carry out the detection of clinical sample
The present embodiment gathers clinical mutations in leukemia patients by peripheral blood or marrow blood sample 150 example, according to this
Bright detection system carries out the qualitative detection of 43 kinds of fusion genes.Wherein 25 example clinical samples are through this
It is positive that bright multiplex nested RT-PCR is detected as fusion gene, and 125 examples are that fusion gene is negative.
Utilize the multiplex nested RT-PCR detection system detection clinical sample of the present invention, operating procedure bag
Include in blood sample leukocyte RNA extract, RNA reverse transcription be cDNA, nest-type PRC the 1st takes turns
Reaction, nest-type PRC the 2nd are taken turns reaction, are separated PCR product, agarose gel electrophoresis.Tool
The operational approach of body is with the embodiment of the present invention 1.The 2nd agarose gel electricity taking turns PCR amplification afterproduct
Swimming result sees Figure of description 4.Wherein Fig. 4 A is the multiplex nested RT-PCR inspection using the present invention
The electrophoresis result of the 5 kinds of Fusional gene isomerides measured；Fig. 4 B is the multiplex nested using the present invention
The electrophoresis result of other 4 kinds of Fusional gene isomerides that RT-PCR detects.Swimming lane 1 in Fig. 4 A, 3,
5,7,9 fusion genes represented successively be respectively CBFB-MYH11 (A type), E2A-PBX1,
SIL-TAL1, AML1-ETO and BCR-ABL1 (p190 type), swimming lane 2,4,6,8,10
Represent corresponding negative control successively.The fusion base that in Fig. 4 B, swimming lane 1,3,5,7 represents successively
Because of respectively BCR-ABL1 (p210 type), PML-RARA (L-type), PML-RARA (S type)
And DEK-CAN, swimming lane 2,4,6,8 represents corresponding negative control successively.M1 is 2000bp
Standard molecular weight, M2 is 500bp standard molecular weight.Fig. 4 A and 4B expands not in negative control
Go out reference gene E2A and fusion gene, positive sample can amplify reference gene E2A.
As can be seen here, the multiplex nested RT-PCR detection system of the present invention, the white blood detected are utilized
Sick fusion gene is consistent with clinical diagnosis, shows the multiplex nested RT-PCR detection system of the present invention
Can be completely applied to Clinical detection.
Result above shows, the multiplex nested RT-PCR detection system of the present invention can be carried out all sidedly
The qualitatively screening of 43 kinds of fusion genes of leukemia.The detection system of the present invention not only saves reagent and sample
This consumption, reduces testing cost simultaneously, has clinical value.
Claims (10)
1. one kind is combined for detecting the primer of leukemia fusion gene, and it is by SEQ ID NO.1-138
Shown primer composition.
Primer the most according to claim 1 combines, the wherein primer shown in SEQ ID NO.1-69
Composition first organizes greatly detection primer, the primer second largest group of detection of composition shown in SEQ ID NO.70-138
Primer；First group detection primer greatly is little by first shown in SEQ ID NO.1-8,68,69 further
Group detection primer, second group's detection primer shown in SEQ ID NO.9-15,68,69, SEQ ID
The 3rd group's detection primer shown in NO.16-21,68,69, SEQ ID NO.22-26,68,69
The 4th shown group's detection primer, the 5th group inspection shown in SEQ ID NO.27-33,68,69
Survey primer, the 6th group's detection primer shown in SEQ ID NO.34-38,68,69, SEQ ID
The 7th group's detection primer shown in NO.39-46,68,69, SEQ ID NO.47-50,68,69
The 8th shown group's detection primer, the 9th group inspection shown in SEQ ID NO.51-54,68,69
Survey primer, the tenth group's detection primer shown in SEQ ID NO.55-59,68,69, SEQ ID
The tenth a small group detection primer composition shown in NO.60-67,68,69；Second largest group of detection primer is entered
One step is by first group's detection primer shown in SEQ ID NO.70-77,137,138, SEQ ID
Second group's detection primer shown in NO.78-84,137,138, SEQ ID NO.85-90,137,
The 3rd group's detection primer shown in 138, the 4th shown in SEQ ID NO.91-95,137,138 is little
Group detection primer, the 5th group's detection primer shown in SEQ ID NO.96-102,137,138, SEQ
The 6th group's detection primer shown in ID NO.103-107,137,138, SEQ ID NO.108-115,
137, the 7th group's detection primer shown in 138, shown in SEQ ID NO.116-119,137,138
The 8th group's detection primer, shown in SEQ ID NO.120-123,137,138 the 9th group inspection
Survey primer, the tenth group's detection primer shown in SEQ ID NO.124-128,137,138, SEQ ID
The tenth a small group detection primer composition shown in NO.129-136,137,138.
3., for detecting a test kit for leukemia fusion gene, it comprises claim 1 or 2 institute
The primer combination stated.
Test kit the most according to claim 3, wherein the primer shown in SEQ ID NO.1-67 is dense
Degree is 3.5pmol/ μ l for the primer concentration shown in 2.5pmol/ μ l, SEQ ID NO.70-136, SEQ
Primer concentration shown in ID NO.68,69,137 and 138 is 2.5pmol/ μ l.
5., according to the test kit described in claim 3 or 4, it comprises the DMSO of 5% further.
6. the primer sets described in claim 1 or 2 is combined in preparation detection leukemia fusion gene test kit
In application.
7. the primer combination described in claim 1 or 2, or according to any one of claim 3-5
Test kit detection leukemia fusion gene in application.
