CN105838792B - For the primer of qualitative detection leukemia fusion gene, probe, kit and method - Google Patents
For the primer of qualitative detection leukemia fusion gene, probe, kit and method Download PDFInfo
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Abstract
The invention belongs to gene engineering technology fields, disclose a kind of primer and probe combination, the kit containing primer and probe combination and multi-fluorescence RT-PCR method for using primer and probe combination or kit to carry out leukemia fusion gene detection for detecting leukemia fusion gene.It is simple, quick the present invention is based on multi-fluorescence RT-PCR technology, high sensitivity.In addition, the present invention is arranged in pairs or groups by reasonable primer and probe, the interaction between primer and primer, between primer and probe and between probe and probe is effectively prevented, detection error is reduced.Qualitative detection comprehensively can be carried out to 45 kinds of leukemia fusion genes using detection method of the invention, effectively shorten detection time, diagnosis, therapeutic evaluation and the prognosis for clinical leukaemia provide very important detection means.
Description
Technical field
The present invention relates to gene engineering technology fields, and in particular to for qualitative detection leukemia fusion gene primer,
Probe, 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, especially can be improved the sensitivity of detection.
Fluorescence RT-PCR is that real-time fluorescence PCR detection is carried out after reverse transcription, and main advantage was with real-time fluorescence PCR platform generation
For common PCR and electrophoretic analysis.Real-time fluorescence PCR has the advantages that the operation of (1) stopped pipe, closed state detection, reduces
The possibility of template pollution and false positive；(2) it without being post-processed to PCR product, operates easier to be quick；(3) sensitivity
Height can detecte single copy gene；(4) specificity is high, can specifically be detected by the combination of probe and target sequence specificity
Target gene；(5) quantitative analysis is carried out by standard curve and Ct value.25 European laboratories pass through EAC project within 2003
(Europe Against Cancer Program) cooperation, establishes the detection of 10 fusions common in leukaemia
Standardized real-time fluorescence RT-PCR method, including fluorescence PCR primer and probe (Leukemia, 2003,17,2318-
2357).Many fluorescence detection methods for leukemia fusion gene are grown up on the basis of this research later
's.
However, multipair primer is added in multi-fluorescence RT-PCR technology needs in the same PCR reaction system, it is even more
Probe, which adds multipair primers and a plurality of probe to interact and be formed in the reaction system the several of dimer
Rate to reduce the specificity and efficiency of PCR reaction, or even cannot amplify specific targets product.In addition, with people
It deepens continuously to leukaemia research, more and more leukemia fusion genes have been found, current detection leukaemia merges base
The method of cause be far from satisfying people for and meanwhile detect the demands of most fusions, need it is a kind of quickly,
Method that is effective and being easy to standardized energy while qualitative detection overwhelming majority leukemia fusion gene.
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
The new detection primer devised for detecting leukemia fusion gene and probe and multi-fluorescence RT-PCR method.Of the invention
Detection method is quickly, effectively and the highly sensitive qualitative detection that is easy to be standardized 45 kinds of leukemia fusion genes.
For this purpose, one aspect of the present invention provides a kind of primer and probe combination for detecting leukemia fusion gene,
The primer and probe shown in SEQ ID NO.1-114 forms.
In a preferred embodiment of the present invention, primer and probe combination is further by 1-12 group primer and probe group
At, wherein probe shown in the 1st group of primer shown in SEQ ID NO.1-9 and SEQ ID NO.10-11 forms, the 2nd group by
The composition of probe shown in primer shown in SEQ ID NO.12-16 and SEQ ID NO.17-18, the 3rd group by SEQ ID NO.19-
The composition of probe shown in primer shown in 27 and SEQ ID NO.28-29, the 4th group of primer as shown in SEQ ID NO.30-35
It is formed with probe shown in SEQ ID NO.36-38, the 5th group of primer as shown in SEQ ID NO.39-43 and SEQ ID
Shown in the composition of probe shown in NO.44-45, the 6th group of primer as shown in SEQ ID NO.46-50 and SEQ ID NO.51-52
Probe composition, probe shown in the 7th group of primer shown in SEQ ID NO.53-59 and SEQ ID NO.60-62 forms, the
Probe shown in 8 groups of primers shown in SEQ ID NO.63-66 and SEQ ID NO.67-88 forms, and the 9th group by SEQ ID
The composition of probe shown in primer shown in NO.69-76 and SEQ ID NO.77-78, the 10th group as shown in SEQ ID NO.79-87
Primer and SEQ ID NO.88-89 shown in probe composition, the 11st group of primer and SEQ as shown in SEQ ID NO.90-99
The composition of probe shown in ID NO.100-101, the 12nd group of primer as shown in SEQ ID NO.102-111 and SEQ ID
The composition of probe shown in NO.112-114.
