CN112430647A - Primer, probe, kit and detection method for detecting ASXL1 gene c.1934dupG mutation - Google Patents
Primer, probe, kit and detection method for detecting ASXL1 gene c.1934dupG mutation Download PDFInfo
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Abstract
The invention discloses a primer, a probe, a kit and a detection method for detecting C.1934dupG mutation of ASXL1 gene, wherein the primer comprises an upstream primer and a downstream primer: the upstream primer is as follows: 5'-CGAGAGGTCACCACTGCCATAG-3' (SEQ ID NO:1), the downstream primer is: 5'-ACAGGCCTCACCACCATCAC-3' (SEQ ID NO: 2). Compared with the prior art, the detection method is simple and convenient to operate, and the result can be qualitative and quantitative. The method has no special requirements on the amplification enzyme, has high accuracy, and solves the problem of high false positive of the existing method. Can be used as a verification method of the existing method. In addition, the detection method of the invention has high sensitivity.
Description
Technical Field
The invention relates to a primer, a probe, a kit and a detection method for detecting ASXL1 gene c.1934dupG mutation, and belongs to the technical field of ASXL1 gene mutation detection.
Background
The ASXL1 gene encodes a chromatin binding protein which is required for the normal determination of the characteristics of the developing embryo fragments. This protein is a member of the polycomb group and is required for maintenance of homeostasis and stable inhibition of other loci. Such proteins are thought to disrupt chromatin in localized areas, enhance transcription of certain genes, while inhibiting transcription of other genes, and play important roles in chromatin recombination and gene transcription regulation.
ASXL1 gene mutation occurs in various blood tumors, and ASXL1 mutation is found in hematopoietic cells of patients with various myeloid tumors, including chronic myelomonocytic leukemia (43%), myelodysplastic syndrome (14%), myeloproliferative tumors (10%), acute myeloid leukemia (20%), and the like. The ASXL1 mutation is mainly based on frameshift mutation and nonsense mutation, and the universality of the ASXL1 mutation indicates that the ASXL1 mutation possibly plays an important role in the pathogenesis and malignant transformation of myeloid tumors. Most mutations of ASXL1 occur in exon 12, and frame shift mutation and nonsense mutation result in truncation of C terminal, and finally affect the function of the protein.
One of the most common mutations of ASXL1 is ASXL1 NM-015338.5: c.1934dup; p.Gly646Trpffs 12(ASXL1 c.1934dupG), mutated from 8G repeats to 9G repeats, accounting for almost half of somatic truncation mutations. Such mutations in the repeat sequence may be due to replication slip during replication. In 2010 Abdel-Wahab, O.et al reported that ASXL1 c.1934dupG was not a true somatic mutation, and they used Sanger sequencing methods, which were analyzed as being caused by false positive results. In 2017, Yannakou, C.K. and the like adopt a fluorescence quantification method, and ASXL1 c.1934dupG is determined to be a real somatic mutation, but the detection sensitivity is 3%. Analysis it was believed that high fidelity enzymes were necessary to reduce false positives using the Sanger sequencing method. In addition, there are also literature reports of second-generation sequencing to detect this mutation (Alberti, M.O. et al 2018, Montes-Moreno, S. et al 2018), although the sensitivity is typically 5%. Therefore, the detection of the ASXL1 c.1934dupG mutation is not only influenced by replicase, but also has lower detection sensitivity, and cannot meet the current clinical requirement.
Disclosure of Invention
The purpose of the invention is as follows: aiming at solving the defects of low accuracy and sensitivity and the like in the prior art, the invention provides a primer, a probe, a kit and a detection method for detecting the C.1934dupG mutation of the ASXL1 gene.
The technical scheme is as follows: in order to achieve the purpose, the invention adopts the following technical scheme:
a pair of PCR amplification primers for detecting the c.1934dupG mutation of the ASXL1 gene comprises an amplification fragment containing the c.1934 site.
