CN114410783A - Oligonucleotide for detecting fusion type of fusion gene FNDC3B/RARA and application - Google Patents
Oligonucleotide for detecting fusion type of fusion gene FNDC3B/RARA and application Download PDFInfo
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Abstract
The invention relates to an oligonucleotide for detecting fusion type of fusion gene FNDC3B/RARA and application thereof. The initial detection is carried out by adopting the fluorescent PCR of the multiplex primers containing the three types of primers. If positive, specific type identification is carried out by using specific primer fluorescence PCR, so that the initial detection cost is reduced, and the detection flux is improved. The kit has the advantages of good specificity, high sensitivity and high flux, and is suitable for large-batch sample detection. Has important significance for leukemia diagnosis, adjustment of treatment scheme, evaluation of treatment effect, prognosis prediction and clinical relapse prevention.
Description
Technical Field
The invention belongs to the fields of life science and biotechnology, and particularly relates to a primer, a probe and a detection kit for leukemia fusion gene FNDC3B/RARA detection for screening purposes.
Background
Acute Promyelocytic Leukemia (APL) is a special subtype M3 of (acute myeloloid 1eukemia, AML), accounts for about 15% of AML, is mainly developed in middle and young years above 20 years old, has a violent course, and has a high bleeding tendency and early mortality. More than 95% of APL patients can see the translocation of chromosome 15 and chromosome 17 to form characteristic t (15; 17) (q 22; q21), namely PML-RARa fusion gene, which is the APL characteristic genetic marker. However, about 5% of patients lack the fusion gene and are referred to as atypical APL patients. Wherein FNDC3B/RARA fusion is one of the atypical fusions. Fusion of RARa and other partner genes is found in atypical APL patients, the partner genes are different, and the responsiveness of the RARa and the ATO is different, for example, ATRA can target degradation of PML-RARa fusion protein, recover the functions of wild RARa and PML genes, and re-induce granulocyte differentiation and maturation. ATO can act on PML to induce rapid degradation of PML-RARa fusion protein and wild-type PML protein. The two medicines have synergistic effect, no cross drug resistance and good treatment effect, so that the APL becomes clinically curable acute myeloid leukemia. APL patients with PLZF-RARa and STAT5B-RARa fusion genes are insensitive or tolerant to ATRA treatment, and have poor overall treatment effect and poor prognosis. APL patients with a BCOR-RARa fusion gene are sensitive to ATRA but tolerant to ATO. APL with FNDC3B/RARA fusion was reported to be sensitive to ATRA. Therefore, the identification of different fusion types is beneficial to personalized treatment, the effective rate of treatment can be improved, the medical cost is reduced, and the survival rate of patients is improved.
In the clinical treatment of acute leukemia, especially for APL, one prognosis evaluation is stratified, and different treatment schemes are selected according to different prognosis stratification. Meanwhile, the expression level of the fusion gene in the treatment process needs to be monitored so as to predict the curative effect and the possibility of relapse. Therefore, the screening of fusion genes related to RARa rearrangement is particularly important.
The FNDC3B/RARA has various related fracture sites and generates a plurality of fusion genes, so that certain difficulties exist in clinical detection. The current methods for FNDC3B/RARA screening are mainly as follows: chromosome karyotype analysis, and multiplex nested PCR combined electrophoresis. The chromosome karyotype analysis is judged by visual observation, nucleated cells in a sample need to be cultured, and observation can be carried out only if a nuclear fission phase exists; in the process of cell culture proliferation, the possibility of 'flooding' diseased cells due to the dominant growth of certain cells can occur, and the false impression of normal karyotype is caused. Secondly, in some cases, chromosomal translocations are complex and microscopic and cannot be analyzed by visual observation. Furthermore, the sensitivity of karyotyping cannot meet the requirements for detection of MRD (Minimal residual Disease). The method of multiple nested PCR combined electrophoresis consumes long time, has complex process and is easy to pollute, and the judgment of results is subjective. And the PCR reaction system is more, the cost is high, and the method is not suitable for high-flux sample detection. Can only carry out qualitative detection, and can not meet the requirements of high sensitivity and good specificity at the same time.
