CN112522403A - Primer and probe for detecting ZNF384 related fusion gene by using multiple fluorescence PCR technology and application - Google Patents
Primer and probe for detecting ZNF384 related fusion gene by using multiple fluorescence PCR technology and application Download PDFInfo
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Abstract
The invention relates to a primer and a probe for detecting ZNF384 related fusion genes by using a multiple fluorescence PCR technology and application in a kit. The screening method integrates ZNF384 fusion genes related to the ALL disease for screening, can assist diagnosis, treatment and prognosis monitoring of the ALL disease more comprehensively, has higher precision compared with the traditional methods such as FISH and the like, and is convenient for judging results. In addition, the invention reasonably matches and optimizes the primers and the probes, and optimizes the detection conditions to be optimal, thereby greatly improving the efficiency. In addition, the multiplex fluorescence PCR technology has high sensitivity, high specificity, less sample requirement and lower cost, and is suitable for screening large-batch clinical samples.
Description
Technical Field
The invention belongs to the fields of life science and biotechnology, and relates to a primer and a probe for detecting ZNF384 related fusion genes by using a multiple fluorescence PCR technology, and application of the primer and the probe in a kit. The ZNF384 related fusion gene screened by the invention comprises: EP300-ZNF384, ARID1B-ZNF384, CREBP-ZNF 384, TCF3-ZNF384, TAF15-ZNF384 and BMP2K-ZNF384, wherein the total number is 6.
Background
Acute Lymphoblastic Leukemia (ALL) is the most common malignancy in children and is also the leading cause of death in children. Genetic abnormalities (gene fusions, aneuploidies, etc.) are the major cause of ALL development, and molecular typing based on this is of great importance for clinical diagnosis, risk stratification, and targeted therapies. Still 20% to 30% of ALL are unknown in genetic characteristics, and with the development of genomics technology, fusion positive ALL of zinc-finger protein 384 (ZNF 384) as transcription factor attracts attention in unique clinical and biological characteristics.
ZNF384 is easy to generate gene fusion and is mostly related to children ALL, and case reports are mostly seen in Asian countries, and the incidence rate of Asian population accounts for about 4% of the total number of ALL. There are 9 fusion partners that have been discovered so far: ARID1B, BMP2K, E1A binding protein P300(E1A binding protein P300, EP300), EP300 paralogous CREB binding protein (CREB binding protein, CREBP), SMARCA2, SYNRG, TATA box binding protein related factor 15(TATA-box binding protein associated factor 15, TAF15), EWS RNA binding protein 1(EWS RNA binding protein 1, EWSR 1), transcription factor 3(tranion factor 3, TCF 3). ZNF384 fusion partners are different, but have similar transcription feature profiles and are tightly clustered into a group, and are different from other known ALL subtypes, and the ALL subtypes are further found to be accompanied by obvious epigenetic regulation gene change by combining with whole exon sequencing analysis. The leukemia cells of ZNF384 fusion positive ALL patients are found to have immunophenotype characteristic of progenitor B cells, namely CD10 is weakly positive or negative and has abnormal expression of one or more myeloid antigens (CD13, CD33), which indicates that B cell differentiation is blocked at the stage, and the subtype of the children is poorer than EVT6-RUNX1+ subtype according to survival analysis data.
Because the chromosomal regions of the ZNF384 gene and some fusion partners (TCF3, EWSR1, EP300, ARID1B) are located near the terminal telomere position of their respective chromosome arms, the difficulty of conventional G-band karyotyping is greatly increased; the FISH reagent has high cost and poor sensitivity compared with PCR; the PCR method is time-consuming and easy to pollute, results are difficult to judge, and the reaction system has more reaction systems and correspondingly higher cost, so that the PCR method is not suitable for clinical large-scale screening.
Disclosure of Invention
In view of the fact that the selectivity of the methodology for screening and detecting the ZNF384 related fusion gene is small at present and the problems of cost, specificity and clinical popularization are comprehensively considered, the invention designs a set of primers and probes for detecting the ZNF384 related common fusion gene by the multiplex fluorescence PCR technology and designs a detection method which has high sensitivity and good specificity and is suitable for large-flux screening.
