CN105838792A - Primer, probe, kit and method for qualitatively detecting fusion genes of leukemia - Google Patents
Primer, probe, kit and method for qualitatively detecting fusion genes of leukemia Download PDFInfo
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Abstract
The invention belongs to the technical field of gene engineering and discloses a prime-probe combination for detecting fusion genes of leukemia, a kit containing the prime-probe combination and a multiplex fluorescent RT-PCR method for detecting the fusion genes of the leukemia by virtue of the prime-probe combination or the kit. Based on a multiplex fluorescent RT-PCR technique, the method is simple, rapid and high in sensitivity. Besides, by virtue of the reasonable prime-probe combination, the mutual action between the two primers, the prime and the probe as well as two probes are effectively avoided, and the detection error is reduced. By virtue of the method, 45 fusion genes of the leukemia can be comprehensively and qualitatively detected, the detection time is effectively shortened, and very important detection measures are provided for the evaluation of clinical diagnosis and treatment and the prognosis of the leukemia.
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, probe, 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, especially can
The enough sensitivity improving detection.Fluorescence RT-PCR is to carry out real-time fluorescence PCR inspection after reverse transcription
Surveying, its main advantage is to replace common PCR and electrophoretic analysis with real-time fluorescence PCR platform.
Real-time fluorescence PCR has the advantage that the operation of (1) stopped pipe, closed state detection, decreases template
Pollute and false-positive possibility；(2) without PCR primer being carried out post processing, operate the easiest quickly；
(3) highly sensitive, single copy gene can be detected；(4) specificity is high, can be by probe and target sequence
Arrange specific combination, specifically testing goal gene；(5) carried out by standard curve and Ct value
Quantitative analysis.Within 2003,25 European laboratorys are by EAC project (Europe Against Cancer
Program) cooperation, the detection to 10 fusion genes common in leukemia establishes 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 all at this afterwards
Grow up on the basis of item research.
But, multi-fluorescence RT-PCR technology needs add multipair in same PCR reaction system
Primer, the most a plurality of probe, which adds multipair primer and a plurality of probe in reaction system
Middle interaction and form dimeric probability, thus reduce the specificity and efficiency of PCR reaction,
Even can not amplify specific targets product.Additionally, along with constantly deep to leukemia research of people
Entering, increasing leukemia fusion gene has been observed that, the side of current detection leukemia fusion gene
Method can not meet the most far away people for detect simultaneously the overwhelming majority fusion gene demand, need one badly
Plant quick, effective and be prone to standardized energy qualitative detection overwhelming majority leukemia fusion gene simultaneously
Method.
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 probe and multiple
Fluorescence RT-PCR method.The detection method of the present invention is quick, effective and is prone to melt 45 kinds of leukemia
Close the high sensitivity qualitative detection that gene is standardized.
To this end, one aspect of the present invention provides a kind of primer for detecting leukemia fusion gene and probe
Combination, it is made up of the primer shown in SEQ ID NO.1-114 and probe.
In a preferred embodiment of the present invention, this primer and probe combinations are drawn by 1-12 group further
Thing and probe composition, wherein the 1st group by the primer shown in SEQ ID NO.1-9 and SEQ ID
Probe composition shown in NO.10-11, the 2nd group by the primer shown in SEQ ID NO.12-16 and SEQ
Probe composition shown in ID NO.17-18, the 3rd group by the primer shown in SEQ ID NO.19-27 and
Probe composition shown in SEQ ID NO.28-29, the 4th group by drawing shown in SEQ ID NO.30-35
Probe composition shown in thing and SEQ ID NO.36-38, the 5th group by shown in SEQ ID NO.39-43
Primer and SEQ ID NO.44-45 shown in probe composition, the 6th group by SEQ ID NO.46-50
Probe composition shown in shown primer and SEQ ID NO.51-52, the 7th group by SEQ ID
Primer shown in NO.53-59 and the composition of the probe shown in SEQ ID NO.60-62, the 8th group by SEQ
Primer shown in ID NO.63-66 and the probe shown in SEQ ID NO.67-88 composition, the 9th group by
Primer shown in SEQ ID NO.69-76 and the composition of the probe shown in SEQ ID NO.77-78, the 10th
Group is made up of the primer shown in SEQ ID NO.79-87 and the probe shown in SEQ ID NO.88-89,
11st group by the primer shown in SEQ ID NO.90-99 and the probe shown in SEQ ID NO.100-101
Composition, the 12nd group by the primer shown in SEQ ID NO.102-111 and SEQ ID NO.112-114 institute
The probe composition shown.