Disclosure of Invention
In view of the fact that the existing methodology for screening FNDC3B/RARA fusion genes cannot simultaneously meet the requirements of economy, high efficiency, accurate result, MRD monitoring and the like, the invention designs a set of primers and probes for detecting common fusion genes by a multiple fluorescence PCR technology, and designs a scheme for quickly and accurately screening and identifying types with high sensitivity, good specificity and large detection flux.
The invention designs primers and probes for detecting an internal reference/target gene, and detects the fusion type of the internal reference gene ABL and the target gene FNDC3B/RARA by adopting real-time fluorescence RT-PCR. The kit can ensure that the amplification efficiency and the amplification rate are both optimal by adjusting the primer probe ratio of the internal reference/target gene and the RT-PCR reaction conditions.
The detection Kit for FNDC3B/RARA contains erythrocyte lysate, TRIzol, chloroform, absolute ethyl alcohol, ReverTra Ace qPCR RT Kit (TOYOBO company), detection system PCR reaction liquid 1, detection system PCR reaction liquid 2, detection system PCR reaction liquid 3, detection system PCR reaction liquid 4, positive control 1, positive control 2, positive control 3, positive control 4, negative control and internal reference ABL reaction liquid.
The detection system PCR reaction solution 1 comprises THUNDERBIRD qPCR MIX (TOYOBO, QPS-101), and upstream and downstream primers for detecting target genes are respectively as follows: RARA/FNDC3B 2-F, RARA/FNDC3B 2-25-R, RARA/FNDC3B2-26-R, FNDC3B/RARA24-3-F, FNDC3B/RARA 24-3-R and Probe RARA/FNDC3B2-Probe, FNDC3B/RARA 24-3-Probe, for initial examination, the sequence of the primers and probes is as follows:
RARA/FNDC3B 2-F:CAGCACCAGCTTCCAGTTAGTG
RARA/FNDC3B 2-25-R:CCTTCTAACTGTGTTACTCGAGG
RARA/FNDC3B 2-26-R:TGTGGCTTCTTCTCCCTTGTACA
FNDC3B/RARA 24-3-F:AACCTATACCTTCAGCACAACC
FNDC3B/RARA 24-3-R:GTGGTAGCCTGAGGACTTGT
RARA/FNDC3B 2-Probe:HEX-ATATAGCACACCATCCC-TAMRA
FNDC3B/RARA 24-3-Probe:FAM-AGACCCAGAGCAGCAGTTCTGAAGAGA–TAMRA
the PCR reaction liquid 2 of the detection system comprises THUNDERBIRD qPCR MIX (TOYOBO, QPS-101), a primer RARA/FNDC3B 2-F, RARA/FNDC3B 2-25-R and a Probe RARA/FNDC3B2-Probe, which are used for identifying RARA/FNDC3B 2-25. The sequences of the probe and the primer are as follows,
RARA/FNDC3B 2-F:CAGCACCAGCTTCCAGTTAGTG
RARA/FNDC3B 2-25-R:CCTTCTAACTGTGTTACTCGAGG
RARA/FNDC3B 2-Probe:HEX-ATATAGCACACCATCCC-TAMRA
the PCR reaction liquid 3 of the detection system comprises THUNDERBIRD qPCR MIX (TOYOBO, QPS-101), a primer RARA/FNDC3B 2-F, RARA/FNDC3B2-26-R and a Probe RARA/FNDC3B2-Probe, which are used for identifying RARA/FNDC3B 2-26. The probe and primer sequences are as follows, RARA/FNDC3B 2-F CAGCACCAGCTTCCAGTTAGTG
RARA/FNDC3B 2-26-R:TGTGGCTTCTTCTCCCTTGTACA
RARA/FNDC3B 2-Probe:HEX-ATATAGCACACCATCCC-TAMRA
The PCR reaction solution 4 of the detection system comprises THUNDERBERBD qPCR MIX (TOYOBO, QPS-101), a primer FNDC3B/RARA24-3-F, FNDC3B/RARA 24-3-R and a Probe FNDC3B/RARA 24-3-Probe for identifying FNDC3B/RARA 24-3. The sequences of the probe and the primer are as follows,
