CN107385080A - A kind of leukemia of children correlation fusion gene multiple PCR detection kit and method - Google Patents
A kind of leukemia of children correlation fusion gene multiple PCR detection kit and method Download PDFInfo
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Abstract
The present invention provides a kind of leukemia of children correlation fusion gene multiple PCR detection kit and method, described kit includes being used to expand 8 kinds of leukemia of children correlation fusion gene C BFB MYH11, SIL TAL1, TEL AML1, BCR ABL, E2A PBX1, PML RARa, AML1 ETO, MLL AF4 chimeric primers, chimeric primers IC and universal primer for amplification interior label, kit provided by the invention can disposably detect 8 kinds of leukemia of children correlation fusion genes, reduce testing cost, save detection time, it is adapted to the conventional screening of leukemia of children patient, and amplification flux and detection sensitivity are higher；And the present invention uses specific primer sequence, reliability, the detection method for ensureing testing result are simple to operate, time saving and energy saving, detection flux is big, reagent consumables cost is low, and directly the nucleic acid of leukemia of children peripheral blood in patients sample extraction can be detected；It is low to detection platform and artificial technology's level requirement, it can be widely popularized in routine screening and detection.
Description
Technical field
The present invention relates to a kind of detection kit technical field, especially a kind of leukemia of children correlation fusion gene is multiple
PCR detection kit and method.
Background technology
Leukaemia is the most common malignant tumour of children, wherein ALL
(AcuteLymphoblastic Leukemia, ALL) accounts for 80% or so, and crowd's incidence of disease reaches 0.3~0.4/ ten thousand in children
People, so far, the fusion that report is related to the related chromosome translocation of leukaemia, inversion is formed is up to kind more than 50, including t
(9；22)、t(12；21)、t(1；19)、t(4；11)、t(9；9)、t(16；21) etc., common leukemia of children correlation fusion base
Because mainly there is t (12；21) TEL-AML1 fusions, the t (1 of transposition formation；19) transposition formed E2A-PBX1 fusions,
t(4；11) MLL-AF4 fusions, the t (9 of transposition formation；22) m-BCR-ABL fusions, the t (8 of transposition formation；21) easily
AML1-ETO fusions, the t (15 of position formation；17) PML-RAR alpha fusion genes, the inv (16)/t (16 of transposition formation；16)
CBF β-MYH11 the fusions that inversion is formed；Wherein, first 4 kinds are the related main fusions of children ALL, and leukaemia is related
Fusion is one of specific molecular marker of leukaemia, is the independent indication of leukemia of children disease parting.
At present, fusion is detected both at home and abroad conventional method mainly include leukaemia's chromosome karyotype analysis,
Chromosome fluorescence in-situ hybridization (Fluorescent In Situ Hybridazation, FISH) technology and polymerase chain
Reaction technology (Polymerase Chain Reaction, PCR).
The positive rate of chromosomal G-banding karyotyping technology depends on the hyperplasia rate of leukaemia to be checked, usually
Influenceed by detection technique condition and manual operation factor, every part of detection sample needs at least to analyze 10-30 metaphase
Cell, and because the cell in sample is also all not homogeneous tumour cell, therefore can not then enter when cell concentration is very few
Row karyotyping, at the same the identification of some complexity chromosome translocations technically exist certain difficulty (XU Li Xia,
2009), but karyotyping technology has advantage in terms of the chromosome abnormality such as detection missing and numerical aberration, is that detection ALL suffers from
The essential means of person's chromosome abnormality；
Chromosome fluorescence in-situ hybridization technology be according to nucleic acid base complementary pairing principle, with hapten-marked DNA or
Person's rna probe and the single strand nucleotide sequence complementary pairing of denaturation, go to identify that haptens is carried out by the antibody with fluorophor
Detection, or probe is directly marked with fluorophor and combined with target sequence, finally directly observe mesh using fluorescence microscope
Mark sequence in nucleus, chromosome or biopsy tissues distribution situation (HE Shi-Bin, 2014；Krauter J, 1998),
FISH technology development early stage, is to apply labelled with radioisotope probe, fluorescence in situ hybridization detection fusion technology is 20
A kind of nonradioactivein situhybridization technology that the latter stage eighties in century grows up on the basis of radioactive in situ hybridization, the skill
Art have the characteristics that detection signal is strong, hybrid specificities are high and can multiple staining mark, be widely used in the assignment of genes gene mapping,
In the research such as chromosome identification, the structure of physical map, evolutionary analysis.But due to fluorescence probe is expensive, experimental implementation compared with
To be cumbersome and batch Samples detection can not be carried out, thus application of the technology in clinic also receive it is a certain degree of
Limitation.