The present invention, by selecting related gene broken site upstream sequence and downstream sequence, carries out specificity
Multi-fluorescence RT-PCR primer and the design of probe.Designed primer Tm all near 60 DEG C,
Integrate each primer of arranging in pairs or groups, thus avoid the generation of primer dimer as far as possible；Probe Tm value all exists
Near 70 DEG C, it is to avoid combination between probe and other non-specific sequences, it also avoid spy simultaneously
Pin forms the dimer of complexity each other, improves the specificity and efficiency of PCR amplification.
For 45 kinds of leukemia fusion genes, the concrete primer of present invention design and probe conditions such as table 1
Shown in.
Table 1, the primer of the present invention and probe conditions table
In a preferred embodiment of the present invention, 5 ' ends of part probe connect fluorescent reporter group FAM,
3 ' ends connect fluorescent quenching group TAMRA；5 ' ends of remaining probe connect fluorescent reporter group JOE,
3 ' ends connect fluorescent quenching group TAMRA；5 ' ends of reference gene GAPDH probe connect fluorescence report
Accusing group JOE, 3 ' ends connect fluorescent quenching group TAMRA.
Another aspect of the present invention provides a kind of test kit for detecting leukemia fusion gene, its bag
Containing primer of the present invention and probe combinations.
In a preferred embodiment of the present invention, in test kit, the concentration of whole primers is 3pmol/ μ l,
All the concentration of probe is 2pmol/ μ l.
Another aspect of the present invention provides primer of the present invention and probe combinations and detects white blood in preparation
Application in sick fusion gene test kit.
Further aspect of the present invention provides primer of the present invention and probe combinations, or institute of the present invention
The test kit stated application in detection leukemia fusion gene.
Last aspect of the present invention provides a kind of multi-fluorescence RT-PCR detecting leukemia fusion gene
Method, it comprises the steps of
1, the cDNA of testing sample is obtained.
CDNA synthesis can use Promega Reverse Transcriptase kit, and brief flow process is as follows:
Take 1～5ug total serum IgE for reverse transcription, take Random Prime 1.5ul and 15ul of 50ug/ml
RNA product, 70 DEG C of reaction 5min, to promote the opening of RNA secondary structure, place the most extremely
Few 2min.Add 5 × Reaction Buffer 6ul, 25mM MgCl2 3.8ul、PCR Nucleotide Mix
1.5ul、Ribonuclease Inhibitor 0.3ul、GoScriptTM Reverse Transcriptase
1ul, Nuclease-Free Water 0.9ul is to final volume 30ul.
Reverse transcription program is: 25 DEG C of 5min；42℃60min；70℃15min.The cDNA that will obtain
Correspondingly dilute (calculating according to total rna concentration) with Nuclease-Free Water, dilute
CDNA after releasing is for carrying out the multi-fluorescence RT-PCR qualitative detection of fusion gene.
2, use primer of the present invention and probe combinations, or use test kit of the present invention,
The cDNA obtained in step 1, as template, carries out multi-fluorescence RT-PCR amplification, and collects glimmering
Optical signal.
In the multi-fluorescence RT-PCR of the present invention detects, the reaction system of 20ul includes: 10ul's
Fluorescence PCR liquid, 1ul ROX Reference Dye, 5ul dilution after cDNA template
(Nuclease-Free Water is as negative control), the primed probe mixed liquor of 2ul and 2ul's
Nuclease-Free Water。
Program of running on ABI 7500 fluorescent PCR instrument is: first pass through 50 DEG C of 2min, 95 DEG C of 10min；
The most again through 95 DEG C of 15sec, 60 DEG C of 30sec, totally 45 circulations.
3, the amplification of reference gene GAPDH is analyzed, if CTValue is less than 30, and detection process has
Effect, if CTValue, more than 35, re-starts checking detection.
In the multi-fluorescence RT-PCR of the present invention detects, internal reference comparison primer and probe use people
GAPDH gene, is not added with cDNA template in negative control (water).Only reference gene GAPDH
CTWhen value is less than 30, detection process is just effective, just enters result interpretation step.
4, the C of reference gene GAPDHTWhen value is less than 30, carry out the result of leukemia fusion gene
Interpretation.
After sample to be tested is detected by the multi-fluorescence RT-PCR detection method using the present invention, only
Have when reference gene GAPDH has a JOE fluorescence signal, and wherein 1 pipe is capable of detecting when FAM
Or JOE fluorescence signal, during no template control product no signal, the testing result of sample to be tested just effectively and
It is positive.
