CN107746881A - Primer and probe and application using real-time fluorescence PCR method detection TBLR1 RAR alpha fusion genes - Google Patents
Primer and probe and application using real-time fluorescence PCR method detection TBLR1 RAR alpha fusion genes Download PDFInfo
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Abstract
The invention provides a kind of primer and probe and application that TBLR1 RAR alpha fusion genes are detected using real-time fluorescence PCR method.It is entirely capable of meeting clinical the needs of being monitored to high sensitivity, testing process simplicity and MRD using the primer, probe and method.
Description
Technical field
It is more particularly to a kind of to use TAQMAN probes and real-time fluorescence the invention belongs to life science and biological technical field
Round pcr quantitatively detects method, primer, probe and the kit of TBLR1-RAR alpha fusion genes.Real-time fluorescence PCR technology maturation
And flux is big, suitable for adjuvant clinical diagnosis and the monitoring of MRD.
Technical background
Acute promyelocytic leukemia (Acute promyelocytic leukemia, APL) is the white blood of acute myeloid
A kind of special disease in sick (Acute myeloid leukemia, AML).Because its therapeutic scheme is different from other kinds of
AML, and the usual easy bleeding of state of an illness crisis of such sufferer, often die of illness because of disseminated intravascular coagulation (DIC)；Therefore must in diagnosis
It must distinguish from AML, timely be treated.
APL diagnosis key is to find special chromosome or gene unconventionality.Almost all of APL is directed to chromosome 17
(q21) the rearrangement fusion that transposition, i.e. RAR α genes participate in.Wherein ＞ 95% case is t (15；17)(q22；Q21), produce
PML-RAR alpha fusion genes；But still the case that is not true to type for having minority can be related to other chromosomes and gene and RAR α genes
Fusion.Such as PLZF the and NuMA genes on No. 11 chromosomes, the TBLR1 on NPM1 genes and No. 3 chromosomes on No. 5 chromosomes
Gene etc..In addition, different genes merges with RAR α genes, the reaction to APL medicine vitamin A acid is different
's.Patient such as PML-RAR α, NPM1-RAR α and the NuMA-RAR α positives has reaction to retinoic acid treatment；And PLZF-RAR α are positive
The patient of property is to retinoic acid treatment resistance.So being precisely quickly found the fusion of RAR α genes participation, and confirm that participation is melted
The partner gene of conjunction is extremely important to APL quick diagnosis and the determination of therapeutic scheme.
TBLR1-RAR alpha fusion genes, it is the 10th kind of rare fusion for being related to RAR α so far；Document report only has 4
Example.Because sample size is few, its reactivity to retinoic acid treatment is still not clear.Clinic there is no the fluorescence for the fusion to determine
Detection reagent is measured, is chanced on when only passing through chromosome karyotype analysis.But due to lacking for chromosome karyotype analysis technology itself
Point：If desired for cell culture is carried out, detection cycle is grown；It is how many and quality is influenceed by mitosis figures；And the low grade of sensitivity
Limitation, the requirement of clinical fast and effective Clinics and Practices can not be met, also be unable to reach MRD (MRD) monitoring institute
The sensitivity requirement needed.
The content of the invention
In view of there is no the reagent of detection TBLR1-RAR alpha fusion genes at present, the present invention considers to use TAQMAN probes and reality
When Fluorescence PCR assay method quantitative detection is carried out to TBLR1-RAR alpha fusion genes, it is clinical to high sensitivity, detection to meet
Simple flow and the demand of MRD monitorings.
One kind uses TAQMAN probe for real-time fluorescence PCR technologies, detects the primer and probe of TBLR1-RAR alpha fusion genes,
The nucleotide sequence of the primer and probe is as follows：
TBLR1-F：5’-GGGAGGAGAATGGAGCACA-3’
RARA-R：5’-AGGGCTGGGCACTATCTCTTC-3’
RARA-P：5’-FAM-CCATTGAGACCCAGAGCAGCAGTTC-TAMRA-3’.