PCR (Polymerase Chain Reaction, PCR) is to utilize DNA 95 DEG C of high temperature in vitro
Time variation can become single-stranded, and primer is combined with single-stranded by the principle of base pair complementarity during low temperature (often 60 DEG C or so), then
Temperature regulating is to archaeal dna polymerase optimal reactive temperature (72 DEG C or so), and archaeal dna polymerase is along phosphoric acid to pentose (5'-3') side
To synthesis complementary strand, two primers of two complementary strand difference complementary pairings of design and template DNA can be to the DNA between primer
Fragment carries out amplification in vitro.PCR detection techniques high sensitivity, high specificity, simple to operate, it is effective that it has become gene diagnosis
And sensitive method, there is unrivaled advantage in the context of detection of leukaemia correlation fusion gene, leukaemia is melted at present
The research trend of genetic test is closed in round pcr, amplified production by sequencing can also obtain sequence information (Natini,
2009), this fusion research complicated to situation is helpful, and quantitative fluorescent PCR passes through fluorescent dye or fluorescence labeling
Specific probe, PCR primer is marked tracking, real time and on line monitoring course of reaction can be right with reference to corresponding software
Product is analyzed.Sharon is drawn by fluorescence quantitative PCR detection ALL using fluorescence probe and two
One amplified fragments of determination, sensitivity and specificity are all very high (Sharon, 2005) simultaneously for thing.But for increasing new
Leukemia fusion gene broken site, it is meant that changeable expanding fragment length, it is desirable to cover fusion feelings as much as possible
Condition, fragment length excursion is very big, but fluorescent quantitative PCR fragment is preferably within 200bp, therefore can only detect common
Type, the fluorescent PCR detection currently for fusion is mostly single fusion.It is white using multiple PCR method detection at present
Blood disease fusion has a small amount of report, multiplex PCR (multiplex PCR), also known as Multiplex PCR, is by two pairs or more
Specific primer add in same PCR reaction systems, while expand the different target genes in a sample, have high efficiency,
The advantages of systematicness, accuracy and economical and convenient, meet the clinical requirement to molecular diagnostic techniques, such as screening can divide simultaneously
The chromosome translocation of virgin ALL risk factors, including t (I；I9)、t(4；II)、t(9；22)、t(I2；2I) etc., Fei et al. is sharp first
, finally can by flow cytometer with multiplex RT-PCR amplification, then the fluorogenic probe hybridzation by amplified production from different passages
The PCR fragment combined with probe is detected, so as to detect corresponding fusion (Fei Ye, 2012), but whole process
Need 5-6 hour, the time is relatively long, and due to multiplex PCR be added in same PCR reaction systems multipair primer with
The PCR reactions of multiple DNA fragmentations are expanded, therefore the multipair primer in same reaction system easily interacts, and such as forms hair
Card structure, dimeric structure etc., primer pair and primer amount are more, and the interaction between primer is more obvious, expand so as to influence PCR
Increasing Efficiency, and then have impact on the extensive use of multiplex PCR.
The content of the invention
In view of the shortcomings of the prior art, the present invention provides a kind of high specificity, high sensitivity, flux high leukemia of children
Correlation fusion gene multiple PCR detection kit and method, the kit can disposably detect leukemia of children patient peripheral
8 kinds of leukemia of children correlation fusion gene Cs BFB-MYH11, SIL-TAL1, TEL-AML1, BCR-ABL, E2A- in blood sample
PBX1, PML-RARa, AML1-ETO and MLL-AF4, so as to realize Multiple detection.
