CN105838793B - Primer, kit and method for qualitative detection leukemia fusion gene - Google Patents
Primer, kit and method for qualitative detection leukemia fusion gene Download PDFInfo
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Abstract
The invention belongs to gene engineering technology fields, disclose a kind of primer combination, the kit containing primer combination and multiplex nested RT-PCR method for using primer combination or kit to carry out leukemia fusion gene detection for detecting leukemia fusion gene.The present invention is based on multiplex nested RT-PCR technologies, therefore simple, quick, high sensitivity.In addition, the present invention is arranged in pairs or groups by reasonable primer, the interaction between multipair primer is effectively prevented, detection error is reduced.Qualitative detection comprehensively can be carried out to 43 kinds of leukemia fusion genes using detection method of the invention, reagent dosage can be not only saved, testing cost be reduced, and detection range is wide, suitable for detecting to clinically high-volume sample.
Description
Technical field
The present invention relates to gene engineering technology fields, and in particular to for qualitative detection leukemia fusion gene primer,
Kit and method.
Background technique
Leukaemia is one of the most common type malignant clone disease in children and youth.Multiple studies have shown that most white
There are the distortion of certain chromosome structure (missing, repetition, inversion, transposition etc.) in blood patient, these are that leukaemia is caused to be sent out
Raw and development major reason.The distortion of chromosome structure will lead to the structure variation of proto-oncogene and tumor suppressor gene, so that
Oncogene, Oncogene Mutation activation, tumor suppressor gene missing or inactivation, generate new fusion, encoding fusion protein.Thus
Different fusions has become the molecular biology specificity marker of different type leukaemia.Such as chronic myelocytic leukemia
(CML) the BCR-ABL1 fusion in, the PML-RARA fusion in acute promyelocytic leukemia (APL) have become
Respective molecular marker and the target for further becoming treatment.WHO in 2000 about in the standard of leukemia classification be even more by its
In some common exceptions be summarized as one of the standard of leukaemia basic diagnosis.In view of the detection pair of leukaemia correlation fusion gene
Clinical valence in leukemia diagnosis, parting, clinical treatment selection, prognosis therapeutic evaluation and minimal residual disease (MRD) detection
The exploitation of value, fusion detection technique has great clinical meaning and market value.
Currently, the detection method of fusion mainly includes chromosome karyotype analysis, fluorescence in situ hybridization technique and gathers
Polymerase chain reacts (PCR).Compared with chromosome karyotype analysis and fluorescence in situ hybridization technique, PCR detection method has quick
Effectively, the advantages that high sensitivity, clinically there is wider application.Multiple PCR technique refers to be added in the same PCR system
Multipair primer can amplify multiple DNA fragmentations simultaneously.The deriving technology important as PCR, multiplex PCR can expand simultaneously
Multiple target gene have the advantages that save the time, reduce cost, improve efficiency.Nest-type PRC is a kind of round pcr of variation,
It uses two pairs of complete segments of primer amplification.First pair of primer amplification segment is similar with regular-PCR.Second pair of primer is known as nest
Formula primer is incorporated in the inside of first time PCR product, expands for the first time so that the second amplified fragments are shorter than.Nest-type PRC it is excellent
Point is, if amplification produces false segments for the first time, can carry out primer pairing in false segments for the second time and expand
Probability it is extremely low.Therefore, nest-type PRC can be improved the sensitivity of PCR and reduce non-specific amplification.
However, multipair primer is added in the same PCR reaction system in multiplex-nested PCR technology needs, it is increased by this way
Multipair primer interacts and is formed in the reaction system the probability of primer dimer, to reduce the special of PCR reaction
Property and efficiency, or even specific targets product cannot be amplified.In addition, as people deepen continuously to leukaemia research, more
It is had been found come more leukemia fusion genes, the method for current detection leukemia fusion gene has been far from satisfying people
For detecting the demands of most fusions simultaneously, need it is a kind of quickly, effectively and be easy to standardized and can determine simultaneously
Property detect most leukemia fusion genes method.