The upstream primer is as follows: 5'-CGAGAGGTCACCACTGCCATAG-3' (SEQ ID NO:1),
the downstream primer is: 5'-ACAGGCCTCACCACCATCAC-3' (SEQ ID NO: 2).
A pair of taqman probes which can be specifically combined with the C.1934 site 8G and 9G repeated sequences of ASXL1 gene:
probes capable of binding to wild-type 8G: 5 '-VIC-CCACCCCCCCCTCCGATGG-BHQ 1-3' (SEQ ID NO:3),
probe capable of binding to mutant 9G: 5 '-FAM-CACCCCCCCCCTCCGATGG-BHQ 1-3' (SEQ ID NO: 4).
The 5 'end of the probe is respectively modified by FAM and VIC fluorescent groups, and the 3' end is modified by BHQ1 quenching groups.
A kit for detecting ASXL1 gene c.1934dupG mutation comprises the primer and the probe.
The primer, the probe or the kit can be applied to detection of ASXL1 gene c.1934dupG mutation.
A method for detecting ASXL1 gene c.1934dupG mutation based on a digital PCR technology comprises the following steps:
(1) and mixing the PCR premix with a DNA template to be detected, the primers and the probes to prepare a digital PCR mixed solution.
(2) Preparing the digital PCR mixed solution into droplets by using a droplet generator, and then carrying out PCR amplification reaction;
(3) and (3) performing signal analysis on the product after the PCR reaction by using a droplet reader, judging the mutation condition of the sample to be detected according to different fluorescent signals in analysis software, and calculating the content of the mutation template.
Preferably, in the step (2), the conditions of the PCR amplification reaction are: at 95 ℃ for 10 min; 30s at 94 ℃; 60 ℃ for 60 s; at 98 ℃ for 10 min; terminating at 4 ℃; wherein, 40 cycles are carried out at 94 ℃ and 60 ℃, and the temperature rising and reducing speed is 2 ℃/s.
The digital PCR is an absolute nucleic acid molecule quantitative method, and nucleic acid molecules are dispersed into microreactors of a chip by utilizing a microfluidic or microdroplet method, wherein the number of nucleic acid templates in each reactor is less than or equal to 1. After PCR amplification, a reactor with a nucleic acid molecule template will produce a fluorescent signal, and a reactor without a template will not. Thus, the nucleic acid content of the original solution can be deduced from the number of reactors and the ratio of reactors containing the fluorescent signal. As a third-generation PCR technology, the digital PCR has the advantages of high quantification speed, high accuracy and the like, and is widely used for copy number variation, mutation detection, gene relative expression research (such as allele unbalanced expression), second-generation sequencing result verification, miRNA expression analysis, single-cell gene expression analysis and the like.
The Taqman fluorescent probe is an oligonucleotide probe, the 5 'end of the Taqman fluorescent probe carries a fluorescent group such as FAM, TET, VIC, HEX and the like, and the 3' end of the Taqman fluorescent probe carries a quenching group such as BHQ, MGB and the like. During PCR amplification, a pair of primers and a pair of specific fluorescent probes are added at the same time, and when the probes are complete, a fluorescent signal emitted by a reporter group is absorbed by a quenching group; during PCR amplification, the 5 '-3' exonuclease activity of Taq enzyme cuts and degrades the probe, so that the reporter fluorescent group and the quenching fluorescent group are separated, and the fluorescence monitoring system can receive a fluorescence signal. Since the 5' end fluorescent groups of the two specific probes are different, different template DNAs can be detected.
Has the advantages that: compared with the prior art, the invention has the following advantages:
1) the method is simple and convenient to operate, and the result can be qualitative and quantitative.
2) The method has no special requirements on the amplification enzyme, has high accuracy, and solves the problem of high false positive of the existing method. Can be used as a verification method of the existing method.
3) The method has high sensitivity, and can effectively detect the C.1934dupG mutation of the ASXL1 gene.
Drawings
FIG. 1 shows the negative results of the detection of the c.1934dupG mutation of the ASXL1 gene by the method according to the invention.