FNDC3B/RARA 24-3-F:AACCTATACCTTCAGCACAACC
FNDC3B/RARA 24-3-R:GTGGTAGCCTGAGGACTTGT
FNDC3B/RARA 24-3-Probe:FAM-AGACCCAGAGCAGCAGTTCTGAAGAGA–TAMRA
the internal reference ABL reaction solution comprises THUNDERBIRD qPCR MIX (TOYOBO, QPS-101), a primer ABL-F, ABL-R for detecting ABL and a Probe ABL-Probe. The sequences of the probe primers are as follows,
ABL-F:GATACGAAGGGAGGGTGTACCA
ABL-R:CTCGGCCAGGGTGTTGAA
ABL-Probe:CY5-GCTTCTGATGGCAAGCTCTACGTCTCCT-BHQ
in addition, the negative control is a solution without MLL/CBP fusion gene. The positive control product 1 is a plasmid solution containing fusion genes RARA/FNDC3B 2-25, RARA/FNDC3B2-26 and FNDC3B/RARA 24-3; the positive control 2 is fusion gene RARA/FNDC3B 2-25 plasmid solution; the positive control 3 is fusion gene RARA/FNDC3B2-26 plasmid solution; positive control 4 is FNDC3B/RARA24-3 fusion gene plasmid solution.
The design idea of the invention is as follows:
1. design of primers and probes
Primers and probes are designed aiming at RARA/FNDC3B 2-25, RARA/FNDC3B2-26 and FNDC3B/RARA24-3 with high FNDC3B/RARA fusion probability. The primers are designed according to FNDC3B exon24, exon25 and exon26 sequences, and then the primers are designed according to RARA exon2 and exon3 sequences, wherein RARA/FNDC3B 2-25 and RARA/FNDC3B2-26 share the same upstream primer and probe, but in order to ensure the reaction efficiency and specificity in a multi-primer fluorescence reaction system, the upstream primer and the downstream primer are independently used by FNDC3B/RARA 24-3. The invention can be said to consider reaction efficiency and specificity to the utmost extent, and simultaneously greatly reduce cost.
2. Detection and identification System design
Because the FNDC3B/RARA has low occurrence probability, the invention firstly adopts multiple primers (one reaction system contains the primers corresponding to 3 fusion types) to combine with real-time fluorescence RT-PCR to carry out primary detection on a sample. If positive results appear in the initial detection result, specific primers (only 1 primer of the fusion type is contained in one reaction system, and the primers are one of multiple primer reaction systems) are used for identifying the specific type.
The invention provides an oligonucleotide for detecting fusion type of fusion gene FNDC3B/RARA, which comprises a primer RARA/FNDC3B 2-F, RARA/FNDC3B 2-25-R, RARA/FNDC3B2-26-R, FNDC3B/RARA24-3-F, FNDC3B/RARA 24-3-R and a Probe RARA/FNDC3B2-Probe and FNDC3B/RARA 24-3-Probe, wherein the sequences of the primer and the Probe are as follows:
RARA/FNDC3B 2-F:CAGCACCAGCTTCCAGTTAGTG
RARA/FNDC3B 2-25-R:CCTTCTAACTGTGTTACTCGAGG
RARA/FNDC3B 2-26-R:TGTGGCTTCTTCTCCCTTGTACA
FNDC3B/RARA 24-3-F:AACCTATACCTTCAGCACAACC
FNDC3B/RARA 24-3-R:GTGGTAGCCTGAGGACTTGT
RARA/FNDC3B 2-Probe:HEX-ATATAGCACACCATCCC-TAMRA
FNDC3B/RARA 24-3-Probe:FAM-AGACCCAGAGCAGCAGTTCTGAAGAGA–TAMRA
further, primers RARA/FNDC3B 2-F, RARA/FNDC3B 2-25-R and probes RARA/FNDC3B2-Probe for identifying RARA/FNDC3B 2-25; primers RARA/FNDC3B 2-F, RARA/FNDC3B2-26-R and probes RARA/FNDC3B2-Probe for identifying RARA/FNDC3B 2-26; primer FNDC3B/RARA24-3-F, FNDC3B/RARA 24-3-R and Probe FNDC3B/RARA 24-3-Probe for identifying FNDC3B/RARA 24-3.