In order to solve the above technical problems, the present invention adopts the following technical scheme that：
A kind of leukemia of children correlation fusion gene multiple PCR detection kit, including following primer pair：
(1) chimeric primers of CBFB-MYH11 genes are detected：
CBFB-MYH11_F, its nucleotide sequence such as SEQ ID NO：Shown in 1；
CBFB-MYH11_R, its nucleotide sequence such as SEQ ID NO：Shown in 2；
(2) chimeric primers of SIL-TAL1 genes are detected：
SIL-TAL1_F, its nucleotide sequence such as SEQ ID NO：Shown in 3；
SIL-TAL1_R, its nucleotide sequence such as SEQ ID NO：Shown in 4；
(3) chimeric primers of TEL-AML1 genes are detected：
TEL-AML1_F, its nucleotide sequence such as SEQ ID NO：Shown in 5；
TEL-AML1_R, its nucleotide sequence such as SEQ ID NO：Shown in 6；
(4) chimeric primers of BCR-ABL genes are detected：
M-bcr_F, its nucleotide sequence such as SEQ ID NO：Shown in 7；
M-bcr_F, its nucleotide sequence such as SEQ ID NO：Shown in 8；
Bcr_R, its nucleotide sequence such as SEQ ID NO：Shown in 9；
(5) chimeric primers of E2A-PBX1 genes are detected：
E2A-PBX1_F, its nucleotide sequence such as SEQ ID NO：Shown in 10；
E2A-PBX1_R, its nucleotide sequence such as SEQ ID NO：Shown in 11；
(6) chimeric primers of PML-RARa genes are detected：
PML-RARa_F, its nucleotide sequence such as SEQ ID NO：Shown in 12；
PML-RARa_R, its nucleotide sequence such as SEQ ID NO：Shown in 13；
(7) chimeric primers of AML1-ETO genes are detected：
AML1-ETO_F, its nucleotide sequence such as SEQ ID NO：Shown in 14；
AML1-ETO_R, its nucleotide sequence such as SEQ ID NO：Shown in 15；
(8) chimeric primers of MLL-AF4 genes are detected：
MLL-AF4_F, its nucleotide sequence such as SEQ ID NO：Shown in 16；
MLL-AF4_R, its nucleotide sequence such as SEQ ID NO：Shown in 17；
(9) the chimeric primers IC of internal standard gene：
IC_F, its nucleotide sequence such as SEQ ID NO：Shown in 18；
IC_R, its nucleotide sequence such as SEQ ID NO：Shown in 19；
(10) universal primer：
Tongyong_F, its nucleotide sequence such as SEQ ID NO：Shown in 20；
Tongyong_R, its nucleotide sequence such as SEQ ID NO：Shown in 21；
Above-mentioned primer is used in conjunction with one-time detection.
Described multiple PCR detection kit also include positive control, negative control, enzyme system, PCR reaction buffers and
DEPC water；
Described positive control is 8 kinds of detection targets CBFB-MYH11, SIL-TAL1, TEL-AML1, BCR-ABL, E2A-
The 10 of PBX1, PML-RARa, AML1-ETO and MLL-AF47Copy/mL PMD19-T clone DNA；
Described negative control is physiological saline；
Described enzyme system is made up of thermal starting archaeal dna polymerase, reverse transcriptase and RNase inhibitor；
Described PCR reaction buffers are 5 × one step RT-PCR Buffer, Mg2+With dNTPs mixed liquor.
The method of the offer of the present invention, comprises the following steps：
1) collecting sample and nucleic acid is extracted；
2) multiplexed PCR amplification reaction is carried out to detection sample using mentioned reagent box, obtains amplified production；
3) sample, positive control and negative control pcr amplification product are detected simultaneously using analysis system of capillary electrophoresis, if
Pcr amplification product corresponding to sample to be detected has the characteristic peaks of corresponding fusion, then shows in the sample containing corresponding
The fusion of leukaemia.
The system of described multiplexed PCR amplification is 25 μ L, wherein, the μ L of PCR reaction buffers 17, the μ L of enzyme system 2, template (bag
Include the nucleic acid, negative control and positive control of sample extraction) 3 μ L, all μ L of primer 1 in table 1, mend DEPC water to 25 μ L；
Further, described multiplexed PCR amplification system is using the sample of nucleic acid, negative control and positive control extracted as mould
Plate；
Further, final concentration of 400nM~1 μM of the described universal primer in multiplexed PCR amplification system, it is described embedding
Close final concentration of 20nM~100nM of the primer in multiplexed PCR amplification system；
Further, final concentration of 500nM of the universal primer in multiplexed PCR amplification system, described be fitted together to are drawn
Final concentration of 60nM of the thing in amplification system；
The condition of described multiplexed PCR amplification system is：1,50 DEG C of program, 25min, 1 circulation；2,95 DEG C of program,
10min, 1 circulation；3,95 DEG C of program, 15s, 58 DEG C, 30s, 72 DEG C, 50s, 10 circulations；4,95 DEG C of program, 15s, 65 DEG C,
30s, 72 DEG C, 50s, 10 circulations；5,95 DEG C of program, 15s, 48 DEG C, 30s, 72 DEG C, 50s, 20 circulations；6,72 DEG C of program,
5min, 1 circulation；7,4 DEG C of insulations of program.