FIG. 2 shows the results of the detection of the c.1934dupG mutation in ASXL1 gene by the method according to the invention.
FIG. 3 shows the sensitivity results of the detection of the c.1934dupG mutation of ASXL1 gene by the method according to the invention.
Detailed Description
The following examples are intended to illustrate the invention but are not intended to limit the scope of the invention. Modifications or substitutions to methods, procedures, or conditions of the invention may be made without departing from the spirit and scope of the invention.
Unless otherwise specified, the chemical reagents used in the examples are all conventional commercially available reagents, and the technical means used in the examples are conventional means well known to those skilled in the art.
Example 1 primer and Probe design
1. Material
The digital PCR instrument used was a Bio-Rad QX-200 instrument, and the ddPCR Supermix for Probes (no dUTP) and related consumables were purchased from Bio-Rad.
2. Blood genomic DNA extraction
2ml of whole blood of leukemia patients is collected. 200ul of whole blood was taken, and genomic DNA of blood was extracted using a blood/cell/tissue genomic DNA extraction kit (DP304) from Tiangen corporation, and the DNA was quantified using Qubit3.0 from Thermo Fisher corporation.
3. Primer and probe sequence design
ASXL1-F upstream primer: 5'-CGAGAGGTCACCACTGCCATAG-3' (SEQ ID NO:1)
ASXL1-R downstream primer: 5'-ACAGGCCTCACCACCATCAC-3' (SEQ ID NO:2)
ASXL1-wt wild-type probe 5 '-VIC-CCACCCCCCCCTCCGATGG-BHQ 1-3' (SEQ ID NO:3)
ASXL1-mt mutant probe 5 '-FAM-CACCCCCCCCCTCCGATGG-BHQ 1-3' (SEQ ID NO:4)
Primer probe synthesis was purchased from shanghai bio corporation.
Example 2 detection of C.1934dupG mutation of ASXL1 Gene by the method
The specific operation process comprises the following steps:
1. the PCR reaction system was configured as shown in Table 1:
TABLE 1
2. Adding 20ul of the prepared sample reaction system into 8 wells in the middle row of the DG8 cartridge, adding 70ul of microdroplet generating Oil (DG Oil) into each of the 8 wells in the bottom row of the DG8 cartridge, covering with a rubber pad (gasket), and paying attention to the fact that the small wells on both sides are hooked tightly; the droplet generation card base is placed into a droplet generator for droplet generation. The resulting droplets were transferred from the wells on the upper row of reaction wells to the wells of a 96-well plate by pipetting the droplets from the wells on the upper row of reaction wells, and then sealing the 96-well plate using a PX1 heat sealer.
3. The membrane-sealed 96-well plate was placed in a PCR instrument and the following reaction program was run:
note: the temperature rising and falling speed is 2 ℃/s
4. And (4) analyzing results: after the PCR reaction procedure was completed, the 96-well plate was placed in a QX200 droplet reader for droplet reading, and the results were viewed and analyzed on the software QuantaSoft. The axis channel1 is the FAM signal and channel2 is the VIC signal. Droplets of different fluorescence signals were divided into 4 types, black template-free droplet (bottom left)/green wild-type-containing droplet (bottom right)/blue mutant-containing droplet (top left)/red wild-type-containing + mutant droplet (between top left and bottom right). When the number of blue droplets containing the mutant is more than or equal to 3, the blue droplets are judged to be the mutant, and the mutation rate is calculated as Fractional Absundance (a/a + b). In FIG. 1 only the black template-free droplet and the green wild-type-containing droplet are present, and thus no mutations are detected. Fig. 2 contains 4 types of droplets, and thus a mutation was detected with a mutation rate of 43%.
Example 3 sensitivity of the method for detecting c.1934dupG mutation of ASXL1 gene
1. Preparing a DNA sample to be tested: a mixture containing 40ng of wild type DNA and 10-fold gradient of mutant DNA was prepared. The wild type DNA is derived from the blood DNA of a healthy person, and the mutant type DNA is derived from a synthetic mutant type plasmid.