In a preferred embodiment of the present invention, 5 ' ends of part probe connect fluorescent reporter group FAM,
3 ' ends connect fluorescent quenching group TAMRA；5 ' ends of remaining probe connect fluorescent reporter group JOE,
3 ' ends connect fluorescent quenching group TAMRA；5 ' ends of reference gene GAPDH probe connect fluorescence
Reporter group JOE, 3 ' ends connect fluorescent quenching group TAMRA.
In further preferred embodiment of the present invention, the concentration of whole primers is 3pmol/ μ l, entirely
The concentration of portion's probe is 2pmol/ μ l.
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 multi-fluorescence RT-PCR technology, the most simply, fast, highly sensitive.Meanwhile, originally
The multi-fluorescence RT-PCR technology that invention uses, compared with multiplex nested RT-PCR, has higher
Sensitivity and specificity.
2, the detection method of the present invention is arranged in pairs or groups by rational primer and probe, can be prevented effectively from primer
And the interaction between primer, between primer and probe and between probe and probe, thus improve
Specificity, reduces false positive, decreases detection error.
3, the detection method of the present invention can carry out high sensitivity to 45 kinds of leukemia fusion genes all sidedly
Qualitative detection, be effectively shortened the detection time, for clinical leukemic diagnosis, therapeutic evaluation and
Prognosis provides very important detection means.
Accompanying drawing explanation
Fig. 1: representative leukemia fusion gene (BCR-ABL1, CBFB-MYH11,
PML-RARA and AML1-ETO) broken site and primer and probe design site structure signal
Figure.
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 amplified fluorescence curve chart.
Fig. 3: embodiment 2 use leukemia fusion gene positive cell line detect detection bodies of the present invention
The amplified fluorescence curve chart of system's repeatability.
Fig. 4: embodiment 3 uses leukemia fusion gene positive cell line and the detection of negative cells system
The amplified fluorescence curve chart of detection system susceptiveness of the present invention.
Fig. 5: embodiment 4 use detection system of the present invention carry out the amplified fluorescence of clinical sample detection
Curve chart.
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 (p210) 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 fluorescence
RT-PCR detects.
4, multi-fluorescence RT-PCR detection
(1) multi-fluorescence RT-PCR detection
The reaction system of 20ul is sequentially added into the cDNA after Nuclease-Free Water 2ul, dilution
Template 5ul, ROX Reference Dye 1ul, primed probe mixed liquor 2ul, Fluorescence PCR liquid
10ul。
Program of running on ABI 7500 fluorescent PCR instrument is: first pass through 50 DEG C of 2min, 95 DEG C of 10min；
The most again through 95 DEG C of 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 amplification terminates, first analyze the amplification of internal reference GAPDH, if its
CTValue, less than 30, shows that whole detection process is effective；If its CTValue more than 35, then needs again
Checking detection.
(3) result detection
Multi-fluorescence RT-PCR detection system of the present invention specific amplification curve result sees description
Accompanying drawing 2.Wherein Fig. 2 A is the fusion gene testing result of K562 positive cell line, and Fig. 2 B is
The fusion gene testing result of Kasumi positive cell line, Fig. 2 C is melting of HL60 negative cells system
Closing gene test result, Fig. 2 D is the testing result of negative control (water).
From accompanying drawing 2 it can be seen that Fig. 2 A, 2B, 2C may detect that internal reference GAPDH's
Fluorescence signal, and the C of GAPDHTValue both less than 30, illustrates that whole detection process is effective.K562
Positive cell line contains the fusion gene of BCR-ABL1, R3 specifically detecting, FAM marks
The BCR-ABL1 fluorescence signal of note, as shown in Figure 2 A；Kasumi positive cell line contains
The fusion gene of AML1-ETO, specifically detects the AML1-ETO of HEX labelling in R9
Fluorescence signal, as shown in Figure 2 B；Without fusion gene in HL60 negative cells system, except internal reference
Outside the fluorescence signal of GAPDH, R1-R12 all can't detect other fluorescence signal, such as Fig. 2 C
Shown in；Negative control, without template, can not expand reference gene GAPDH and fusion gene, thus also
Can't detect fluorescence signal, 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 to detect the repeatability of detection system of the present invention
The present embodiment equally with determine K562 positive cell line containing BCR-ABL1 fusion gene,
The Kasumi cell line of AML1-ETO fusion gene verifies the repeatability of detection system of the present invention.