Above-mentioned primer or PCR detection kit in testing sample is detected whether containing leukaemia correlation fusion gene should
With being also the scope of protection of the invention, leukaemia correlation fusion gene therein be CBFB-MYH11 genes, SIL-TAL1 genes,
At least one of TEL-AML1 genes, BCR-ABL genes, E2A-PBX1 genes, PML-RARa genes, AML1-ETO genes.
Beneficial effects of the present invention are：
1st, the kit can disposably detect 8 kinds of leukemia of children correlation fusion genes, reduce testing cost, save detection
Time, it is adapted to the conventional screening of leukemia of children patient；
2nd, multiplexed PCR amplification method disclosed by the invention and primer concentration are avoided in multiplex PCR procedure in the prior art
Primer interphase interaction and the amplification Preference of primer pair template, improve multiplexed PCR amplification flux and detection sensitivity, especially
Be adapted to amplification to low concentration template, and simple to operate, clinical detection specificity is good, and sensitivity is suitable with quantitative PCR, with compared with
Strong clinical practice promotional value；
3rd, detection method provided by the present invention is detected using capillary electrophoresis technique, and detection sensitivity is compared with agarose
Gel electrophoresis is high, and flux is big, at most can 96 samples of one-time detection,
Also, the present invention uses specific primer sequence, ensure the reliability of testing result；Detection method operation letter
It is single, it is time saving and energy saving；Detection flux is big, and reagent consumables cost is low；Can be directly to leukemia of children peripheral blood in patients sample extraction
Nucleic acid is detected；It is low to detection platform and artificial technology's level requirement, it can be widely popularized in routine screening and detection.
Brief description of the drawings
Fig. 1 is the characteristic peak schematic diagram that the positives control of embodiment 2 is detected using this kit, in figure, is from left to right divided
Not Wei reference gene (IC, 177bp), E2A-PBX1 (223bp), PML-RARa (267bp), CBFB-MYH11 (306bp),
AML1-ETO(360bp)、M-bcr(406bp)、MLL-AF4(539bp)、m-bcr(591bp)、TEL-AML1(699bp)、SIL-
TAL1(812bp)；
Fig. 2 is that concentration is 10 in embodiment 42Copies/ μ L 8 kinds of leukemia of children correlation fusion gene recombination plasmids
Characteristic peak schematic diagram, from left to right respectively reference gene (IC, 177bp), E2A-PBX1 (223bp), PML-RARa
(269bp)、CBFB-MYH11(307bp)、AML1-ETO(359bp)、M-bcr(408bp)、MLL-AF4(540bp)、m-bcr
(592bp)、TEL-AML1(699bp)、SIL-TAL1(810bp)。
Embodiment
The embodiment of the present invention is described further below in conjunction with the accompanying drawings：
The multiple PCR detection kit of embodiment 1
The present embodiment provides a kind of leukemia of children correlation fusion gene multiple PCR detection kit, including
1), for common leukemia of children correlation fusion gene C BFB-MYH11, SIL-TAL1, TEL-AML1, BCR- in 8
The relevant primer of ABL, E2A-PBX1, PML-RARa, AML1-ETO and MLL-AF4 design, specific primer are shown in Table 1, wherein, table 1
Described in primer synthesized by Sangon Biotech (Shanghai) Co., Ltd.：
Table 1
Wherein, described chimeric primers are formed by universal primer and specific primer sets, i.e., in specific primer sequence
5 ' end addition one section of universal primer sequence, by specific primer 5 ' end addition one section with template to be measured without any amplification
Oligonucleotide sequence and carry out PCR amplification, 5 ' end addition oligonucleotide sequence be universal primer, PCR amplification when be fitted together to
Expanded in the first few circulation that primer concentration expands for 1/10, PCR of universal primer by the specific sequence of chimeric primers
Increase, enriched nucleic acid template, with the progress that PCR reacts, chimeric primers gradually use up, and universal primer plays master because concentration is larger
Act on, in remainder circulation, all amplifications are expanded by universal primer, and the amplification for reducing common multiplex PCR is inclined
Good property so that the nucleic acid-templated of low concentration is effectively expanded, and because chimeric primers concentration is relatively low, avoids two level between primer
The influence of structure；
2), positive control, negative control, enzyme system, PCR reaction buffers and DEPC water.