The present invention is by selection related gene broken site upstream sequence and downstream sequence, to carry out specific multi-fluorescence
The design of RT-PCR primer and probe.Designed primer Tm is near 60 DEG C, each primer of integration collocation, thus as far as possible
Avoid the generation of primer dimer；Probe Tm value avoids between probe and other non-specific sequences near 70 DEG C
Combination, while also avoiding probe and forming complicated dimer between each other, improve the specificity and efficiency of PCR amplification.
For 45 kinds of leukemia fusion genes, the specific primer and probe situation that the present invention designs is as shown in table 1.
Table 1, primer and probe situation table of the invention
In a preferred embodiment of the present invention, 5 ' end connection fluorescent reporter group FAM of part probe, 3 ' end connections are glimmering
Optical quenching group TAMRA；5 ' end connection fluorescent reporter group JOE, 3 ' end connection fluorescent quenching group TAMRA of remaining probe；
5 ' end connection fluorescent reporter group JOE, 3 ' end connection fluorescent quenching group TAMRA of reference gene GAPDH probe.
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 and probe combination stated.
In a preferred embodiment of the present invention, the concentration of whole primers is 3pmol/ μ l in kit, whole probes
Concentration is 2pmol/ μ l.
Another aspect of the present invention provides primer and probe combination of the present invention in preparation detection leukaemia fusion base
Because of the application in kit.
Further aspect of the present invention provides primer and probe combination of the present invention or kit of the present invention
Application in detection leukemia fusion gene.
Last aspect of the present invention provides a kind of multi-fluorescence RT-PCR method for detecting leukemia fusion gene, packet
Containing following steps:
1, the cDNA of sample to be tested is obtained.
CDNA synthesis can use Promega Reverse Transcriptase kit, and brief process is as follows:
It takes 1~5ug total serum IgE for reverse transcription, takes the RNA product of Random the Prime 1.5ul and 15ul of 50ug/ml,
70 DEG C are reacted 5min to promote the opening of RNA secondary structure, place at least 2min on ice.5 × Reaction Buffer is added
6ul、25mM MgCl2 3.8ul、PCR Nucleotide Mix1.5ul、Ribonuclease Inhibitor
0.3ul、GoScriptTMReverse Transcriptase1ul, Nuclease-Free Water 0.9ul to final volume
30ul。
Reverse transcription process are as follows: 25 DEG C of 5min；42℃60min；70℃15min.By the cDNA Nuclease-Free of acquisition
Water is correspondingly diluted and (is calculated according to total rna concentration), and the cDNA after dilution is used to carry out the multiple of fusion
Fluorescence RT-PCR qualitative detection.
2, it is combined using primer and probe of the present invention, or uses kit of the present invention, to be obtained in step 1
The cDNA taken is template, carries out multi-fluorescence RT-PCR amplification, and collect fluorescence signal.
In multi-fluorescence RT-PCR detection of the invention, the reaction system of 20ul includes: the Fluorescence PCR of 10ul
Liquid, ROX Reference Dye of 1ul, 5ul dilution after cDNA template (Nuclease-Free Water is as negative right
According to), the Nuclease-Free Water of the primed probe mixed liquor of 2ul and 2ul.