Concrete detection method is as follows:
Use multi-fluorescence RT-PCR reaction system same as in Example 1 and reaction condition, with
K562cDNA and Kasumi cDNA is template, each sample duplicate detection three times, repeatability inspection
The fluorescent PCR amplification curve surveyed sees Figure of description 3.Wherein Fig. 3 A is K562 positive cell line
Fusion gene testing result, Fig. 3 B is the fusion gene testing result of Kasumi positive cell line.
As shown in Figure 3A, the positive cell line of K562 carries out three duplicate detection, internal reference all detected
GAPDH gene and the fluorescence signal of fusion gene BCR-ABL1, and GAPDH and BCR-ABL1
CTValue is coincide very well；Similarly, three repetition experimental results of the positive cell line of Kasumi
Also match.By this example demonstrates that, utilize the detection system of the present invention to carry out leukemia and merge base
The multi-fluorescence RT-PCR qualitative detection of cause has good repeatability.
Embodiment 3: 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 is positive to determine the K562 containing BCR-ABL1 (p210) fusion gene equally
Cell line, the Kasumi cell line of AML1-ETO fusion gene and the HL60 without fusion gene
Negative cells 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 multi-fluorescence RT-PCR inspection
Survey.
Use multi-fluorescence RT-PCR reaction system same as in Example 1 and reaction condition, multiple
The amplification curve of fluorescence RT-PCR sees Figure of description 4.Fig. 4 A represent 100%K562,10%
K562,1%K562,1 ‰ K562,0.5 ‰ K562,0.25 ‰ K562 and 0.125 ‰ K562
Fluoroscopic examination result；Fig. 4 B represent 100%Kasumi, 10%Kasumi, 1%Kasumi, 1 ‰
The fluoroscopic examination knot of Kasumi, 0.5 ‰ Kasumi, 0.25 ‰ Kasumi and 0.125 ‰ Kasumi
Really.
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 multi-fluorescence RT-PCR of the present invention
The C of fusion gene BCR-ABL1 and AML1-ETO that detection system detectsTIt is worth still less than 35,
And RT-Nested PCR method can't detect corresponding fusion gene accordingly.It is indicated above that and nest
Formula RT-PCR is compared, and the multi-fluorescence RT-PCR detection system of the present invention is to leukemia fusion gene
Detection sensitivity far above corresponding RT-Nested PCR detection method.
Embodiment 4: 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, according to the inspection of the present invention
Survey system carries out the qualitative detection of 45 kinds of fusion genes.The isomer bag of the fusion gene wherein detected
Include 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..
Utilize the multi-fluorescence RT-PCR detection system detection clinical sample of the present invention, operating procedure bag
Including leukocyte RNA extraction, RNA reverse transcription in blood sample is cDNA and multi-fluorescence RT-PCR
Detection.Concrete operational approach is with the embodiment of the present invention 1.The amplified fluorescence curve of clinical sample detection
See Figure of description 5.Wherein Fig. 5 A is that the multi-fluorescence RT-PCR using the present invention detects
The fluorescence results of 4 kinds of Fusional gene isomerides, including BCR-ABL1 (p190), BCR-ABL1 (p210),
PML-RARA (L-type) and PML-RARA (S type)；Fig. 5 B is the multi-fluorescence using the present invention
The fluorescence results of 3 kinds of Fusional gene isomerides that RT-PCR detects, including CBFB-MYH11 (A
Type), SIL-TAL1 and E2A-PBX1；Fig. 5 C is the multi-fluorescence RT-PCR inspection using the present invention
The fluorescence results of the 4 kinds of Fusional gene isomerides measured, including AML1-ETO, SET-CAN,
DEK-CAN and MLL-ELL.
As can be seen here, the multi-fluorescence RT-PCR detection system of the present invention, the clinic detected are utilized
The type of samples fusion gene almost covers the most common leukemia fusion gene, and detection knot
Fruit is consistent with clinical diagnosis.
Result above shows, the multi-fluorescence RT-PCR detection system of the present invention can be carried out all sidedly
The qualitatively screening of 45 kinds of fusion genes of leukemia.And compared with RT-Nested PCR detection method, this
The multi-fluorescence RT-PCR detection system of invention has higher sensitivity and specificity, detects operation
Simply, substantially reduce the detection time, provide one more for leukemic diagnosis, chemotherapy and prognosis
Add the most fast and convenient gene diagnosis technology, there is clinical value widely.