Summary of the invention
The present invention in view of the above defects of the prior art, is based on newest leukemia fusion gene database, weight
Newly devise the detection primer and multiplex nested RT-PCR method for detecting leukemia fusion gene.Detection side of the invention
Method is quickly, effectively and the qualitative detection that is easy to be standardized 43 kinds of leukemia fusion genes.
For this purpose, one aspect of the present invention provides a kind of primer combination for detecting leukemia fusion gene, by SEQ
The composition of primer shown in ID NO.1-138.
In a preferred embodiment of the present invention, primer composition first shown in SEQ ID NO.1-69 in primer combination
Big to organize detection primer, primer shown in SEQ ID NO.70-138 forms second largest group of detection primer；First big group detection primer
Further first group's detection primer as shown in SEQ ID NO.1-8,68,69, shown in SEQ ID NO.9-15,68,69
Second group's detection primer, third group detection primer shown in SEQ ID NO.16-21,68,69, SEQ ID NO.22-26,
68, the 4th group's detection primer shown in 69, the 5th group's detection primer shown in SEQ ID NO.27-33,68,69, SEQ
6th group's detection primer shown in ID NO.34-38,68,69, the inspection of the 7th group shown in SEQ ID NO.39-46,68,69
Survey primer, the 8th group's detection primer shown in SEQ ID NO.47-50,68,69, shown in SEQ ID NO.51-54,68,69
The 9th group's detection primer, the tenth group's detection primer shown in SEQ ID NO.55-59,68,69, SEQ ID NO.60-
67, the tenth a small group detection primer shown in 68,69 forms；Second largest group of detection primer further by SEQ ID NO.70-77,
137, first group's detection primer shown in 138, second group's detection primer shown in SEQ ID NO.78-84,137,138,
Third group detection primer shown in SEQ ID NO.85-90,137,138, shown in SEQ ID NO.91-95,137,138
Four group's detection primers, the 5th group's detection primer shown in SEQ ID NO.96-102,137,138, SEQ ID NO.103-
107, the 6th group's detection primer shown in 137,138, the detection of the 7th group shown in SEQ ID NO.108-115,137,138
Primer, the 8th group's detection primer shown in SEQ ID NO.116-119,137,138, SEQ ID NO.120-123,137,
9th group's detection primer shown in 138, the tenth group's detection primer shown in SEQ ID NO.124-128,137,138, SEQ
The composition of tenth a small group detection primer shown in ID NO.129-136,137,138.
The present invention is by selection related gene broken site upstream sequence and downstream sequence, to carry out specific multiplex PCR
The design of primer.Designed primer Tm is near 60 DEG C, each primer of integration collocation, to avoid primer two as far as possible
Aggressiveness generates, and improves the specificity and efficiency of PCR amplification.
For 43 kinds of leukemia fusion genes, the specific primer situation that the present invention designs is as shown in table 1.
Table 1, primer situation table of the invention
Another aspect of the present invention provides a kind of for detecting the kit of leukemia fusion gene, and it includes institutes of the present invention
The primer combination stated.
In a preferred embodiment of the present invention, primer concentration shown in SEQ ID NO.1-67 is in kit
Primer concentration shown in 2.5pmol/ μ l, SEQ ID NO.70-136 is 3.5pmol/ μ l, the SEQ and of ID NO.68,69,137
Primer concentration shown in 138 is 2.5pmol/ μ l.
5% DMSO is further included in further preferred embodiment of the present invention, in kit.
Another aspect of the present invention provides primer combination of the present invention and detects leukemia fusion gene reagent in preparation
Application in box.
Further aspect of the present invention provides primer combination of the present invention or kit of the present invention is detecting
Application in leukemia fusion gene.
Last aspect of the present invention provides a kind of multiplex nested RT-PCR method for detecting leukemia fusion gene, packet
Containing following steps:
1, the cDNA template of sample to be tested is obtained.