A primer and a probe for screening and identifying 6 relatively common ZNF384 related fusion genes of EP300-ZNF384, ARID1B-ZNF384, CREBP-ZNF 384, TCF3-ZNF384, TAF15-ZNF384 and BMP2K-ZNF384 by using a fluorescence PCR technology are disclosed, wherein three groups of downstream primers existing in the ZNF384 gene and the corresponding probes are matched with a plurality of upstream primers on different upstream genes by using a multiplex fluorescence PCR technology. The nucleotide sequence is as follows:
the primer probe for screening the fusion gene also comprises a primer and a probe for amplifying housekeeping gene ABL serving as an internal reference, and the nucleotide sequence of the primer and the probe is as follows:
ABL1-F：5’-GATACGAAGGGAGGGTGTACCA-3’
ABL1-R：5’-CTCGGCCAGGGTGTTGAA-3’
ABL1-P：5’-FAM-TGCTTCTGATGGCAAGCTCTACGTCTCCT-TAMRA-3’。
the fusion reported in literature occurs on exons 3, 4 and 7 of ZNF384, TCF3 is exons 11, 13, 16 and 17, TAF15 is exons 6 and 11, CREBP comprises exons 4, 5, 6, 7 and 10, BMP2K is exons 14 and 15, EP300 mainly occurs in exon 6, and ARID1B is exon 4. The primer probes are schematically arranged as shown in FIGS. 1 and 2. Meanwhile, in order to ensure good fluorescent amplification effect, the sequencing fragment is controlled within 200 bp. In addition, in order to ensure the efficiency of PCR amplification, the design of primers needs to follow some principles of primer design: such as avoiding consecutive 4G and 4C, avoiding regions with too high GC content, etc.
Combining the above factors, the final designed primer is as described above.
The invention also provides a primer probe for identifying 6 fusion genes of EP300-ZNF384, ARID1B-ZNF384, CREBP-ZNF 384, TCF3-ZNF384, TAF15-ZNF384 and BMP2K-ZNF384, which is characterized in that three groups of primer probes in the 3 rd to 7 th exons of the ZNF384 gene are commonly used to match with different upstream primers for detection. The primer probe sequence is as follows:
an EP300-ZNF384 fusion gene detection primer probe:
ARID1B-ZNF384 fusion gene detection primer probe:
3, detecting a primer probe for detecting CREBP-ZNF 384 fusion gene:
TCF3-ZNF384 fusion gene detection primer probe:
TAF15-ZNF384 fusion gene detection primer probe:
BMP2K-ZNF384 fusion gene detection primer probe:
the primer probe for identifying the fusion gene also comprises a primer and a probe for amplifying a housekeeping gene ABL1 serving as an internal reference, and the nucleotide sequence of the primer and the probe is as follows:
ABL1-F：5’-GATACGAAGGGAGGGTGTACCA-3’
ABL1-R：5’-CTCGGCCAGGGTGTTGAA-3’
ABL1-P：5’-FAM-TGCTTCTGATGGCAAGCTCTACGTCTCCT-TAMRA-3’。
the invention also provides application of primers and probes for screening and identifying 6 ZNF384 related fusion genes, namely EP300-ZNF384, ARID1B-ZNF384, CREBP-ZNF 384, TCF3-ZNF384, TAF15-ZNF384 and BMP2K-ZNF384, in preparing a kit for screening the 6 ZNF384 related fusion genes by using a real-time fluorescent quantitative PCR technology.
The invention also provides a method for screening and identifying 6 ZNF384 related fusion genes of EP300-ZNF384, ARID1B-ZNF384, CREBP-ZNF 384, TCF3-ZNF384, TAF15-ZNF384 and BMP2K-ZNF384 by using the primer and the probe, which is characterized by comprising the following steps:
(1) extracting RNA in a human whole blood sample;
(2) the whole detection system can be divided into two steps, wherein the first step is primary screening, and 6 common ZNF384 related chaperone genes are involved and comprise: EP300, ARID1B, CREBBP, TCF3, TAF15, BMP2K, and the second step is to confirm the fusion type, i.e. to further type the first positive sample.