Further, the kit also comprises a primer ABL-F, ABL-R and a Probe ABL-Probe for detecting the reference gene ABL, wherein the primer and the Probe have the following sequences:
ABL-F:GATACGAAGGGAGGGTGTACCA
ABL-R:CTCGGCCAGGGTGTTGAA
ABL-Probe:CY5-GCTTCTGATGGCAAGCTCTACGTCTCCT-BHQ。
the invention also provides a kit for detecting the fusion type oligonucleotide of the fusion gene FNDC3B/RARA, which comprises a primer RARA/FNDC3B 2-F, RARA/FNDC3B 2-25-R, RARA/FNDC3B2-26-R, FNDC3B/RARA24-3-F, FNDC3B/RARA 24-3-R and a Probe RARA/DC 3B 2-FN 2-Probe and FNDC3B/RARA 24-3-Probe, wherein the sequences of the primer and the Probe are as follows:
RARA/FNDC3B 2-F:CAGCACCAGCTTCCAGTTAGTG
RARA/FNDC3B 2-25-R:CCTTCTAACTGTGTTACTCGAGG
RARA/FNDC3B 2-26-R:TGTGGCTTCTTCTCCCTTGTACA
FNDC3B/RARA 24-3-F:AACCTATACCTTCAGCACAACC
FNDC3B/RARA 24-3-R:GTGGTAGCCTGAGGACTTGT
RARA/FNDC3B 2-Probe:HEX-ATATAGCACACCATCCC-TAMRA
FNDC3B/RARA 24-3-Probe:FAM-AGACCCAGAGCAGCAGTTCTGAAGAGA–TAMRA
further, the composition also contains erythrocyte lysate.
Furthermore, the kit also comprises a positive control substance and a negative control substance.
A method for reducing the detection cost of fusion gene FNDC3B/RARA comprises the following steps:
(1) extracting total RNA in a blood sample;
(2) reverse transcribing the RNA extracted in step 1 into cDNA;
(3) combining the detection system PCR reaction solution 1 with real-time fluorescence PCR to carry out preliminary detection on the sample, and stopping detection if the preliminary detection result shows negative;
(4) if the detection result in the step 3 shows positive, identifying the specific type of the detection sample by combining the detection system PCR reaction liquid 2, the detection system PCR reaction liquid 3 and the detection system PCR reaction liquid 4 with real-time fluorescence PCR;
(5) and (5) judging a result: adjusting the threshold line to be above the background signal and the negative amplification line to obtain a Ct value;
1) when the internal reference is positive, the detection result is considered to be effective; 2) positive judgment standard: ct <36, positive; ct is more than or equal to 35 and less than or equal to 38, is suspected positive and needs to be verified again; ct > 38, negative.