Program is run on 7500 fluorescent PCR instrument of ABI are as follows: first passes through 50 DEG C of 2min, 95 DEG C of 10min；Then using 95
DEG C 15sec, 60 DEG C of 30sec, totally 45 circulations.
3, the amplification for analyzing reference gene GAPDH, if CTValue is less than 30, and detection process is effective, if CTValue is greater than
35, re-start verifying detection.
In multi-fluorescence RT-PCR detection of the invention, internal reference compares primer and probe and uses people GAPDH gene, negative
CDNA template is not added in control (water).The only C of reference gene GAPDHTValue less than 30 when, detection process just it is effective, just into
Enter result interpretation step.
4, the C of reference gene GAPDHTWhen value is less than 30, the result interpretation of leukemia fusion gene is carried out.
After being detected using multi-fluorescence RT-PCR detection method of the invention to sample to be tested, only when internal reference base
FAM or JOE fluorescence signal, no template control product no signal are capable of detecting when because GAPDH has JOE fluorescence signal, and wherein in 1 pipe
When, the testing result of sample to be tested just effectively and is positive.
In a preferred embodiment of the present invention, 5 ' end connection fluorescent reporter group FAM of part probe, 3 ' end connections are glimmering
Optical quenching group TAMRA；5 ' end connection fluorescent reporter group JOE, 3 ' end connection fluorescent quenching group TAMRA of remaining probe；
5 ' end connection fluorescent reporter group JOE, 3 ' end connection fluorescent quenching group TAMRA of reference gene GAPDH probe.
In further preferred embodiment of the present invention, the concentration of whole primers is 3pmol/ μ l, whole probes it is dense
Degree is 2pmol/ μ l.
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
Weight fluorescent RT-PCR technology, therefore simple, quick, high sensitivity.Meanwhile the multi-fluorescence RT-PCR technology that uses of the present invention with
Multiplex nested RT-PCR is compared, and has higher sensitivity and specificity.
2, detection method of the invention is arranged in pairs or groups by reasonable primer and probe, it is possible to prevente effectively from primer and primer it
Between, between primer and probe and the interaction between probe and probe is to improve specificity reduces false positive,
Reduce detection error.
3, detection method of the invention can comprehensively carry out highly sensitive qualitative inspection to 45 kinds of leukemia fusion genes
It surveys, effectively shortens detection time, diagnosis, therapeutic evaluation and the prognosis for clinical leukaemia provide very important detection
Means.
Detailed description of the invention
Fig. 1: representative leukemia fusion gene (BCR-ABL1, CBFB-MYH11, PML-RARA and AML1-
ETO broken site and primer and probe) designs site structure schematic diagram.
Fig. 2: using leukemia fusion gene positive cell line and negative cells system detection present invention detection body in embodiment 1
It is the fluorescent amplification curve figure of specificity.
Fig. 3: the glimmering of detection architecture repeatability of the present invention is detected using leukemia fusion gene positive cell line in embodiment 2
Light amplification curve diagram.
Fig. 4: using leukemia fusion gene positive cell line and negative cells system detection present invention detection body in embodiment 3
It is the fluorescent amplification curve figure of sensitivity.
Fig. 5: the fluorescent amplification curve figure of clinical sample detection is carried out in embodiment 4 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 the K562 positive cell line containing BCR-ABL1 (p210) fusion, AML1-ETO fusion
The Kasumi cell line of gene and HL60 negative cells system without fusion verify the feasible of detection method
Property and specificity.Wherein K562 cell line, Kasumi cell line, HL60 cell line are purchased from Beijing ancient cooking vessel state prosperity biotechnology and have
Limit responsible 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 fluorescence RT-PCR detection.
4, multi-fluorescence RT-PCR is detected
(1) multi-fluorescence RT-PCR is detected
CDNA template 5ul after sequentially adding Nuclease-Free Water 2ul, dilution in the reaction system of 20ul,
ROX Reference Dye 1ul, primed probe mixed liquor 2ul, Fluorescence PCR liquid 10ul.