2, using primer SEQ ID NO.1-69 of the present invention, or kit of the present invention is used, with step 1
The cDNA of middle acquisition is template, carries out first round PCR amplification.
The primer of first round PCR amplification is Outside primer, is divided into 11 pipes, is multi-primers in every pipe, using 20 μ l's
Reaction system: 8.18 μ l of deionized water, 10 × Stanard Taq Reaction Buffer, 2 μ l, 1.6 μ of 2.5mM dNTPs
l、25mM MgCl2 0.12μl、2.5pmol/μl Primes 2μl、DMSO 1μl、cDNA Template 5μl、Hot
0.1 μ l of Start Taq archaeal dna polymerase, reaction condition are as follows: 95 DEG C of 30s first；Secondly 95 DEG C of 30s, 58 DEG C of 30s, 68 DEG C of 50s,
Totally 25 circulations.
3, using primer SEQ ID NO.70-138 of the present invention, or kit of the present invention is used, with step
The amplified production obtained in rapid 2 is template, carries out the second wheel PCR amplification.
The primer of second wheel PCR amplification is inner primer, similarly corresponding to be divided into 11 pipes, is also multiple draw in every pipe
Object, the same reaction system for using 20 μ l: 12.3 μ l of deionized water, 10 × Stanard Taq Reaction Buffer, 2 μ l,
1.6 μ l of 2.5mM dNTPs, 2 μ l of 3.5pmol/ μ l Primes, 1 DMSO μ l, the 1st wheel 1 μ l of PCR product, Hot Start
0.1 μ l of Taq archaeal dna polymerase, reaction condition are as follows: 95 DEG C of 30s first；Secondly 95 DEG C of 30s, 58 DEG C of 30s, 68 DEG C of 50s, totally 20
Circulation.
4, the second wheel pcr amplification product carries out result interpretation after electrophoresis.
In the detection of multiplex nested RT-PCR, internal reference compares primer and is directed to people E2A gene, and the first round and the present invention
When two wheel PCR amplifications, reference gene E2A primer is added in every Guan Zhongjun, does not add any cDNA template then in negative control (water).
Therefore, when being detected using electrophoresis, only when reference gene amplifies the purpose band of 690bp, and wherein can in 1 pipe
Corresponding fusion purpose band is amplified, when no template control product are without amplified signal, the testing result of sample to be tested just has
It imitates and is positive.
In a preferred embodiment of the present invention, primer concentration shown in SEQ ID NO.1-67 is 2.5pmol/ μ l, SEQ
Primer concentration shown in ID NO.70-136 is primer concentration shown in 3.5pmol/ μ l, SEQ ID NO.68,69,137 and 138
For 2.5pmol/ μ l.
In further preferred embodiment of the present invention, in the first round and the second wheel PCR amplification, it is added in system
The DMSO that final volume is 5%, to improve PCR amplification efficiency and specificity.
Seen from the above description, compared with prior art, the present invention has following advantage.
1, compared with existing chromosome karyotype analysis and fluorescence in situ hybridization technique, detection method of the invention is based on more
Nest type RT-PCR technology, because the method is simple, quick, high sensitivity.
2, detection method of the invention is arranged in pairs or groups by reasonable primer, it is possible to prevente effectively from the phase interaction between multipair primer
With to reduce detection error.
3, detection method of the invention comprehensively can carry out qualitative detections to 43 kinds of leukemia fusion genes, not only can be with
Reagent dosage is saved, testing cost is reduced, and detection range is wide, suitable for detecting to clinically high-volume sample.
Detailed description of the invention
The operation principle schematic diagram of Fig. 1: 43 kinds of leukemia fusion gene RT-Nested PCRs.
Fig. 2: using leukemia fusion gene positive cell line and negative cells system detection present invention detection body in embodiment 1
It is the PCR reaction product result figure of specificity.
Fig. 3: using leukemia fusion gene positive cell line and negative cells system detection present invention detection body in embodiment 2
It is the PCR reaction product result figure of sensitivity.
Fig. 4: the result figure of clinical sample detection is carried out in embodiment 3 using detection architecture of the present invention.