The invention also provides a method for detecting the fusion type of the fusion gene FNDC3B/RARA, which comprises the following steps:
(1) extracting total RNA in a blood sample;
(2) reverse transcribing the RNA extracted in step 1 into cDNA;
(3) combining the detection system PCR reaction solution 1 with real-time fluorescence PCR to carry out preliminary detection on the sample, and stopping detection if the preliminary detection result shows negative; if the preliminary detection result shows positive, judging the gene type of the MLL/CBP fusion gene;
(4) if the detection result in the step 3 shows positive, identifying the specific type of the detection sample by combining the detection system PCR reaction liquid 2, the detection system PCR reaction liquid 3 and the detection system PCR reaction liquid 4 with real-time fluorescence PCR;
(5) and (5) judging a result: adjusting the threshold line to be above the background signal and the negative amplification line to obtain a Ct value; 1) when the internal reference is positive, the detection result is considered to be effective; 2) positive judgment standard: ct <36, positive; ct is more than or equal to 35 and less than or equal to 38, is suspected positive and needs to be verified again; ct > 38, negative.
The invention has the beneficial effects that: the invention adopts a real-time fluorescence PCR Tapman probe method to detect and identify the fusion gene FNDC3B/RARA in vivo of a detected person, and compared with the prior fluorescence in situ hybridization and the traditional reverse transcription PCR method, the kit has the advantages of whole-process monitoring, high sensitivity, pollution prevention, convenient result interpretation and the like. And the kit adopts a method of combining the primary detection of a multiple primer system and a specific primer, so that the primary detection cost is reduced, and the detection flux is improved. In addition, the kit reasonably matches and optimizes primers and probes required by a reaction system, so that the experimental conditions are optimal, a complex condition exploration link is omitted, and the experimental efficiency is greatly improved. Therefore, the kit can quickly and accurately detect FNDC3B/RARA in a large number of samples, and has important significance for leukemia diagnosis, adjustment of treatment schemes, evaluation of treatment effects, prognosis prediction and clinical relapse prevention.
Drawings
FIG. 1 sample 3, sample 2, was screened for FNDC3B/RARA negative results, all positive controls were positive lines, sample 2 had only ABL positive lines, and the 3-fold fusion type FNDC3B/RARA did not have lines, indicating negative results.
Detailed Description
Example 1
The method comprises the following operation processes:
(1) extracting total RNA from blood by adding 1ml of erythrocyte lysate into a clean 1.5ml centrifuge tube, and uniformly mixing 0.5ml of anticoagulation blood. Standing at room temperature for 10 min; centrifuging at 5000rpm for 5min, discarding supernatant, and collecting cells at bottom; adding 0.5ml of erythrocyte lysate again, centrifuging at 5000rpm for 5min, discarding the supernatant, and collecting the cells at the bottom; adding 1ml of TRIzol into the cells, repeatedly blowing and beating until the precipitate is completely dissolved, and standing for 5min at room temperature; adding 0.2ml of chloroform, and shaking uniformly; centrifuging at 14000rpm and 4 ℃ for 10min, sucking the supernatant and transferring to another new centrifuge tube; adding isopropanol with the same volume, mixing thoroughly, standing at room temperature for 10 min; centrifuging at 14000rpm and 4 ℃ for 10min, removing the supernatant, adding 1ml of 75% ethanol, and slightly reversing the upper part and the lower part to wash the tube wall; centrifuging at 14000rpm and 4 ℃ for 5min, and removing ethanol; drying at room temperature for 10-15min, adding 20 μ l RNase-free water to dissolve precipitate.
(2) RNA was inverted to cDNA by referring to the Rever Tra Ace qPCR RT Kit instructions from TOYOBO.
(3) Preparing a reagent: preparing a detection system PCR reaction solution X ul according to the requirement of initial detection or type identification, and subpackaging 23 mu l of each person:
x ═ 23 μ l reaction X (n specimens +1 positive control +1 negative control +1 blank);
(4) sample adding: adding 2 mu lcDNA into the PCR reaction solution of the detection system; when the positive control or the negative control is detected, 2 mul of positive control or negative control is added into the PCR reaction solution of the detection reaction system; in the case of blank control detection, 2. mu.l of physiological saline was added to the PCR reaction solution of the detection reaction system or nothing was added.
The detection reaction system PCR reaction solution is selected from one of a detection system PCR reaction solution 1, a detection system PCR reaction solution 2, a detection system PCR reaction solution 3 and a detection system PCR reaction solution 4 (the specific composition is shown in Table 1).