Program is run on 7500 fluorescent PCR instrument of ABI are as follows: first passes through 50 DEG C of 2min, 95 DEG C of 10min；Then using 95
DEG C 15sec, 60 DEG C of 30sec, totally 45 circulations.
Amplification procedure Instrumental collects fluorescence signal automatically.
(2) data collection process and analysis
After fluorescent PCR expands, the amplification of internal reference GAPDH is analyzed first, if its CTValue shows whole less than 30
A detection process is effective；If its CTValue is greater than 35, then needs to verify detection again.
(3) result detects
The amplification curve result of multi-fluorescence RT-PCR detection architecture specificity of the present invention is referring to Figure of description 2.Wherein
Fig. 2A is the fusion testing result of K562 positive cell line, and Fig. 2 B is that the fusion of Kasumi positive cell line detects knot
Fruit, Fig. 2 C are the fusion testing result of HL60 negative cells system, and Fig. 2 D is the testing result of negative control (water).
From attached drawing 2 as can be seen that may detect that the fluorescence signal of internal reference GAPDH in Fig. 2A, 2B, 2C, and GAPDH
CTValue both less than 30, illustrates that entire detection process is effective.Containing the fusion of BCR-ABL1 in K562 positive cell line, in R3
Specifically detect the BCR-ABL1 fluorescence signal of FAM label, as shown in Figure 2 A；Contain in Kasumi positive cell line
The fusion of AML1-ETO specifically detects the AML1-ETO fluorescence signal of HEX label in R9, as shown in Figure 2 B；
It can't detect other in R1-R12 other than the fluorescence signal of internal reference GAPDH without fusion in HL60 negative cells system
Fluorescence signal, as shown in Figure 2 C；Negative control can not expand reference gene GAPDH and fusion without template, thus also examine
Fluorescence signal is not detected, as shown in Figure 2 D.
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 repeatability of detection architecture of the present invention is detected using fusion positive cell line
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 verifies the repeatability of detection architecture of the present invention.
The specific detection method is as follows:
Using multi-fluorescence RT-PCR reaction system and reaction condition same as Example 1, with K562cDNA and
Kasumi cDNA is template, and each sample repeats detection three times, and the fluorescent PCR amplification curve of repeatability detection is referring to specification
Attached drawing 3.Wherein Fig. 3 A is the fusion testing result of K562 positive cell line, and Fig. 3 B is the fusion of Kasumi positive cell line
Genetic test result.As shown in Figure 3A, the positive cell line of K562 carries out repeating to detect three times, detects internal reference GAPDH base
The fluorescence signal of cause and fusion BCR-ABL1, and the C of GAPDH and BCR-ABL1TValue is coincide very well；Similarly,
The three repeated experiments result of the positive cell line of Kasumi also matches.Show to utilize inspection of the invention through this embodiment
The multi-fluorescence RT-PCR qualitative detection that survey system carries out leukemia fusion gene has repeatability well.
Embodiment 3: 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 the K562 positive cell line, the AML1-ETO that contain BCR-ABL1 (p210) fusion
The Kasumi cell line of fusion and HL60 negative cells system without fusion verify detection architecture of the present invention
Sensitivity.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 multi-fluorescence RT-PCR detection.
Using multi-fluorescence RT-PCR reaction system and reaction condition same as Example 1, multi-fluorescence RT-PCR's
Amplification curve is referring to Figure of description 4.Fig. 4 A represent 100%K562,10%K562,1%K562,1 ‰ K562,0.5 ‰ K562,
The fluorescence detection result of 0.25 ‰ K562 and 0.125 ‰ K562；Fig. 4 B represents 100%Kasumi, 10%Kasumi, 1%
The fluorescence detection result of Kasumi, 1 ‰ Kasumi, 0.5 ‰ Kasumi, 0.25 ‰ Kasumi and 0.125 ‰ Kasumi.
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, fusion BCR-ABL1 that multi-fluorescence RT-PCR detection architecture of the invention detects
With the C of AML1-ETOTValue is still less than 35, and corresponding RT-Nested PCR method can't detect corresponding fusion.Thus
Show compared with RT-Nested PCR, the detection spirit of multi-fluorescence RT-PCR detection architecture of the invention to leukemia fusion gene
Sensitivity is much higher than corresponding RT-Nested PCR detection method.