Wherein, the enzyme system is to contain reverse transcriptase (200U/ul), RNase inhibitor (80U/ul), thermal starting Taq
The mixture of archaeal dna polymerase (5U/ul)；
The PCR reaction buffers are to contain 5 × one step RT-PCR Buffer (100mM Tris-HCl
pH8.5、500nM KCl、15nM MgCl2)、Mg2+(25mM), dNTPs (25mM) mixed liquor；
Positive control is by 8 kinds of targets CBFB-MYH11, SIL-TAL1, TEL-AML1, BCR-ABL, E2A-PBX1, PML-
The 10 of RARa, AML1-ETO and MLL-AF47Copy/mL PMD19-T clones DNA is formed；
Negative control is physiological saline.
The system of described multiplexed PCR amplification is 25 μ L, wherein, the μ L of PCR reaction buffers 17, the μ L of enzyme system 2, template (bag
Include the nucleic acid, negative control and positive control of sample extraction) 3 μ L, all μ L of primer 1 in table 1, mend DEPC water to 25 μ L；
Detecting step is as follows：
1) collecting sample and nucleic acid is extracted；
2) using the sample of nucleic acid, negative control and positive control of extraction as template, and using mentioned reagent box to detecting sample
Product carry out multiplexed PCR amplification reaction, obtain amplified production；
3) PCR primer of sample, positive control and negative control is detected simultaneously using analysis system of capillary electrophoresis；
The program of above-mentioned multiplexed PCR amplification is：
1,50 DEG C of program, 25min, 1 circulation；2,95 DEG C of program, 10min, 1 circulation；3,95 DEG C of program, 15s, 58
DEG C, 30s, 72 DEG C, 50s, 10 circulation；4,95 DEG C of program, 15s, 65 DEG C, 30s, 72 DEG C, 50s, 10 circulations；Program 5,95
DEG C, 15s, 48 DEG C, 30s, 72 DEG C, 50s, 20 circulation；6,72 DEG C of program, 5min, 1 circulation；7,4 DEG C of insulations of program.
If pcr amplification product corresponding to sample to be detected has the characteristic peaks of corresponding fusion, show the sample
In contain corresponding leukaemia fusion.
The detection of the multiple PCR detection kit of embodiment 2
Specific detection method is as follows：
(1) sample collection
2 milliliters of person under inspection's venous blood is extracted with disposable sterilized injector, injects (disodium ethylene diamine tetraacetate) containing EDTA
Or the glass tube of sodium citrate anticoagulant, glass tube is gently overturned immediately and is mixed 5~10 times, makes anti-coagulants fully mixed with venous blood
It is even, take 25 μ L whole bloods to carry out nucleic acid extraction；
(2) extraction of genomic DNA
Carried using the whole blood DNA rapid extraction kit (article No. L0109) of the sincere bio tech ltd of Nanjing Mei Ningkang
The nucleic acid of above-mentioned sample is taken, and using the sample of nucleic acid of extraction and negative control and positive control as template；
(3) preparation of amplification reaction system
Detected using the kit of embodiment 1, after candidate agent box PCR reaction solutions are completely dissolved at ambient temperature,
Quick concussion mixes, and PCR reaction systems are 25 μ L, wherein, the μ L of PCR reaction buffers 17, the μ L of enzyme system 2, template (including sample carries
Nucleic acid, negative control and the positive control taken) 3 μ L, all μ L of primer 1 in table 1, mend DEPC water to 25 μ L；
(4) PCR is expanded
PCR pipe is put into regular-PCR instrument, after opening heat lid, requires to set PCR instrument response procedures according to following：
1,50 DEG C of program, 25min, 1 circulation；2,95 DEG C of program, 10min, 1 circulation；3,95 DEG C of program, 15s, 58
DEG C, 30s, 72 DEG C, 50s, 10 circulation；4,95 DEG C of program, 15s, 65 DEG C, 30s, 72 DEG C, 50s, 10 circulations；Program 5,95
DEG C, 15s, 48 DEG C, 30s, 72 DEG C, 50s, 20 circulation；6,72 DEG C of program, 5min, 1 circulation；7,4 DEG C of insulations of program；
(5) using Qiagen QIAxcel Advanced capillary electrophoresis systems simultaneously analyze sample, negative control and
The PCR primer of positive control, sample analysis result such as table 2, as shown in Figure 1, negative control result is without table 2 for positive control result
The characteristic peak of shown peak value size, show that the kit can detect 8 kinds of leukemia of children correlation fusions in sample simultaneously
Gene.