Specific embodiment
Below by embodiment, the present invention is described in further detail, it is intended to limit this for illustrating rather than
Invention.It should be pointed out that those skilled in the art, it without departing from the principle of the present invention, can also be to this hair
Bright some improvement and modification can also be carried out, these improvement and modification are similarly fallen under the scope of the present invention.
Embodiment 1: the specificity of detection architecture of the present invention is detected using fusion positive cell line and negative cells system
The present embodiment is to determine K562 positive cell line containing BCR-ABL1 fusion, AML1-ETO fusion
Kasumi cell line and HL60 negative cells system without fusion verify feasibility and the spy of detection method
It is anisotropic.Wherein K562 cell line, Kasumi cell line, HL60 cell line are purchased from Beijing ancient cooking vessel state prosperity biotechnology Limited Liability
Company.
The specific detection method is as follows for the present embodiment:
1, cell culture
According to standard operation culture K562 cell line, Kasumi cell line and the HL60 cell line of cell culture, it is placed in
37 DEG C, 5%CO2Culture in incubator.
2, nucleic acid extraction
It is recommended that using TIANGEN RNAprep Pure Blood Kit kit, mentioned according to kit specification operation
The RNA in cell line is taken, carries out reverse transcription immediately later.
3, reverse transcription
It is recommended that using Promega GoScriptTMReverse Transcriptase kit, illustrates according to kit
Book operates to obtain cDNA.The cDNA of reverse transcription is correspondingly diluted (dense according to total serum IgE with Nuclease-Free Water
Degree is calculated), the cDNA after dilution is used to carry out RT-Nested PCR detection.
4, multiplex nested RT-PCR is detected
(1) wheel of nest-type PRC the 1st reaction
Deionized water 8.18ul, 10 × Stanard Taq Reaction are sequentially added in the reaction system of 20ul
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 are as follows: 95 DEG C of initial denaturation 30s；95 DEG C of denaturation 30s, 58 DEG C of annealing 30s, 68 DEG C of extension 50s, totally 25
A circulation.Template of the product that PCR terminates as the 2nd wheel reaction.
(2) wheel of nest-type PRC the 2nd reaction
Deionized water 12.3ul, 10 × Stanard Taq Reaction are sequentially added in the reaction system of 20ul
Buffer 2ul, 2.5mM dNTPs 1.6ul, 3.5pmol/ul Primes 2ul, DMSO 1ul, the 1st wheel PCR product 1ul,
Hot Start Taq archaeal dna polymerase 0.1ul.
PCR reaction condition are as follows: 95 DEG C of initial denaturation 30s；95 DEG C of denaturation 30s, 58 DEG C of annealing 30s, 68 DEG C of extension 50s, totally 20
A circulation；Last 68 DEG C sufficiently extend 5min.
(3) electrophoresis detection
With 2% the 2nd wheel amplified production of agarose gel electrophoresis detection, testing result is referring to Figure of description 2.Wherein scheme
2A is the fusion testing result of K562 positive cell line；Fig. 2 B is that the fusion of Kasumi positive cell line detects knot
Fruit；Fig. 2 C is the fusion testing result of HL60 negative cells system；Fig. 2 D is the testing result of negative control (water).Swimming lane 1-
11 respectively represent the reaction product in 1-11 pipe (R1-R11), and M is 500bp standard molecular weight.
From attached drawing 2 as can be seen that amplifying the reference gene of 690bp in each swimming lane of R1-R11 in Fig. 2A, 2B and 2C
E2A.Containing the fusion of BCR-ABL1 in K562 positive cell line, the purpose item of BCR-ABL1 can be amplified in R6 swimming lane
Band, as shown in Figure 2 A；Containing the fusion of AML1-ETO in Kasumi positive cell line, can be amplified in R4 swimming lane
The purpose band of AML1-ETO, as shown in Figure 2 B；Fusion is free of in HL60 negative cells system, is all expanded in R1-R11 swimming lane
Do not go out purpose band, as shown in Figure 2 C；Negative control does not go out reference gene E2A and fusion without template amplification, such as Fig. 2 D institute
Show.