8. detecting a multiplex nested RT-PCR method for leukemia fusion gene, it comprises following step
Rapid:
(1) the cDNA template of testing sample is obtained；
(2) use the primer SEQ ID NO.1-69 described in claim 1 or 2, or use right to want
Seek the test kit according to any one of 3-5, as template, carry out with the cDNA of acquisition in step (1)
First round PCR expands；
(3) use the primer SEQ ID NO.70-138 described in claim 1 or 2, or use right
Require the test kit according to any one of 3-5, with the amplified production of acquisition in step (2) as template,
Carry out second and take turns PCR amplification；
(4) second take turns pcr amplification product after electrophoresis, carry out result interpretation.
Method the most according to claim 8, the wherein primer concentration shown in SEQ ID NO.1-67
It is 3.5pmol/ μ l, SEQ ID for the primer concentration shown in 2.5pmol/ μ l, SEQ ID NO.70-136
Primer concentration shown in NO.68,69,137 and 138 is 2.5pmol/ μ l.
The most according to claim 8 or claim 9, method, wherein take turns PCR in the first round and second and expand
During increasing, in system, all add the DMSO that final volume is 5%.
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107217104A (en) * | 2017-07-17 | 2017-09-29 | 北京陆道培干细胞生物技术有限公司 | Leukemia fusion gene examination detection method |
| CN107245529A (en) * | 2017-08-08 | 2017-10-13 | 杭州千麦医学检验所有限公司 | Blood disease fusion screening method |
| CN107385042A (en) * | 2017-07-28 | 2017-11-24 | 广州永诺健康科技有限公司 | A kind of multiple PCR primer and method of grappling Nest multiplex PCR joint high-flux sequence detection Gene Fusion |
| CN107385031A (en) * | 2017-07-17 | 2017-11-24 | 北京陆道培干细胞生物技术有限公司 | Leukemia fusion gene examination detection kit |
| CN110820051A (en) * | 2018-12-28 | 2020-02-21 | 广州表观生物科技有限公司 | High-sensitivity fusion gene detection method and application thereof |
| CN114487212A (en) * | 2022-03-31 | 2022-05-13 | 深圳荻硕贝肯精准医学有限公司 | Detection method for detecting concentration of posaconazole in blood by adopting liquid chromatography-mass spectrometry |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO1998024928A3 (en) * | 1996-12-06 | 1999-03-25 | Niels Pallisgaard | Detection of chromosomal abnormalities |
| CN102533740A (en) * | 2011-09-07 | 2012-07-04 | 杭州艾迪康医学检验中心有限公司 | Reverse transcription-polymerase chain reaction (RT-PCR) primer of human leukemia fusion gene BCR-ABL and application method thereof |
| CN104178557A (en) * | 2013-05-28 | 2014-12-03 | 上海生物芯片有限公司 | Detection method for leukemia fusion genes, primers, probes and gene chip thereof |
-
2016
- 2016-04-22 CN CN201610254800.5A patent/CN105838793B/en active Active
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO1998024928A3 (en) * | 1996-12-06 | 1999-03-25 | Niels Pallisgaard | Detection of chromosomal abnormalities |
| CN102533740A (en) * | 2011-09-07 | 2012-07-04 | 杭州艾迪康医学检验中心有限公司 | Reverse transcription-polymerase chain reaction (RT-PCR) primer of human leukemia fusion gene BCR-ABL and application method thereof |
| CN104178557A (en) * | 2013-05-28 | 2014-12-03 | 上海生物芯片有限公司 | Detection method for leukemia fusion genes, primers, probes and gene chip thereof |
Non-Patent Citations (2)
| Title |
|---|
| NIELS PALLISGAARD等: "Multiplex Reverse Transcription-Polymerase Chain Reaction for Simultaneous Screening of 29 Translocations and Chromosomal Aberrations in Acute Leukemia", 《BLOOD》 * |
| 肖恒等: "白血病融合基因及检测方法研究进展", 《医学综述》 * |
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|---|---|---|---|---|
| CN107217104A (en) * | 2017-07-17 | 2017-09-29 | 北京陆道培干细胞生物技术有限公司 | Leukemia fusion gene examination detection method |
| CN107385031A (en) * | 2017-07-17 | 2017-11-24 | 北京陆道培干细胞生物技术有限公司 | Leukemia fusion gene examination detection kit |
| CN107385042A (en) * | 2017-07-28 | 2017-11-24 | 广州永诺健康科技有限公司 | A kind of multiple PCR primer and method of grappling Nest multiplex PCR joint high-flux sequence detection Gene Fusion |
| CN107385042B (en) * | 2017-07-28 | 2021-01-01 | 广州永诺健康科技有限公司 | Multi-PCR primer and method for detecting gene fusion by combining anchoring nest type multi-PCR with high-throughput sequencing |
| CN107245529A (en) * | 2017-08-08 | 2017-10-13 | 杭州千麦医学检验所有限公司 | Blood disease fusion screening method |
| CN110820051A (en) * | 2018-12-28 | 2020-02-21 | 广州表观生物科技有限公司 | High-sensitivity fusion gene detection method and application thereof |
| CN110820051B (en) * | 2018-12-28 | 2023-04-28 | 广州表观生物科技有限公司 | High-sensitivity fusion gene detection method and application thereof |
| CN114487212A (en) * | 2022-03-31 | 2022-05-13 | 深圳荻硕贝肯精准医学有限公司 | Detection method for detecting concentration of posaconazole in blood by adopting liquid chromatography-mass spectrometry |
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