Further, the primer and probe also includes being used for primer and the spy for expanding the house-keeping gene ABL as internal reference
Pin, its nucleotide sequence are as follows：
ABL-F：5’-GATACGAAGGGAGGGTGTACCA-3’
ABL-R：5’-CTCGGCCAGGGTGTTGAA-3’
ABL-P：5’-FAM-TGCTTCTGATGGCAAGCTCTACGTCTCCT-TAMRA-3’.
Present invention also offers a kind of method of detection TBLR1-RAR alpha fusion genes, comprise the following steps：
(1) karyocyte in whole blood sample is extracted；
(2) total serum IgE in extraction step 1 in karyocyte, and by RNA reverse transcriptions into cDNA；
(3) using the cDNA of step 2 as template, the quantitative detection of TBLR1-RAR alpha fusion genes is carried out.
Fusion quantitatively detects the sequence for including sense primer TBLR1-F, anti-sense primer RARA-R and probe RARA-P,
It includes：
TBLR1-F：5’-GGGAGGAGAATGGAGCACA-3’
RARA-R：5’-AGGGCTGGGCACTATCTCTTC-3’
RARA-P：5’-FAM-CCATTGAGACCCAGAGCAGCAGTTC-TAMRA-3.
During detection, the primer and probe for expanding the house-keeping gene ABL as internal reference may also include.
More specifically, the erythrocyte cracked liquid in step 1 is 10 × erythrocyte cracked liquid, and its formula is：NH4Cl
82g, NaHCO38.4g, EDTA-Na23.72g, add ddH2O is settled to 1000ml.
Step 2 is using the total serum IgE in TRIzol RNA extraction methods extraction karyocyte
The reaction condition of the quantitative detection of step 3 fusion is：95 DEG C of pre-degeneration 1min；95 DEG C of 10s, 58 DEG C of 50s, altogether
40 circulations；Fluorescence is gathered at 58 DEG C.
Present invention also offers a kind of kit of detection TBLR1-RAR alpha fusion genes, including for detecting TBLR1-
The primer and probe of RAR alpha fusion genes, the sense primer TBLR1-F, anti-sense primer RARA-R and probe RARA-P sequence
Including：
TBLR1-F：5’-GGGAGGAGAATGGAGCACA-3’
RARA-R：5’-AGGGCTGGGCACTATCTCTTC-3’
RARA-P：5’-FAM-CCATTGAGACCCAGAGCAGCAGTTC-TAMRA-3；
Kit can also further comprise also including being used for the primer and probe for expanding the house-keeping gene ABL as internal reference.
Erythrocyte cracked liquid is additionally provided in kit.The erythrocyte cracked liquid is 10 × erythrocyte cracked liquid, and it is matched somebody with somebody
Fang Wei：NH4Cl 82g, NaHCO38.4g, EDTA-Na23.72g, add ddH2O is settled to 1000ml.
Beneficial effects of the present invention are：This is using the method for TAQMAN probes and real-time fluorescence PCR technology to TBLR1-RAR
Alpha fusion gene carries out quantitative detection, is entirely capable of meeting clinical the needs of being monitored to high sensitivity, testing process simplicity and MRD.
Brief description of the drawings
Fig. 1 is primer and probe design attitude figure of the present invention.
Fig. 2 is the fluorescent amplification curve figure detected using primed probe of the present invention and method to clinical doubtful sample 1.
Fig. 3 is the fluorescent amplification curve figure detected using primed probe of the present invention and method to clinical doubtful sample 2.
Embodiment
Embodiment 1：
Whole blood RNA extraction：1 × erythrocyte cracked liquids of 1ml are added in 1.5ml centrifuge tubes, take whole blood sample to be checked
0.5ml, overturn and mix.4000rpm centrifuges 3min, and suction abandons supernatant, adds erythrocyte cracked liquid washed once, obtain required cell；Add l
Ml Total RNA Isolation Reagent, pressure-vaccum is until without obvious cell mass, the addition μ l of chloroform 200, whirlpool repeatedly
30s is mixed, stands 10min on ice.14,000rpm, 4 DEG C of centrifugation 10min.It is transferred to separately with the μ l of pipettor Aspirate supernatant 450
In one centrifuge tube, isometric pre- cold isopropanol is added, 10min is stood on ice after reverse mixing.14,000rpm, 4 DEG C of centrifugations
10min.Then centrifugation is washed respectively once with 75% ethanol and absolute ethyl alcohol.Drying at room temperature 5min, add 50 μ l DEPC-
H2O dissolves.10 × erythrocyte splitting formula of liquid is：NH4Cl 82g, NaHCO38.4g, EDTA-Na23.72g, add ddH2O determines
Hold to 1000ml.