The sample analysis result of table 2
Embodiment 3, kit specific detection
Every kind of leukemia of children correlation fusion gene is detected using kit in embodiment 1 and method respectively, as a result only
The characteristic peak of corresponding peak value size is amplified, testing result is as shown in table 3, and each result without non-specific amplification, shows amplification knot
Fruit is good.
38 kinds of leukemia of children correlation fusion genetic test results of table
The kit sensitivity technique of embodiment 4
The use of the 8 kinds of leukemia of children correlation fusion gene recombination plasmids demarcated is template, gradient dilution arrives respectively
105copies/μL、104copies/μL、103copies/μL、102Copies/ μ L, use the kit pair described in embodiment 1
Template after above-mentioned dilution enters performing PCR amplification respectively, and the result judgement standard in embodiment 2 is judged that 8 kinds of children are white
Blood disease correlation fusion genetic test sensitivity results are as shown in table 4, concentration 102Copies/ μ L 8 kinds of leukemia of children correlations
As shown in Figure 2,8 kinds of leukemia of children correlation fusion genes are the positive to the testing result of fusion recombinant plasmid.
48 kinds of leukemia of children correlation fusion genetic test sensitivity results of table
| Detect target | 105copies/μL | 104copies/μL | 103copies/μL | 102copies/μL |
| E2A-PBX1 | + | + | + | + |
| PML-RARa | + | + | + | + |
| CBFB-MYH11 | + | + | + | + |
| M-bcr | + | + | + | + |
| MLL-AF4 | + | + | + | + |
| m-bcr | + | + | + | + |
| TEL-AML1 | + | + | + | + |
| SIL-TAL1 | + | + | + | + |
| E2A-PBX1 | + | + | + | + |
As known from Table 4,9 detection targets of the 8 kinds of leukemia of children correlation fusion genes to more than of kit described in embodiment 1
Target plasmid standard detection sensitivity is 102Copies/ μ L, it is therefore, of the present invention in the case where template concentrations are relatively low
Kit can still provide for detecting, and have very high sensitivity.
Embodiment 5：Clinical sample is detected using 8 kinds of leukemia of children correlation fusion gene multiple PCR detection kits
The clinical sample of 121 share child leukaemics is chosen as sample to be checked, using the kit in embodiment 1 simultaneously
Operated according to the methods described of embodiment 2 and result judgement, this 121 parts of clinical samples detect 21 BCR-ABL, wherein having
9 only detection M-bcr, 8 only detection m-bcr, while detect M-bcr and m-bcr and have 4, detect 13 CBFB-MYH11
Detect, detect 13 SIL-TAL1, detect 16 TEL-AML1, detect 16 E2A-PBX1, detect 15 PML-
RARa, 14 AML1-ETO are detected, detect 13 MLL-AF4, sample confirms it is correct, specific such as table 5 through sequencing
It is shown.
The clinical sample testing result of table 5
| Fusion | Detect number |
| CBFB-MYH11 | 13 |
| SIL-TAL1 | 13 |
| TEL-AML1 | 16 |
| BCR-ABL | 21 |
| E2A-PBX1 | 16 |
| PML-RARa | 15 |
| AML1-ETO | 14 |
| MLL-AF4 | 13 |
Merely illustrating the principles of the invention described in above-described embodiment and specification and most preferred embodiment, this is not being departed from
On the premise of spirit and scope, various changes and modifications of the present invention are possible, and these changes and improvements both fall within requirement and protected
In the scope of the invention of shield.