Claims (10)
1., for detecting primer and the probe combinations of leukemia fusion gene, it is by SEQ ID
Primer shown in NO.1-114 and probe composition.
Primer the most according to claim 1 and probe combinations, it is further by 1-12 group primer
Forming with probe, wherein the 1st group by the primer shown in SEQ ID NO.1-9 and SEQ ID NO.10-11
Shown probe composition, the 2nd group by the primer shown in SEQ ID NO.12-16 and SEQ ID
Probe composition shown in NO.17-18, the 3rd group by the primer shown in SEQ ID NO.19-27 and SEQ
Probe composition shown in ID NO.28-29, the 4th group by the primer shown in SEQ ID NO.30-35 and
Probe composition shown in SEQ ID NO.36-38, the 5th group by drawing shown in SEQ ID NO.39-43
Probe composition shown in thing and SEQ ID NO.44-45, the 6th group by shown in SEQ ID NO.46-50
Primer and SEQ ID NO.51-52 shown in probe composition, the 7th group by SEQ ID NO.53-59
Probe composition shown in shown primer and SEQ ID NO.60-62, the 8th group by SEQ ID
Primer shown in NO.63-66 and the composition of the probe shown in SEQ ID NO.67-88, the 9th group by SEQ
Primer shown in ID NO.69-76 and the probe shown in SEQ ID NO.77-78 composition, the 10th group by
Primer shown in SEQ ID NO.79-87 and the composition of the probe shown in SEQ ID NO.88-89, the 11st
Group is made up of the primer shown in SEQ ID NO.90-99 and the probe shown in SEQ ID NO.100-101,
12nd group by the primer shown in SEQ ID NO.102-111 and the spy shown in SEQ ID NO.112-114
Pin forms.
Primer the most according to claim 1 and 2 and probe combinations, wherein 5 ' ends of part probe
Connecting fluorescent reporter group FAM, 3 ' ends connect fluorescent quenching group TAMRA；The 5 ' of remaining probe
End connects fluorescent reporter group JOE, and 3 ' ends connect fluorescent quenching group TAMRA；Reference gene
5 ' ends of GAPDH probe connect fluorescent reporter group JOE, and 3 ' ends connect fluorescent quenching group
TAMRA。
4., for detecting a test kit for leukemia fusion gene, it comprises in claim 1-3 and appoints
One described primer and probe combinations.
Test kit the most according to claim 4, wherein all the concentration of primer is 3pmol/ μ l,
All the concentration of probe is 2pmol/ μ l.
6. primer and probe combinations according to any one of claim 1-3 are melted in preparation detection leukemia
Close the application in kit gene.
7. the primer according to any one of claim 1-3 and probe combinations, or claim 4 or 5
The application in detection leukemia fusion gene of the described test kit.
8. detecting a multi-fluorescence RT-PCR method for leukemia fusion gene, it comprises following step
Rapid:
(1) cDNA of testing sample is obtained；
(2) use the primer according to any one of claim 1-3 and probe combinations, or use right
Require the test kit described in 4 or 5, as template, carry out multiple with the cDNA of acquisition in step (1)
Fluorescence RT-PCR expands, and collects fluorescence signal；
(3) amplification of reference gene GAPDH is analyzed, if CTValue, less than 30, detects process
Effectively, if CTValue, more than 35, re-starts checking detection；
(4) C of reference gene GAPDHTWhen value is less than 30, carry out the knot of leukemia fusion gene
Really interpretation.
Method the most according to claim 8, wherein 5 ' ends of part probe connect fluorescence report base
Group FAM, 3 ' ends connect fluorescent quenching group TAMRA；5 ' ends of remaining probe connect fluorescence report
Group JOE, 3 ' ends connect fluorescent quenching group TAMRA；The 5 ' of reference gene GAPDH probe
End connects fluorescent reporter group JOE, and 3 ' ends connect fluorescent quenching group TAMRA.
The most according to claim 8 or claim 9, method, wherein all the concentration of primer is 3pmol/
μ l, the concentration of whole probes is 2pmol/ μ l.
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| CN108034721A (en) * | 2017-12-04 | 2018-05-15 | 南昌艾迪康医学检验实验室有限公司 | Detect leukaemia NUP98 series fusions oligonucleotides, 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 |
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| CN112011621A (en) * | 2020-09-28 | 2020-12-01 | 东南大学 | Primer combination and method for screening high-risk subtype of acute lymphocytic leukemia |
| 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 |
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