It can be seen that the characteristic phase one of the testing result of the present embodiment and fusion positive cell line and negative cells system
It causes, shows the qualitative detection that can specifically carry out leukemia fusion gene using detection architecture of the invention.
Embodiment 2: the sensitivity of detection architecture of the present invention is detected using fusion positive cell line and negative cells system
The present embodiment is equally to determine that the K562 positive cell line containing BCR-ABL1 fusion, AML1-ETO merge base
The Kasumi cell line of cause and HL60 negative cells system without fusion verify the sensitivity of detection architecture of the present invention.
The specific detection method is as follows:
(BCR-ABL1 is contained to K562 cell line respectively with HL-60 cell line (the negative cells system without fusion)
The positive cell line of fusion) and Kasumi cell line (positive cell line containing AML-ETO fusion) progress
100%, 10%, 1%, 1 ‰, 0.5 ‰, 0.25 ‰, 0.125 ‰ (ratios of positive cell line and negative cells system after dilution)
Dilution, extracts RNA with the cell mixing system diluted and reverse transcription is at cDNA, carries out RT-Nested PCR detection.
The fine jade of product after PCR amplification is taken turns using nested PCR reaction system and reaction condition same as Example 1, second
Sepharose electrophoresis result is referring to Figure of description 3.Swimming lane 1-7 respectively represents 100%K562,10%K562,1% in Fig. 3 A
K562,1 ‰ K562,0.5 ‰ K562,0.25 ‰ K562 and 0.125 ‰ K562；Swimming lane 1-7 respectively represents 100% in Fig. 3 B
Kasumi, 10%Kasumi, 1%Kasumi, 1 ‰ Kasumi, 0.5 ‰ Kasumi, 0.25 ‰ Kasumi and 0.125 ‰
Kasumi, M are 500bp standard molecular weight.Reference gene E2A, Fig. 3 A of 690bp is amplified in Fig. 3 A and 3B in each swimming lane
Swimming lane 1-6 all amplify the BCR-ABL1 fusion of 472bp, the swimming lane 1-6 of Fig. 3 B amplifies the AML1-ETO of 353bp
Fusion.
It can be seen that (containing 1 in 8000 HL60 cells when reach 0.125 ‰ K562 or 0.125 ‰ Kasumi cells
K562 or 1 Kasumi cell) when, multiplex nested RT-PCR method of the invention can't detect corresponding fusion, show
The detection sensitivity of multiplex nested RT-PCR detection architecture of the invention is between 1/4000-1/8000 (cancer cell and normal cell
Number ratio) between.
Embodiment 3: the detection of clinical sample is carried out using detection architecture of the invention
The present embodiment acquires clinical mutations in leukemia patients by peripheral blood or marrow blood sample 150, detection body according to the invention
System carries out the qualitative detection of 43 kinds of fusions.Wherein 25 clinical samples are detected as melting through multiplex nested RT-PCR of the invention
Gene masculine is closed, 125 negative for fusion.
Clinical sample is detected using multiplex nested RT-PCR detection architecture of the invention, operating procedure includes in blood sample
Leucocyte RNA is extracted, RNA reverse transcription is cDNA, the reaction of the wheel of nest-type PRC the 1st, nest-type PRC the 2nd takes turns reaction, separation PCR reaction produces
Object, agarose gel electrophoresis.Specific operating method is the same as the embodiment of the present invention 1.The agarose of product is solidifying after 2nd wheel PCR amplification
Gel electrophoresis result is referring to Figure of description 4.Wherein Fig. 4 A is the 5 kinds of fusions detected using multiplex nested RT-PCR of the invention
The electrophoresis result of gene isomers；Fig. 4 B is the other 4 kinds of fusions detected using multiplex nested RT-PCR of the invention
The electrophoresis result of isomers.The fusion that swimming lane 1,3,5,7,9 successively represents in Fig. 4 A be respectively CBFB-MYH11 (A type),
E2A-PBX1, SIL-TAL1, AML1-ETO and BCR-ABL1 (p190 type), swimming lane 2,4,6,8,10 successively represent corresponding yin
Property control.The fusion that swimming lane 1,3,5,7 successively represents in Fig. 4 B is respectively BCR-ABL1 (p210 type), PML-RARA (L
Type), PML-RARA (S type) and DEK-CAN, swimming lane 2,4,6,8 successively represent corresponding negative control.M1 is 2000bp standard
Molecular weight, M2 are 500bp standard molecular weight.Reference gene E2A and fusion can not be expanded in negative control in Fig. 4 A and 4B,
Reference gene E2A can be amplified in positive sample.