The positive control substance is selected from one of a positive control substance 1, a positive control substance 2, a positive control substance 3 and a positive control substance 4.
TABLE 1 detection System PCR reaction solution preparation Components
(5) And (3) detection: the detection was performed on a real-time fluorescent PCR instrument, and available instruments include ABI7300, 7500 (Applied Biosystems, USA), and the like. Reaction conditions are as follows: pre-denaturation at 95 ℃ for 1 min; the reaction was carried out at 95 ℃ for 15s and at 58 ℃ for 35s for 40 cycles, and the fluorescence signal was collected at 58 ℃ for 35 s.
If only the expression condition of the fusion gene FNDC3B/RARA in the detection sample needs to be obtained, type identification is not needed, and the detection system PCR reaction liquid 1 is adopted.
If the specific type of the fusion gene FNDC3B/RARA in the detection sample is to be obtained, the sample is preliminarily detected by combining the detection system PCR reaction liquid 1 with real-time fluorescence PCR. And if the preliminary detection result shows negative, stopping the detection. If the preliminary detection result shows positive, the detection system PCR reaction liquid 2, the detection system PCR reaction liquid 3 and the detection system PCR reaction liquid 4 are combined with real-time fluorescence PCR to identify the specific type of the detection sample.
(6) And (5) judging a result: and adjusting the threshold line to be above the background signal and the negative amplification line to obtain the Ct value.
1) When the internal reference is positive, the detection result is considered to be effective; 2) positive judgment standard: ct <36, positive; ct is more than or equal to 35 and less than or equal to 38, is suspected positive and needs to be verified again; ct > 38, negative.
EXAMPLE 2 Primary examination of clinically collected leukemia samples
Randomly taking 9 parts of clinical samples to be detected, extracting nucleic acid RNA according to the method described in the embodiment 1, preparing a reagent according to the PCR reaction solution 1 of the detection system, and detecting. Mu.l of each sample was added to the reaction solution of the detection system. At the same time, positive and negative are made, and blank control is performed respectively. The results are given in table 2 below.
TABLE 1
EXAMPLE 3 preliminary examination of specimens clinically diagnosed as APL
The total of 8 samples of anticoagulant samples of patients who are confirmed to be APL are taken and sent for detection, nucleic acid RNA is extracted according to the method described in the embodiment 1, and reagents are prepared according to the PCR reaction liquid 1 of the detection system and are detected.
Mu.l of each sample is added into the PCR reaction solution of the detection system. And simultaneously, making positive, negative and blank controls. Since the fusion gene FNDC3B/RARA is rare among leukemia patients, 8 leukemia samples are negative results. The results are given in table 1 below:
TABLE 2
Wherein the results of FNDC3B/RARA screening test for sample No. 2 are shown in FIG. 1.