Embodiment 4: 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, and detection architecture according to the invention carries out
The qualitative detection of 45 kinds of fusions.The isomers of the fusion wherein detected includes BCR-ABL1 (p190), BCR-
ABL1 (p210), PML-RARA (L-type), PML-RARA (S type), CBFB-MYH11 (A type), SIL-TAL1, E2A-PBX1,
AML1-ETO, TEL-AML1, SET-CAN, DEK-CAN, MLL-ELL, MLL-AF9 and MLL-AF6 etc..
Clinical sample is detected using multi-fluorescence RT-PCR detection architecture of the invention, operating procedure includes in blood sample
Leucocyte RNA is extracted, RNA reverse transcription is that cDNA and multi-fluorescence RT-PCR is detected.Specific operating method is implemented with the present invention
Example 1.The fluorescent amplification curve of clinical sample detection is referring to Figure of description 5.Wherein Fig. 5 A is using multi-fluorescence of the invention
The fluorescence results for 4 kinds of Fusional gene isomerides that RT-PCR is detected, including BCR-ABL1 (p190), BCR-ABL1 (p210),
PML-RARA (L-type) and PML-RARA (S type)；Fig. 5 B is the 3 kinds of fusions detected using multi-fluorescence RT-PCR of the invention
The fluorescence results of gene isomers, including CBFB-MYH11 (A type), SIL-TAL1 and E2A-PBX1；Fig. 5 C is using the present invention
The fluorescence results of 4 kinds of Fusional gene isomerides that detect of multi-fluorescence RT-PCR, including AML1-ETO, SET-CAN, DEK-
CAN and MLL-ELL.
It can be seen that using multi-fluorescence RT-PCR detection architecture of the invention, the clinical sample fusion detected
Type almost cover clinically common leukemia fusion gene, and testing result is consistent with clinical diagnosis.
Melt the above result shows that multi-fluorescence RT-PCR detection architecture of the invention can comprehensively carry out 45 kinds of leukaemia
Close the qualitatively screening of gene.And compared with RT-Nested PCR detection method, multi-fluorescence RT-PCR detection architecture of the invention
Sensitivity and specificity with higher, detection process is simple, substantially reduces detection time, is diagnosis, the chemotherapy of leukaemia
And prognosis provides a kind of more completely new fast and convenient gene diagnosis technology, has extensive clinical value.
Claims (5)
1. a kind of primer and probe for detecting leukemia fusion gene combines, it is made of 1-12 group primer and probe,
Wherein probe shown in the 1st group of primer shown in SEQ ID NO.1-9 and SEQ ID NO.10-11 forms, and the 2nd group by SEQ
The composition of probe shown in primer shown in ID NO.12-16 and SEQ ID NO.17-18, the 3rd group by SEQ ID NO.19-27 institute
The composition of probe shown in the primer and SEQ ID NO.28-29 shown, the 4th group of primer and SEQ as shown in SEQ ID NO.30-35
The composition of probe shown in ID NO.36-38, the 5th group of primer as shown in SEQ ID NO.39-43 and SEQ ID NO.44-45 institute
Probe shown in the probe composition shown, the 6th group of primer shown in SEQ ID NO.46-50 and SEQ ID NO.51-52 forms,
Probe shown in the 7th group of primer shown in SEQ ID NO.53-59 and SEQ ID NO.60-62 forms, and the 8th group by SEQ ID
The composition of probe shown in primer shown in NO.63-66 and SEQ ID NO.67-88, the 9th group as shown in SEQ ID NO.69-76
Primer and SEQ ID NO.77-78 shown in probe composition, the 10th group of primer and SEQ as shown in SEQ ID NO.79-87
The composition of probe shown in ID NO.88-89, the 11st group of primer as shown in SEQ ID NO.90-99 and SEQ ID NO.100-
Shown in the composition of probe shown in 101, the 12nd group of primer as shown in SEQ ID NO.102-111 and SEQ ID NO.112-114
Probe composition.