It can be seen that using multiplex nested RT-PCR detection architecture of the invention, the leukemia fusion gene detected with
Clinical diagnosis is consistent, shows that multiplex nested RT-PCR detection architecture of the invention can be completely applied to clinical detection.
Melt the above result shows that multiplex nested RT-PCR detection architecture of the invention can comprehensively carry out 43 kinds of leukaemia
Close the qualitatively screening of gene.The detection architecture of the present invention not only saves the dosage of reagent and sample, at the same reduce detection at
This, has clinical value.
Claims (5)
1. a kind of primer for detecting leukemia fusion gene combines, the primer shown in SEQ ID NO.1-138 is formed,
Wherein the composition of primer shown in SEQ ID NO.1-69 first organizes greatly detection primer, primer sets shown in SEQ ID NO.70-138
At second largest group of detection primer；First organizes greatly detection primer further first group as shown in SEQ ID NO.1-8,68,69
Detection primer, second group's detection primer shown in SEQ ID NO.9-15,68,69, shown in SEQ ID NO.16-21,68,69
Third group detection primer, the 4th group's detection primer shown in SEQ ID NO.22-26,68,69, SEQ ID NO.27-
33, the 5th group's detection primer shown in 68,69, the 6th group's detection primer shown in SEQ ID NO.34-38,68,69,
7th group's detection primer shown in SEQ ID NO.39-46,68,69, the 8th is small shown in SEQ ID NO.47-50,68,69
Organize detection primer, the 9th group's detection primer shown in SEQ ID NO.51-54,68,69, SEQ ID NO.55-59,68,69
Shown in the tenth group's detection primer, the composition of the tenth a small group detection primer shown in SEQ ID NO.60-67,68,69；Second
It is big to organize detection primer further first group's detection primer as shown in SEQ ID NO.70-77,137,138, SEQ ID
Second group's detection primer shown in NO.78-84,137,138, third group shown in SEQ ID NO.85-90,137,138
Detection primer, the 4th group's detection primer shown in SEQ ID NO.91-95,137,138, SEQ ID NO.96-102,137,
5th group's detection primer shown in 138, the 6th group's detection primer shown in SEQ ID NO.103-107,137,138, SEQ
7th group's detection primer shown in ID NO.108-115,137,138, shown in SEQ ID NO.116-119,137,138
Eight group's detection primers, the 9th group's detection primer shown in SEQ ID NO.120-123,137,138, SEQ ID NO.124-
128, the tenth group's detection primer shown in 137,138, the inspection of the tenth a small group shown in SEQ ID NO.129-136,137,138
Survey primer composition.
2. a kind of for detecting the kit of leukemia fusion gene, it includes primer described in claim 1 combinations.
3. kit according to claim 2, wherein primer concentration shown in SEQ ID NO.1-67 is 2.5pmol/ μ l,
Primer concentration shown in SEQ ID NO.70-136 is primer shown in 3.5pmol/ μ l, SEQ ID NO.68,69,137 and 138
Concentration is 2.5pmol/ μ l.
4. kit according to claim 2 or 3 further includes 5% DMSO.
5. application of the primer combination described in claim 1 in preparation detection leukemia fusion gene kit.
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| 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 |
| 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 |
| 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 |
| 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 |
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