CN114525339A - Specific probe and detection method for MPL gene mutation detection - Google Patents
Specific probe and detection method for MPL gene mutation detection Download PDFInfo
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Abstract
The invention relates to the technical field of biology, in particular to a specific probe and a detection method for MPL gene mutation detection, wherein the probe consists of a nucleotide chain containing two fragments; a first fragment, all or a part of which has a recognition sequence for molecular recognition of a target nucleic acid; a second fragment, including but not limited to C3 Spacer, C6 Spacer, C12 Spacer, Spacer 9, or Spacer 18. Compared with the prior art, the specific probe and the detection method for MPL gene mutation detection provided by the invention inhibit amplification of a wild type sequence by designing the probe specifically combined with the wild type gene sequence, so that selective amplification and enrichment of the mutant type sequence are realized, the sequencing sensitivity of a Sanger sequencing method can be improved to about 0.5-1% from about 10-20% at present, and the detection rate of the MPL gene mutation is improved.
Description
Technical Field
The invention relates to the technical field of biology, in particular to a specific probe and a detection method for MPL gene mutation detection.
Background
Myeloproliferative tumors are a heterogeneous group of malignant tumors derived from clonal hematopoietic stem cells and are characterized by one or more lines of myelohyperproliferation, wherein the MPN negative to the BCR-ABL1 gene includes Polycythemia Vera (PV), Essential Thrombocythemia (ET), and Primary Myelofibrosis (PMF). MPL is one of the members of the non-receptor tyrosine kinase, JAKs protein (Janus kinases) family, and plays an important role in the proliferation and differentiation of hematopoietic stem cells by mediating the intracellular signal transduction of cytokines through the JAK-STAT signal pathway. Studies have shown that the MPL gene V617F activating mutation is present in approximately 97% of PV patients, 50-60% of ET patients, and 50% of PMF patients. The WHO classification of hematopoietic and lymphoid tissue tumors (4 th edition) in 2017 definitely uses the MPL gene V617F mutation as one of the main diagnostic indicators of PV, ET and PMF in MPN, and the American NCCN medical guideline and the Chinese expert guideline both suggest the adoption of the standard.
A sequencing-by-generation technology represented by Sanger nucleic acid sequencing is widely applied to clinical application, and plays an important role in prevention, diagnosis and differential diagnosis of various diseases, prognosis prediction, medication guidance, curative effect evaluation and the like. The first generation sequencing technology is stable, simple and convenient, has high automation degree, obtains direct genome sequence data, has accurate data and good repeatability, and is recognized as the 'gold standard' of gene detection.
The Sanger method, also known as dideoxy chain termination, is the most widely used nucleic acid sequencing technique and was invented by Sanger et al in 1977. At present, a plurality of gene analyzers are developed and marketed at home and abroad. Based on the principle that the mobility rates of molecules with different sizes in capillary electrophoresis are different, when the molecules marked with fluorescence pass through the capillary one by one, the laser detector can detect the molecules one by one and synchronously image, and then analysis software automatically converts different fluorescence signals into nucleic acid sequences, thereby realizing the purpose of sequencing. However, the sensitivity of the traditional Sanger sequencing method is only about 20%, which greatly limits the application of the traditional Sanger sequencing method in clinical practice.
Disclosure of Invention
Aiming at the defects of the background technology, the invention provides a specific probe and a detection method for MPL gene mutation detection, and solves the problems of low sensitivity and low gene mutation detection rate of the traditional Sanger sequencing method.
In one aspect, the present invention provides a specific probe for MPL gene mutation detection, which is characterized in that: consists of a nucleotide chain comprising two segments;
a first fragment, all or a part of which has a recognition sequence for molecular recognition of a target nucleic acid;
a second fragment including, but not limited to, C3 Spacer, C6 Spacer, C12 Spacer, Spacer 9, or Spacer 18.
The sequence of the first fragment is
Seq ID NO.1：TGCAGGAAACTGCCACCTCAGCAGCAGCAG。
Preferably, the first and second fragments are joined 3-3'.
On the other hand, the invention provides a method for detecting MPL gene mutation, which is characterized by comprising the following steps:
s1, system configuration: preparing an amplification system by using a genome extracted from a blood sample as a template, a PCR amplification premix, the specific probe, the MPL upstream primer and the MPL downstream primer according to claim 1;
s2, performing multiple PCR amplification, and purifying an amplification product;
s3, adding an M13R sequencing primer into the purified product serving as a template to perform sequencing reaction;
s4, sequentially adding a sodium acetate-ethanol mixture and 70% ethanol into the sequencing product for purification;
and S5, carrying out sequencing analysis on the purified sequencing product.
Preferably, the sequence of the MPL upstream primer is as follows:
Seq ID NO.2：CAGGAAACAGCTATGACCGGCCGAAGTCTGACCCTTTT；
the sequence of the MPL downstream primer is as follows:
Seq ID NO.3：GTACCTGTAGTGTGCAGGAAACT；
the sequence of the M13R sequencing primer is as follows:
Seq ID NO.4：CAGGAAACAGCTATGACC。
preferably, the PCR amplification pre-mix in S1 includes Taq DNA polymerase and a fluorescent dye.