2. primer and probe combination according to claim 1,5 ' end connection fluorescent reporter groups of part of probe
FAM, 3 ' end connection fluorescent quenching group TAMRA；5 ' end connection fluorescent reporter group JOE of remaining probe, 3 ' end connection fluorescence
Quenching group TAMRA；5 ' end connection fluorescent reporter group JOE of reference gene GAPDH probe, 3 ' end connection fluorescent quenching groups
TAMRA。
3. a kind of for detecting the kit of leukemia fusion gene, it includes primer and probe groups of any of claims 1 or 2
It closes.
4. kit according to claim 3, wherein the concentration of whole primers is 3pmol/ μ l, the concentration of whole probes is
2pmol/μl。
5. application of the primer and probe combination of any of claims 1 or 2 in preparation detection leukemia fusion gene kit.
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| CN107345244A (en) * | 2016-10-12 | 2017-11-14 | 深圳市儿童医院 | Detect method, primer and the kit of leukaemia TEL AML1 fusions |
| 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 |
| CN108034721B (en) * | 2017-12-04 | 2021-07-09 | 南昌艾迪康医学检验实验室有限公司 | Fusion gene oligonucleotide for detecting leukemia NUP98 series, detection method and kit |
| CN110656177A (en) * | 2019-10-15 | 2020-01-07 | 合肥艾迪康医学检验实验室有限公司 | Primer, probe, method and kit for detecting AF1Q gene relative expression level |
| CN111471770A (en) * | 2020-05-07 | 2020-07-31 | 南京实践医学检验有限公司 | Kit and method for detecting leukemia fusion gene based on multiple fluorescence RT-PCR |
| CN112011621B (en) * | 2020-09-28 | 2022-10-28 | 东南大学 | Primer combination and method for screening high-risk subtype of acute lymphocytic leukemia |
| CN112301129A (en) * | 2020-11-05 | 2021-02-02 | 深圳荻硕贝肯精准医学有限公司 | BCR-ABL fusion gene detection kit and BCR-ABL fusion gene detection method |
| CN112280866A (en) * | 2020-11-24 | 2021-01-29 | 福州艾迪康医学检验所有限公司 | Method, primer and probe for screening 14 fusion genes related to acute promyelocytic leukemia by fluorescent quantitative PCR technology |
Citations (3)
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| CN102251031A (en) * | 2011-06-30 | 2011-11-23 | 北京思尔成生物技术有限公司 | TaqMan MGB probe real-time fluorescence PCR detection method for leukemia fusion genes, and special primers, probes and kit used by same |
| CN103571945A (en) * | 2013-09-27 | 2014-02-12 | 沈阳艾迪康医学检验所有限公司 | Method, primer, probe and kit for screening and identifying BCR-ABL unusual fusion types |
| CN105296637A (en) * | 2015-11-13 | 2016-02-03 | 武汉海吉力生物科技有限公司 | Primers, probes and kit for detecting leukemia-related fusion genes |
-
2016
- 2016-04-22 CN CN201610254799.6A patent/CN105838792B/en active Active
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102251031A (en) * | 2011-06-30 | 2011-11-23 | 北京思尔成生物技术有限公司 | TaqMan MGB probe real-time fluorescence PCR detection method for leukemia fusion genes, and special primers, probes and kit used by same |
| CN103571945A (en) * | 2013-09-27 | 2014-02-12 | 沈阳艾迪康医学检验所有限公司 | Method, primer, probe and kit for screening and identifying BCR-ABL unusual fusion types |
| CN105296637A (en) * | 2015-11-13 | 2016-02-03 | 武汉海吉力生物科技有限公司 | Primers, probes and kit for detecting leukemia-related fusion genes |
Non-Patent Citations (1)
| Title |
|---|
| 常见白血病融合基因筛查在白血病诊断与分型中的意义;祝毓琳等;《北京大学学报（医学版）》;20050630;第37卷(第3期);第236-239页 |
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