Embodiment 2：
Reverse transcription：RNA solution 4ul (concentration about 200ng/ul) plus 1ul Primer mix (ReverTra in Example 1
Ace qPCR RT Kit) and 3ulDEPC-H2O mixings, 70 DEG C of pre-degeneration 5min；5*RT is added after being quenched 1min on ice
Buffer4ul (ReverTra Ace qPCR RT Kit), Enzyme Mix 1ul (ReverTra Ace qPCR RT Kit),
And add DEPC-H20 7ul to cumulative volume be 20ul.98 DEG C of 5min inactivations after 37 DEG C of 60min reactions, gained is sample to be checked
CDNA.
Embodiment 3：
Quantitative fluorescent PCR：Reagent and amount of reagent configuration fluorescence quantitative PCR detection system as shown in table 1.Detection reagent contains altogether
2 reaction tubes：Each pipes of TBLR1-RAR α and ABL；Wherein ABL is internal reference detection pipe, for judging whether RNA extractions quality accords with
Close and require, while introduce 4 groups of ABL standard curves and calibrated, to carry out quantitative detection to TBLR1-RAR α.Add embodiment 2
Middle each 2 μ l of gained cDNA.Detected by following program：95 DEG C of pre-degeneration 30s；95 DEG C of 10s, 58 DEG C of 35s, totally 40 circulations；
Fluorescence is gathered at 58 DEG C.HUNDERBIRD Probe qPCR Mix spin (Shanghai) bio tech ltd purchased from Japan.
Table 1
Embodiment 4：
Specificity confirms：Take 10, the normal physical examination sample of clinical blood routine, patients with chronic myelocytic leukemia whole blood
This 5,5, Patients With Acute Lymphoblastic Leukemia sample, the non-APL patient of acute myeloid leukemia 5, totally 25 samples progress
Specificity confirms.25 samples carry out RNA extractions as described in embodiment 1-3, reverse transcription obtains cDNA, fluorescent PCR detects and knot
Fruit is analyzed.25 pattern detection results are feminine gender, and specificity is 100%.
Embodiment 5：
Clinical doubtful sample 1 detects：Embodiment 1-3 institutes are pressed to the whole blood sample to be checked of first doubtful APL patient of clinic
State and carry out RNA extractions, reverse transcription obtains cDNA, fluorescent PCR detects and interpretation of result.Acquired results are as shown in Fig. 2 be TBLR1-
RAR alpha fusion genes are positive；Its relative quantification result is calculated as 92.42% according to inspection software.Dyed body karyotyping
Results verification is t (3；17)(q26；Q21), it is consistent with real-time PCR detection result.
Embodiment 6：
Clinical doubtful sample 2 detects：Embodiment 1-3 institutes are pressed to the whole blood sample to be checked of second doubtful APL patient of clinic
State and carry out RNA extractions, reverse transcription obtains cDNA, fluorescent PCR detects and interpretation of result.Acquired results are as shown in figure 3, be TBLR1-
RAR alpha fusion genes are negative.Dyed body karyotyping results verification is t (15；17)(q22；Q21), non-TBLR1-RAR α melt
Close, be consistent with real-time PCR detection result.
According to the TBLR1-RAR alpha fusion gene testing results of the gained of embodiment 5 and 6, summarize as shown in table 2.
Table 2
Sequence table
<110>Wuhan Aidikang Medical Lab Ltd.