Example 4FNDC3B/RARA type identification
3 APL samples after the initial detection of the embodiment 3 are randomly taken, nucleic acid RNA is extracted according to the method of the embodiment 1, and reagents are prepared according to the PCR reaction liquid 2,3 and 4 of the detection system and are detected. And adding 2 mul of each sample into the PCR reaction solution of the identification and detection system. At the same time, positive and negative are made, and blank control is performed respectively. Since the probability of occurrence of FNDC3B/RARA was low, the results were consistent with example 3, and none of the results were positive. The results are given in Table 3 below
TABLE 3
Sequence listing
<110> Yidekang laboratory Co., Ltd for medical examination of Hefei Aidikang
<120> oligonucleotide for detecting fusion type of fusion gene FNDC3B/RARA and application
<160> 10
<170> SIPOSequenceListing 1.0
<210> 1
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
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cagcaccagc ttccagttag tg 22
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<211> 23
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<213> Artificial Sequence (Artificial Sequence)
<400> 2
ccttctaact gtgttactcg agg 23
<210> 3
<211> 23
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 3
<210> 4
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
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aacctatacc ttcagcacaa cc 22
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<210> 6
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<213> Artificial Sequence (Artificial Sequence)
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<213> Artificial Sequence (Artificial Sequence)
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agacccagag cagcagttct gaagaga 27
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gatacgaagg gagggtgtac ca 22
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gcttctgatg gcaagctcta cgtctcct 28
Claims (9)
1. The oligonucleotide for detecting the fusion type of the fusion gene FNDC3B/RARA is characterized by comprising a primer RARA/FNDC3B 2-F, RARA/FNDC3B 2-25-R, RARA/FNDC3B2-26-R, FNDC3B/RARA24-3-F, FNDC3B/RARA 24-3-R and a Probe RARA/FNDC3B2-Probe and FNDC3B/RARA 24-3-Probe, wherein the sequences of the primer and the Probe are as follows:
RARA/FNDC3B 2-F:CAGCACCAGCTTCCAGTTAGTG
RARA/FNDC3B 2-25-R:CCTTCTAACTGTGTTACTCGAGG
RARA/FNDC3B 2-26-R:TGTGGCTTCTTCTCCCTTGTACA
FNDC3B/RARA 24-3-F:AACCTATACCTTCAGCACAACC
FNDC3B/RARA 24-3-R:GTGGTAGCCTGAGGACTTGT
RARA/FNDC3B 2-Probe:HEX-ATATAGCACACCATCCC-TAMRA
FNDC3B/RARA 24-3-Probe:FAM-AGACCCAGAGCAGCAGTTCTGAAGAGA–TAMRA。
2. the oligonucleotide according to claim 1, wherein the primers RARA/FNDC3B 2-F, RARA/FNDC3B 2-25-R and the Probe RARA/FNDC3B2-Probe for identifying RARA/FNDC3B 2-25; primers RARA/FNDC3B 2-F, RARA/FNDC3B2-26-R and probes RARA/FNDC3B2-Probe for identifying RARA/FNDC3B 2-26; primer FNDC3B/RARA24-3-F, FNDC3B/RARA 24-3-R and Probe FNDC3B/RARA 24-3-Probe for identifying FNDC3B/RARA 24-3.
3. The oligonucleotide of claim 1, further comprising a primer ABL-F, ABL-R and a Probe ABL-Probe for reference gene ABL detection, wherein the primer and Probe sequences are as follows:
ABL-F:GATACGAAGGGAGGGTGTACCA
ABL-R:CTCGGCCAGGGTGTTGAA
ABL-Probe:CY5-GCTTCTGATGGCAAGCTCTACGTCTCCT-BHQ。
4. the kit for detecting the fusion type oligonucleotide of the fusion gene FNDC3B/RARA is characterized by comprising a primer RARA/FNDC3B 2-F, RARA/FNDC3B 2-25-R, RARA/FNDC3B2-26-R, FNDC3B/RARA24-3-F, FNDC3B/RARA 24-3-R and a Probe RARA/FNDC3B2-Probe and FNDC3B/RARA 24-3-Probe, wherein the sequences of the primer and the Probe are as follows:
RARA/FNDC3B 2-F:CAGCACCAGCTTCCAGTTAGTG
RARA/FNDC3B 2-25-R:CCTTCTAACTGTGTTACTCGAGG
RARA/FNDC3B 2-26-R:TGTGGCTTCTTCTCCCTTGTACA
FNDC3B/RARA 24-3-F:AACCTATACCTTCAGCACAACC
FNDC3B/RARA 24-3-R:GTGGTAGCCTGAGGACTTGT
RARA/FNDC3B 2-Probe:HEX-ATATAGCACACCATCCC-TAMRA
FNDC3B/RARA 24-3-Probe:FAM-AGACCCAGAGCAGCAGTTCTGAAGAGA–TAMRA。
5. the kit of claim 4, further comprising a red blood cell lysate.