2. The detection method was performed according to the procedure of example 2.
3. And (4) analyzing results: in the mutant samples diluted in the gradient, the mutation rates detected were 6.0%, 0.47%, and 0.042% in this order. No mutation was detected in the wild type sample. See fig. 3. Thus, using the method of the invention, 0.05% of mutations in 40ng of genomic DNA can be detected.
Although the invention has been described in detail hereinabove with respect to a general description and specific embodiments thereof, it will be apparent to those skilled in the art that modifications or improvements may be made thereto based on the invention. Accordingly, such modifications and improvements are intended to be within the scope of the invention as claimed.
Sequence listing
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Claims (7)
1. A primer for detecting the c.1934dupG mutation of the ASXL1 gene is characterized by comprising an upstream primer and a downstream primer:
the upstream primer is as follows: 5'-CGAGAGGTCACCACTGCCATAG-3' (SEQ ID NO:1),
the downstream primer is: 5'-ACAGGCCTCACCACCATCAC-3' (SEQ ID NO: 2).
2. A probe for detecting C.1934dupG mutation of ASXL1 gene is characterized by comprising a wild-type probe and a mutant-type probe:
wild-type probe: 5 '-VIC-CCACCCCCCCCTCCGATGG-BHQ 1-3' (SEQ ID NO:3),
mutant probes: 5 '-FAM-CACCCCCCCCCTCCGATGG-BHQ 1-3' (SEQ ID NO: 4).
3. The probe for detecting the C.1934dupG mutation of the ASXL1 gene according to claim 2, wherein the wild-type probe is a taqman probe capable of specifically binding to the wild-type 8G repeat sequence at the c.1934 site of the ASXL1 gene, and the mutant probe is a taqman probe capable of specifically binding to the mutant 9G repeat sequence at the c.1934 site of the ASXL1 gene.
4. A kit for detecting mutations in ASXL1 gene c.1934dupg, comprising the primers of claim 1 and the probe of claim 2.
5. Use of the primer of claim 1, the probe of claim 2, or the kit of claim 4 for detecting a mutation in ASXL1 gene c.1934 dupg.
6. A method for detecting ASXL1 gene c.1934dupG mutation based on a digital PCR technology is characterized by comprising the following steps:
(1) mixing the PCR premix with a DNA template to be detected, the primer of claim 1 and the probe of claim 2 to prepare a digital PCR mixture;
(2) preparing the digital PCR mixed solution into microdroplets, and then carrying out PCR amplification reaction;
(3) and (3) performing signal analysis on the product after the PCR amplification reaction, judging the mutation condition of the sample to be detected in analysis software according to different fluorescent signals, and calculating the content of the mutation template.
7. The method for detecting the c.1934dupG mutation of the ASXL1 gene based on the digital PCR technology as claimed in claim 6, wherein in the step (2), the conditions of the PCR amplification reaction are as follows: at 95 ℃ for 10 min; 30s at 94 ℃; 60 ℃ for 60 s; at 98 ℃ for 10 min; terminating at 4 ℃; wherein, 40 cycles are carried out at 94 ℃ and 60 ℃, and the temperature rising and reducing speed is 2 ℃/s.
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| CN114752667A (en) * | 2022-04-25 | 2022-07-15 | 安徽医科大学第一附属医院 | Primer group, probe and kit for quantitatively detecting heterogeneity of MT-ATP6m.9185 locus |
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2020
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| Title |
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| COSTAS K. YANNAKOU等: "ASXL1 c.1934dup;p.Gly646Trpfs*12-atrue somatic alteration requiring a new approach", 《BLOOD CANCER JOURNAL》 * |
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| CN114752667A (en) * | 2022-04-25 | 2022-07-15 | 安徽医科大学第一附属医院 | Primer group, probe and kit for quantitatively detecting heterogeneity of MT-ATP6m.9185 locus |
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