The method comprises the following steps of carrying out multiple fluorescent PCR amplification by using a primer probe MIX obtained by premixing 21 primers and probes shown in the invention, carrying out detection by using an ABI7500 fluorescent quantitative PCR instrument, wherein a detection channel comprises the following steps: setting the detection probe as FAM-TAMRA; the reference fluorescence was set as ROX. The PCR amplification conditions were: pre-denaturation at 95 ℃ for 1 min; denaturation at 95 ℃ for 10sec, extension at 58 ℃ for 35sec, 40 cycles, and fluorescence signal collection set at 58 ℃ extension.
Analysis of the results: and (4) analyzing and judging the primary screening result by using 7500System SDS software, and if the primary screening result is positive, performing the type analysis of the second step. If the sample is negative and the corresponding reference gene exists, the sample is judged not to contain the ZNF384 related fusion forms.
The positive judgment is: CT value < 32 and amplification curve is true.
The rules are as follows:
the primary screening result shows that the gene is negative and the internal reference ABL gene exists, and the result is negative; as shown in fig. 3.
And (3) performing typing detection on the positive reference ABL gene shown by the preliminary screening result and the presence of the internal reference ABL gene: including 6 fusion types contained in the invention, if the detection hole corresponding to the EP300-ZNF384 is found to be positive, the sample is the EP300-ZNF384 fusion type, such as FIG. 4-1 and FIG. 4-2; if the detection hole corresponding to CREBP-ZNF 384 is found to be positive, the sample is the CREBP-ZNF 384 fusion type, such as the samples shown in FIG. 5-1 and FIG. 5-2; if the corresponding test well of TCF3-ZNF384 is found to be positive, the sample is indicated as a TCF3-ZNF384 fusion type, as shown in FIG. 6-1 and FIG. 6-2.
According to the invention, multiple fluorescence PCR technology is combined with a Tapman probe, so that common or uncommon ZNF384 fusion genes related to the ALL disease are integrated together for screening, diagnosis, treatment and prognosis monitoring of the ALL disease can be comprehensively assisted, and compared with the conventional methods such as FISH and the like, the method has higher precision and is convenient for result interpretation. In addition, the invention reasonably matches and optimizes the primers and the probes, and optimizes the detection conditions to be optimal, thereby greatly improving the efficiency. In addition, the multiplex fluorescence PCR technology has high sensitivity, high specificity, less sample requirement and lower cost, and is suitable for screening large-batch clinical samples.
Drawings
FIG. 1 and FIG. 2 are schematic diagrams of the fusion mode and the primer design position of the present invention.
FIG. 3 is a diagram showing the results of fluorescent PCR screening of a first blood sample to be examined using the primer multiplex fluorescent PCR method of the present invention (ZNF 384-related fusion gene negative).
FIG. 4-1 is a diagram showing the results of fluorescent PCR for preliminary screening of a second blood sample to be examined by the primer multiplex fluorescent PCR method of the present invention; FIG. 4-2 is a diagram showing the results of fluorescent PCR for secondary typing (ZNF 384-related fusion is EP300-ZNF384 fusion).
FIG. 5-1 is a diagram showing the results of fluorescent PCR for preliminary screening of a third blood sample to be examined by the primer multiplex fluorescent PCR method of the present invention; FIG. 5-2 is a graph showing the results of fluorescent PCR for double typing (ZNF384 related fusion is CREBP-ZNF 384 fusion).
FIG. 6-1 is a diagram showing the results of fluorescent PCR for preliminary screening of a fourth blood sample to be examined by the primer multiplex fluorescent PCR method of the present invention; FIG. 6-2 is a diagram showing the results of fluorescent PCR for double typing (ZNF 384-related fusion is TCF3-ZNF384 fusion).