Claims (9)
1. a kind of leukemia of children correlation fusion gene multiple PCR detection kit, it is characterised in that including following primer pair：
(1) chimeric primers of CBFB-MYH11 genes are detected：
CBFB-MYH11_F, its nucleotide sequence such as SEQ ID NO：Shown in 1；
CBFB-MYH11_R, its nucleotide sequence such as SEQ ID NO：Shown in 2；
(2) chimeric primers of SIL-TAL1 genes are detected：
SIL-TAL1_F, its nucleotide sequence such as SEQ ID NO：Shown in 3；
SIL-TAL1_R, its nucleotide sequence such as SEQ ID NO：Shown in 4；
(3) chimeric primers of TEL-AML1 genes are detected：
TEL-AML1_F, its nucleotide sequence such as SEQ ID NO：Shown in 5；
TEL-AML1_R, its nucleotide sequence such as SEQ ID NO：Shown in 6；
(4) chimeric primers of BCR-ABL genes are detected：
M-bcr_F, its nucleotide sequence such as SEQ ID NO：Shown in 7；
M-bcr_F, its nucleotide sequence such as SEQ ID NO：Shown in 8；
Bcr_R, its nucleotide sequence such as SEQ ID NO：Shown in 9；
(5) chimeric primers of E2A-PBX1 genes are detected：
E2A-PBX1_F, its nucleotide sequence such as SEQ ID NO：Shown in 10；
E2A-PBX1_R, its nucleotide sequence such as SEQ ID NO：Shown in 11；
(6) chimeric primers of PML-RARa genes are detected：
PML-RARa_F, its nucleotide sequence such as SEQ ID NO：Shown in 12；
PML-RARa_R, its nucleotide sequence such as SEQ ID NO：Shown in 13；
(7) chimeric primers of AML1-ETO genes are detected：
AML1-ETO_F, its nucleotide sequence such as SEQ ID NO：Shown in 14；
AML1-ETO_R, its nucleotide sequence such as SEQ ID NO：Shown in 15；
(8) chimeric primers of MLL-AF4 genes are detected：
MLL-AF4_F, its nucleotide sequence such as SEQ ID NO：Shown in 16；
MLL-AF4_R, its nucleotide sequence such as SEQ ID NO：Shown in 17；
(9) the chimeric primers IC of internal standard gene：
IC_F, its nucleotide sequence such as SEQ ID NO：Shown in 18；
IC_R, its nucleotide sequence such as SEQ ID NO：Shown in 19；
(10) universal primer：
Tongyong_F, its nucleotide sequence such as SEQ ID NO：Shown in 20；
Tongyong_R, its nucleotide sequence such as SEQ ID NO：Shown in 21；
Above-mentioned primer is used in conjunction with one-time detection.
2. a kind of leukemia of children correlation fusion gene multiple PCR detection kit according to claim 1, its feature exist
In：Described multiple PCR detection kit also includes positive control, negative control, enzyme system, PCR reaction buffers and DEPC water.
3. a kind of leukemia of children correlation fusion gene multiple PCR detection kit according to claim 2, its feature exist
In：Described positive control is above-mentioned 8 kinds detection targets CBFB-MYH11, SIL-TAL1, TEL-AML1, BCR-ABL, E2A-
The 10 of PBX1, PML-RARa, AML1-ETO and MLL-AF47Copy/mLPMD19-T clone DNA；
Described negative control is physiological saline；
Described enzyme system is made up of thermal starting archaeal dna polymerase, reverse transcriptase and RNase inhibitor；
Described PCR reaction buffers are 5 × one step RT-PCR Buffer, Mg2+With dNTPs mixed liquor.
The application of leukemia of children correlation fusion gene multiple PCR detection kit described in 4., it is characterised in that：Described examination
Agent box be mainly used in 8 kinds of leukemia of children correlation fusion gene C BFB-MYH11, SIL-TAL1, TEL-AML1, BCR-ABL,
E2A-PBX1, PML-RARa, AML1-ETO and MLL-AF4 detection.