6. The kit of claim 4, further comprising a positive control and a negative control.
7. A method for reducing the detection cost of fusion gene FNDC3B/RARA comprises the following steps:
(1) extracting total RNA in a blood sample;
(2) reverse transcribing the RNA extracted in step 1 into cDNA;
(3) combining the detection system PCR reaction solution 1 with real-time fluorescence PCR to carry out preliminary detection on the sample, and stopping detection if the preliminary detection result shows negative;
(4) if the detection result in the step 3 shows positive, identifying the specific type of the detection sample by combining the detection system PCR reaction liquid 2, the detection system PCR reaction liquid 3 and the detection system PCR reaction liquid 4 with real-time fluorescence PCR;
(5) and (5) judging a result: adjusting the threshold line to be above the background signal and the negative amplification line to obtain a Ct value; 1) when the internal reference is positive, the detection result is considered to be effective; 2) positive judgment standard: ct <36, positive; ct is more than or equal to 35 and less than or equal to 38, is suspected positive and needs to be verified again; ct > 38, negative.
8. The method of claim 7, wherein the step of applying the coating comprises applying a coating to the substrate
The detection system PCR reaction solution 1 includes: RARA/FNDC3B 2-F, RARA/FNDC3B 2-25-R, RARA/FNDC3B2-26-R, FNDC3B/RARA24-3-F, FNDC3B/RARA 24-3-R and Probe RARA/FNDC3B2-Probe, FNDC3B/RARA 24-3-Probe;
the PCR reaction liquid 2 of the detection system comprises RARA/FNDC3B 2-F, RARA/FNDC3B 2-25-R and a Probe RARA/FNDC3B 2-Probe;
the PCR reaction liquid 3 of the detection system comprises RARA/FNDC3B 2-F, RARA/FNDC3B2-26-R and a Probe RARA/FNDC3B 2-Probe;
the PCR reaction solution 4 of the detection system comprises FNDC3B/RARA24-3-F, FNDC3B/RARA 24-3-R and a Probe FNDC3B/RARA 24-3-Probe
A method for detecting the fusion type of the fusion gene FNDC3B/RARA, comprising
(1) Extracting total RNA in a blood sample;
(2) reverse transcribing the RNA extracted in step 1 into cDNA;
(3) combining the detection system PCR reaction solution 1 with real-time fluorescence PCR to carry out preliminary detection on the sample, and stopping detection if the preliminary detection result shows negative; if the preliminary detection result shows positive, judging the gene type of the MLL/CBP fusion gene;
(4) if the detection result in the step 3 shows positive, identifying the specific type of the detection sample by combining the detection system PCR reaction liquid 2, the detection system PCR reaction liquid 3 and the detection system PCR reaction liquid 4 with real-time fluorescence PCR;
(5) and (5) judging a result: adjusting the threshold line to be above the background signal and the negative amplification line to obtain a Ct value; 1) when the internal reference is positive, the detection result is considered to be effective; 2) positive judgment standard: ct <36, positive; ct is more than or equal to 35 and less than or equal to 38, is suspected positive and needs to be verified again; ct > 38, negative.
9. The method of claim 8,
the detection system PCR reaction solution 1 includes: RARA/FNDC3B 2-F, RARA/FNDC3B 2-25-R, RARA/FNDC3B2-26-R, FNDC3B/RARA24-3-F, FNDC3B/RARA 24-3-R and Probe RARA/FNDC3B2-Probe, FNDC3B/RARA 24-3-Probe;
the PCR reaction liquid 2 of the detection system comprises RARA/FNDC3B 2-F, RARA/FNDC3B 2-25-R and a Probe RARA/FNDC3B 2-Probe;
the PCR reaction liquid 3 of the detection system comprises RARA/FNDC3B 2-F, RARA/FNDC3B2-26-R and a Probe RARA/FNDC3B 2-Probe;
the PCR reaction solution 4 of the detection system comprises FNDC3B/RARA24-3-F, FNDC3B/RARA 24-3-R and a Probe FNDC3B/RARA 24-3-Probe.
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