<120>Primer and probe and application using real-time fluorescence PCR method detection TBLR1-RAR alpha fusion genes
<160> 6
<170> SIPOSequenceListing 1.0
<210> 1
<211> 19
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 1
gggaggagaa tggagcaca 19
<210> 2
<211> 21
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 2
agggctgggc actatctctt c 21
<210> 3
<211> 25
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 3
ccattgagac ccagagcagc agttc 25
<210> 4
<211> 22
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 4
gatacgaagg gagggtgtac ca 22
<210> 5
<211> 18
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 5
ctcggccagg gtgttgaa 18
<210> 6
<211> 29
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 6
tgcttctgat ggcaagctct acgtctcct 29
Claims (6)
1. detect the primer and probe of TBLR1-RAR alpha fusion genes, it is characterised in that the sense primer TBLR1-F, downstream
Primer RARA-R and probe RARA-P sequence include：
TBLR1-F：5’-GGGAGGAGAATGGAGCACA-3’
RARA-R：5’-AGGGCTGGGCACTATCTCTTC-3’
RARA-P：5’-FAM-CCATTGAGACCCAGAGCAGCAGTTC-TAMRA-3’.
2. primer and probe according to claim 1, it is characterised in that also include being used to expand house keeper's base as internal reference
Because ABL primer and probe, its nucleotide sequence are as follows：
ABL-F：5’-GATACGAAGGGAGGGTGTACCA-3’
ABL-R：5’-CTCGGCCAGGGTGTTGAA-3’
ABL-P：5’-FAM-TGCTTCTGATGGCAAGCTCTACGTCTCCT-TAMRA-3’.
A kind of 3. method of detection TBLR1-RAR alpha fusion genes, it is characterised in that comprise the following steps：
(1) karyocyte in whole blood sample is extracted；
(2) total serum IgE in extraction step 1 in karyocyte, and by RNA reverse transcriptions into cDNA；
(3) using the cDNA of step 2 as template, the quantitative detection of TBLR1-RAR alpha fusion genes is carried out；
The fusion quantitatively detects the sequence for including sense primer TBLR1-F, anti-sense primer RARA-R and probe RARA-P,
It includes：
TBLR1-F：5’-GGGAGGAGAATGGAGCACA-3’
RARA-R：5’-AGGGCTGGGCACTATCTCTTC-3’
RARA-P：5’-FAM-CCATTGAGACCCAGAGCAGCAGTTC-TAMRA-3.
4. according to the method for claim 3, it is characterised in that also include being used to expand the house-keeping gene ABL as internal reference
Primer and probe, its nucleotide sequence is as follows：
ABL-F：5’-GATACGAAGGGAGGGTGTACCA-3’
ABL-R：5’-CTCGGCCAGGGTGTTGAA-3’
ABL-P：5’-FAM-TGCTTCTGATGGCAAGCTCTACGTCTCCT-TAMRA-3’.
5. a kind of kit of detection TBLR1-RAR alpha fusion genes, including for detecting the primer of TBLR1-RAR alpha fusion genes
And probe, it is characterised in that the sense primer TBLR1-F, anti-sense primer RARA-R and probe RARA-P sequence include：
TBLR1-F：5’-GGGAGGAGAATGGAGCACA-3’
RARA-R：5’-AGGGCTGGGCACTATCTCTTC-3’
RARA-P：5’-FAM-CCATTGAGACCCAGAGCAGCAGTTC-TAMRA-3.
6. kit according to claim 5, it is characterised in that also include being used to expand the house-keeping gene as internal reference
ABL primer and probe, its nucleotide sequence are as follows：
ABL-F：5’-GATACGAAGGGAGGGTGTACCA-3’
ABL-R：5’-CTCGGCCAGGGTGTTGAA-3’
ABL-P：5’-FAM-TGCTTCTGATGGCAAGCTCTACGTCTCCT-TAMRA-3’.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| 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 (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106906288A (en) * | 2017-03-24 | 2017-06-30 | 福州艾迪康医学检验所有限公司 | Detect the primer and method of leukaemia CDX2 gene expression doses |
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2017
- 2017-11-10 CN CN201711102374.4A patent/CN107746881A/en active Pending
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106906288A (en) * | 2017-03-24 | 2017-06-30 | 福州艾迪康医学检验所有限公司 | Detect the primer and method of leukaemia CDX2 gene expression doses |
Non-Patent Citations (1)
| Title |
|---|
| YIRUI CHEN等: "TBLR1 fuses to retinoid acid receptor a in a variant t(3;17)(q26;q21) translocation of acute promyelocytic leukemia", 《BLOOD》 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| 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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Application publication date: 20180302 |