Detailed Description
Example 1: the amplification primer probes of the ZNF384 related fusion gene are shown in the following table:
example 2:
extraction of blood RNA:
adding 0.5ml of whole blood sample into 1ml of erythrocyte lysate, reversing and uniformly mixing, standing at room temperature for 5min, and centrifuging at 4000rpm for 3 min; discarding the supernatant; the erythrocyte lysate is resuspended, and the cell pellet volume is observed by repeating washing for 2 times, and reaches 10-30 ul. Adding l ml Trizol Reagent, repeatedly blowing and beating until cell-free mass is in the lysate, and standing for 5min at room temperature. And adding 200 mul of chloroform into the Trizol solution, mixing uniformly by vortex until the mixture is fully emulsified, standing on ice for 10min, and then centrifuging at 14000rpm for 10 min. Sucking 450 μ l of the supernatant, transferring into a centrifuge tube filled with pre-cooled isopropanol, turning upside down, mixing, standing at-30 deg.C for 10min on ice, centrifuging at 14000rpm for 10min, discarding the supernatant, adding l ml of 75% ethanol, washing the precipitate, and centrifuging at 14000rpm for 5 min; discarding the supernatant again, adding l ml of absolute ethyl alcohol, washing the precipitate, and centrifuging for 5min at 14000 rpm; the absolute ethyl alcohol is discarded, and the mixture is dried for 5 min. Adding 30-100 μ l RNase-free water according to the amount of precipitate to dissolve. The obtained RNA is the sample RNA.
The formula of the 10 multiplied erythrocyte lysate is as follows: NH (NH)4Cl 82g，NaHCO3 8.4g，EDTA-Na23.72g, plus ddH2O is added to reach the volume of 1000 ml.
Example 3:
multiplex fluorescence PCR amplification:
the reverse transcription system was prepared with the following reagents and amounts, Primer Mix 1ul, 5 × RT buffer 4ul, Enzyme Mix 1ul (tokyo textile biotechnology limited); adding 10ul of deionized water; finally, 4ul of sample RNA template was added. The reverse transcription procedure was: 60min at 37 ℃ and 5min at 98 ℃. Sample cDNA was prepared.
Configuring a PCR amplification system according to the following reagents and reagent amounts, wherein the primers of the primary screening system are 0.1ul respectively, and the probes are 0.05ul respectively; THUNDERBIRD Probe qPCR Mix 12.5ul, 50 × ROX 0.5ul (Toyobo (Shanghai) Biotech Co., Ltd.); adding 8ul of deionized water; finally adding 2ul of cDNA template; the amplification procedure was as follows: pre-denaturation at 95 ℃ for 1 min; denaturation at 95 ℃ for 10sec, extension at 58 ℃ for 35sec, 40 cycles, and fluorescence signal collection set at 58 ℃ extension.
Example 4:
and (3) analyzing the PCR result: the results of the multiplex fluorescent PCR primary screening were analyzed using 7500System SDS software.
Example 5:
if the primary screening result is positive, configuring a PCR amplification system according to the following reagents and reagent amounts, wherein each secondary typing primer is 0.5ul, and each probe is 0.25 ul; THUNDERBIRD Probe qPCR Mix 12.5ul, 50 × ROX 0.5ul (Toyobo (Shanghai) Biotech Co., Ltd.); adding 8ul of deionized water; finally adding a cDNA template obtained by reverse transcription to obtain 2 ul; the amplification procedure was as follows: pre-denaturation at 95 ℃ for 1 min; denaturation at 95 ℃ for 10sec, extension at 58 ℃ for 35sec, 40 cycles, and fluorescence signal collection set at 58 ℃ extension.
Example 6:
and (3) analyzing the PCR result: the results of multiplex fluorescent PCR secondary typing were analyzed using 7500System SDS software.