5. a kind of multi-PCR detection method of leukemia of children correlation fusion gene, it is characterised in that comprise the following steps：
1), collecting sample and nucleic acid is extracted；
2), using the sample of nucleic acid, negative control and positive control of extraction as template, and detection sample is entered using mentioned reagent box
Row multiplexed PCR amplification reacts, and obtains amplified production；
3) pcr amplification product of sample, positive control and negative control is detected simultaneously using analysis system of capillary electrophoresis, if inspection
Pcr amplification product corresponding to test sample sheet has the characteristic peaks of corresponding fusion, then shows in the sample containing corresponding white blood
The fusion of disease.
6. a kind of multi-PCR detection method of leukemia of children correlation fusion gene according to claim 5, its feature exist
In：The system of described multiplexed PCR amplification is 25 μ L, wherein, μ L of enzyme system 2.0, μ L of PCR reaction buffers 17, μ L of template 2~5,
And all μ L of primer pair 1.0 in claim 1, DEPC water is mended to 25 μ L.
7. a kind of multi-PCR detection method of leukemia of children correlation fusion gene according to claim 6, its feature exist
In：Final concentration of 400nM~1 μM of the described universal primer in multiplexed PCR amplification system, the chimeric primers are multiple
Final concentration of 20nM~100nM in PCR amplification system.
8. a kind of multi-PCR detection method of leukemia of children correlation fusion gene according to claim 6, its feature exist
In the condition of described multiplexed PCR amplification system is：1,50 DEG C of program, 25min, 1 circulation；2,95 DEG C of program, 10min, 1
Individual circulation；3,95 DEG C of program, 15s, 58 DEG C, 30s, 72 DEG C, 50s, 10 circulations；4,95 DEG C of program, 15s, 65 DEG C, 30s, 72
DEG C, 50s, 10 circulation；5,95 DEG C of program, 15s, 48 DEG C, 30s, 72 DEG C, 50s, 20 circulations；6,72 DEG C of program, 5min, 1
Circulation；7,4 DEG C of insulations of program.
9. a kind of multi-PCR detection method of leukemia of children correlation fusion gene according to claim 6, its feature exist
In：Final concentration of 500nM of the universal primer in multiplexed PCR amplification system, the chimeric primers are in amplification system
Final concentration of 60nM.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109402259A (en) * | 2018-11-13 | 2019-03-01 | 中山大学达安基因股份有限公司 | A kind of kit detecting leukemia fusion gene and gene mutation |
| CN111793676A (en) * | 2020-07-21 | 2020-10-20 | 北京安智因生物技术有限公司 | Method and kit for detecting gene polymorphism and application thereof |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2000075369A1 (en) * | 1999-06-02 | 2000-12-14 | Reinald Repp | Method for the detection of nucleic acid amplification products, using primers containing primer-integrated reporter sequences (pirs) |
| CN106544437A (en) * | 2016-11-25 | 2017-03-29 | 徐州医科大学 | A kind of multiple fluorescence PCR test kit and method of detection leukemia fusion gene |
-
2017
- 2017-08-31 CN CN201710773199.5A patent/CN107385080A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2000075369A1 (en) * | 1999-06-02 | 2000-12-14 | Reinald Repp | Method for the detection of nucleic acid amplification products, using primers containing primer-integrated reporter sequences (pirs) |
| CN106544437A (en) * | 2016-11-25 | 2017-03-29 | 徐州医科大学 | A kind of multiple fluorescence PCR test kit and method of detection leukemia fusion gene |
Non-Patent Citations (3)
| Title |
|---|
| MARIE-PIERRE LAFORÊT等: "Design and Feasibility of a Novel, Rapid, and Simple Fluorescence 26-Plex RT-PCR Assay for Simultaneous Detection of 24 Fusion Transcripts in Adult Acute Myeloid Leukemia", 《THE JOURNAL OF MOLECULAR DIAGNOSTICS》 * |
| 李伟等: "《分子诊断学》", 30 September 2015 * |
| 陈小文等: "一步法多重 RT-PCR 对儿童急性淋巴细胞白血病常见融合基因的检测及意义", 《中国当代儿科杂志》 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109402259A (en) * | 2018-11-13 | 2019-03-01 | 中山大学达安基因股份有限公司 | A kind of kit detecting leukemia fusion gene and gene mutation |
| CN111793676A (en) * | 2020-07-21 | 2020-10-20 | 北京安智因生物技术有限公司 | Method and kit for detecting gene polymorphism and application thereof |
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