Example 7:
detecting a first whole blood sample to be detected: the first whole blood sample to be tested was subjected to RNA extraction and multiplex fluorescence PCR amplification as described in examples 2-4, and the results of PCR analysis gave the fluorescence results shown in FIG. 3. The result of the primary screening of the sample to be detected is negative.
Example 8:
and (3) detecting a second whole blood sample to be detected: the second whole blood sample to be tested was subjected to RNA extraction and multiplex fluorescence PCR amplification as described in example 2-4, and the results of PCR analysis gave the fluorescence results shown in FIG. 4-1. The primary screening results of the samples to be tested were positive, so the PCR results obtained by secondary typing as described in examples 5-6 are shown in FIG. 4-2. And the secondary typing result of the sample to be detected is an EP300-ZNF384 fusion type.
Example 9:
and (3) detecting a third whole blood sample to be detected: the second whole blood sample to be tested was subjected to RNA extraction and multiplex fluorescence PCR amplification as described in example 2-4, and the results of PCR analysis gave fluorescence results as shown in FIG. 5-1. The primary screening result of the sample to be examined was positive, and the PCR result obtained by the secondary typing as described in example 5-6 is shown in FIG. 5-2. And the secondary typing result of the sample to be detected is CREBP-ZNF 384 fusion type.
Example 10:
and (3) detecting a fourth whole blood sample to be detected: the second whole blood sample to be tested was subjected to RNA extraction and multiplex fluorescence PCR amplification as described in example 2-4, and the results of PCR analysis gave the fluorescence results shown in FIG. 6-1. The primary screening result of the sample to be examined was positive, and the PCR result obtained by the secondary typing as described in example 5-6 is shown in FIG. 6-2. And the secondary typing result of the sample to be detected is TCF3-ZNF384 fusion type.
Sequence listing
<110> Jinan Aidean medical testing center Limited
<120> primers and probes for detecting ZNF384 related fusion gene by using multiplex fluorescence PCR technology and application
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Claims (4)
1. Screening primers and probes of 6 ZNF384 related fusion genes including EP300-ZNF384, ARID1B-ZNF384, CREBP-ZNF 384, TCF3-ZNF384, TAF15-ZNF384 and BMP2K-ZNF384 by using a multiple fluorescence PCR technology, and matching three groups of downstream primers existing in the ZNF384 gene and the corresponding probes with a plurality of upstream primers on different upstream genes by using the multiple fluorescence PCR technology; the nucleotide sequences of the primers and the probes are as follows:
TCF3-11F：GGAGCAGAGGTGAACGGT
TCF3-13F：TCTCGTCCAGCCCTTCTACC
TCF3-16F：CAGCACCAGCCTCATGC
TCF3-17F：CGGAGGAGGAGAAGAAGGA
TAF15-6F：AGGGAAAACTACAGCCACCA
TAF15-11F：CATTTGATGACCCTCCTTCAG
CREBBP-4F：AATCACATGACGCATTGTCAG
CREBBP-5F：GCCAGTGACAAGCGAAACC
CREBBP-6F：CCACCAGCAGATGAGGACTC
CREBBP-7F：CAGAATCAGCTCTTCCGACTTC
CREBBP-10F：GGCACAACCTGTGAGACCTC
BMP2K-14F：TTGGACAGAGAATTTGACCTTC
BMP2K-15F：TCCTTTTGGTTCTGTTCCTTTC
EP300-6F：CCAGCAGAATCAGCAGCCT
ARID1B-4F：CAGCAAGAAAGACCATCAAGTT
ZNF384-3P：5'-FAM-TCTCACTTCAATTCTAACCCGTACTTCTGGC-TAMRA-3'
ZNF384-3R：GACCTGAGACTGTGGGGATAG
ZNF384-7P：5'-FAM-CCGGATGTGCTCACTGACATTCTACTCC-TAMRA-3'
ZNF384-7R：GTGTGTGACTTGGAGTGGATCT
ZNF384-4P：5'-FAM-AGCTGTTGCCAGAGAAGGGCTGTGGT-TAMRA-3'
ZNF384-4R：CAAGGTGGGGTAGTGAGGTG。
2. the primer and probe according to claim 1,
the nucleotide sequences of the detection primers and the detection probes for the EP300-ZNF384 fusion gene are as follows: EP300-6F, NF384-3P, ZNF384-3R, ZNF384-7P, ZNF384-7R, ZNF384-4P, ZNF 384-4R;
the nucleotide sequences of the detection primers and the detection probes for the ARID1B-ZNF384 fusion gene are as follows: ARID1B-4F, ZNF384-3P, ZNF384-3R, ZNF384-7P, ZNF384-7R, ZNF384-4P, ZNF 384-4R;
the nucleotide sequences of the detection primer and the detection probe for the CREBP-ZNF 384 fusion gene are as follows: CREBP-4F, CREBP-5F, CREBP-6F, CREBP-7F, CREBP-10F, ZNF384-3P, ZNF384-3R, ZNF384-7P, ZNF384-7R, ZNF384-4P, ZNF 384-4R;
the nucleotide sequences of the detection primers and the detection probes for the TCF3-ZNF384 fusion gene are as follows: TCF3-11F, TCF3-13F, TCF3-16F, TCF3-17F, ZNF384-3P, ZNF384-3R, ZNF384-7P, ZNF384-7R, ZNF384-4P and ZNF 384-4R; the nucleotide sequences of the detection primers and the detection probes for the TAF15-ZNF384 fusion gene are as follows: TAF15-6F, TAF15-11F, ZNF384-3P, ZNF384-3R, ZNF384-7P, ZNF384-7R, ZNF384-4P and ZNF 384-4R;
the nucleotide sequences of the detection primer and the probe for the BMP2K-ZNF384 fusion gene are as follows: BMP2K-14F, BMP2K-15F, ZNF384-3P, ZNF384-3R, ZNF384-7P, ZNF384-7R, ZNF384-4P and ZNF 384-4R.
3. The primers and probes according to claim 1 or 2, further comprising primers and probes for amplifying housekeeping gene ABL as a reference, the nucleotide sequences of which are as follows:
ABL1-F：5’-GATACGAAGGGAGGGTGTACCA-3’
ABL1-R：5’-CTCGGCCAGGGTGTTGAA-3’
ABL1-P：5’-FAM-TGCTTCTGATGGCAAGCTCTACGTCTCCT-TAMRA-3’。
4. use of the primers and probes as claimed in any one of claims 1 to 3 for the preparation of a kit for screening 6 ZNF384 related fusion genes.
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| CN113684261A (en) * | 2021-09-02 | 2021-11-23 | 济南艾迪康医学检验中心有限公司 | Primer, probe and kit for detecting ZNF384 gene rearrangement by using fluorescent quantitative PCR |
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| WO2015194524A1 (en) * | 2014-06-16 | 2015-12-23 | 国立研究開発法人 国立成育医療研究センター | B-precursor acute lymphoblastic leukemia novel chimeric gene |
| CN109609632A (en) * | 2018-12-21 | 2019-04-12 | 北京优迅医学检验实验室有限公司 | Reagent, kit and the application of detection fusion gene |
| CN111321227A (en) * | 2020-03-09 | 2020-06-23 | 南京实践医学检验有限公司 | Multiplex fluorescence RT-PCR detection method for leukemia MEF2D gene and ZNF384 gene |
| CN111826375A (en) * | 2019-04-17 | 2020-10-27 | 北京大学人民医院(北京大学第二临床医学院) | Kit for detecting ZNF384 related fusion gene and application thereof |
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| WO2015194524A1 (en) * | 2014-06-16 | 2015-12-23 | 国立研究開発法人 国立成育医療研究センター | B-precursor acute lymphoblastic leukemia novel chimeric gene |
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| CN113684261B (en) * | 2021-09-02 | 2024-04-30 | 济南艾迪康医学检验中心有限公司 | Primer, probe and kit for detecting ZNF384 gene rearrangement by utilizing fluorescence quantitative PCR |
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