Preferably, the amplification procedure of S2 is: 95 ℃/10 min; 40 cycles of 95 ℃/15s, 60 ℃/20s, 72 ℃/30 s; 72 ℃/5 min.
Preferably, the purification conditions of S2 are specifically: taking 6.0 mu L of PCR amplification product, 1.0 mu L of exonuclease ExoI, 0.2 mu L of calf intestinal alkaline phosphatase and 2.8 mu L of purified water, and carrying out enzymolysis purification reaction on the PCR amplification product; the purification procedure was: 37 ℃/60 min; 80 ℃/15 min; 25 ℃/1 min.
Preferably, the sequencing reaction procedure of S3 is: 37 ℃/10 min; 96 ℃/1 min; at 96 deg.C/10 s, at 50 deg.C/5 s, at 60 deg.C/2 min for 35 cycles; 60 ℃/2 min; 25 ℃/1 min.
Preferably, the purification conditions of S4 are specifically: adding 16 mu L of sodium acetate-ethanol mixture into a sequencing product, performing vortex oscillation, standing in the dark, performing centrifugal separation at 4 ℃, removing the upper layer liquid, adding 70 mu L of precooled 70% ethanol, performing vortex oscillation, performing centrifugal separation at 4 ℃, removing the upper layer liquid, and adding formamide to dissolve DNA.
Has the advantages that: compared with the prior art, the specific probe and the detection method for MPL gene mutation detection provided by the invention inhibit amplification of a wild type sequence by designing the probe specifically combined with the wild type gene sequence, so that selective amplification and enrichment of the mutant type sequence are realized, the sequencing sensitivity of a Sanger sequencing method can be improved to about 0.5-1% from about 10-20% at present, and the detection rate of the MPL gene mutation is improved.
Drawings
FIG. 1 is a comparison graph of the detection results of the present invention and the common Sanger sequencing method.
Detailed Description
In order to make the technical solutions of the present invention better understood, the present invention will be described in detail below with reference to the accompanying drawings and specific embodiments.
Example 1A specific Probe for MPL Gene mutation detection
The probe sequence is as follows:
TGCAGGAAACTGCCACCTCAGCAGCAGCAG-C3 Spacer。
example 2A specific Probe for MPL Gene mutation detection
The probe sequence is as follows:
TGCAGGAAACTGCCACCTCAGCAGCAGCAG-C6 Spacer。
example 3A specific Probe for MPL Gene mutation detection
The probe sequence is as follows:
TGCAGGAAACTGCCACCTCAGCAGCAGCAG-C12 Spacer。
example 4A specific Probe for MPL Gene mutation detection
The probe sequence is as follows:
TGCAGGAAACTGCCACCTCAGCAGCAGCAG-Spacer 9。
example 5A specific Probe for MPL Gene mutation detection
The probe sequence is as follows:
TGCAGGAAACTGCCACCTCAGCAGCAGCAG-Spacer 18。
example 6 detection method of MPL Gene mutation
1. Taking genomes extracted from a cell line and a clinical blood sample as templates, wherein the sample loading amount of the templates is 2 mul, adding 12.5 mul of PCR amplification premix, MPL upstream primer, MPL downstream primer and the specific probe of example 1, and supplementing the rest of the PCR amplification premix with purified water to a 25 mul amplification system, wherein the final concentrations of the MPL upstream primer, the MPL downstream primer and the MPL probe are 200nM, 200nM and 100nM respectively, and quickly centrifuging after mixing;
wherein, the PCR amplification PreMix is SuperReal PreMix Plus (purchased from Tiangen Biochemical technology, Beijing) Co., Ltd.) containing Taq DNA polymerase and fluorescent dye;
the sequences of the MPL upstream primer and the MPL downstream primer are as follows:
Seq ID NO.2：CAGGAAACAGCTATGACCGGCCGAAGTCTGACCCTTTT；
Seq ID NO.3：GTACCTGTAGTGTGCAGGAAACT；
2. and (3) placing the amplification system in a PCR instrument for amplification, wherein the amplification procedure is as follows: 95 ℃/10 min; 40 cycles of 95 ℃/15s, 60 ℃/20s, 72 ℃/30 s; 72 ℃/5min, then preparing 6.0 mu L of PCR amplification product, 1.0 mu L of exonuclease ExoI, 0.2 mu L of calf intestinal alkaline phosphatase and 2.8 mu L of purified water into a mixed system, placing the mixed system into a PCR instrument for carrying out enzymolysis purification reaction on the PCR amplification product, wherein the purification procedure is as follows: 37 ℃/60 min; 80 ℃/15 min; 25 ℃/1 min;
3. taking 3.5 mu L of enzymolysis purified product, 2.5 mu L of BigDye Terminator 3.1Ready Reaction Mix (from Life Technologies), 1.0 mu L of BigDye Terminator v1.1& v3.15XSqueuing Buffer (from Life Technologies), and 3 mu L of M13R sequencing primer to prepare a sequencing mixed system, and placing the mixed system in a PCR instrument for sequencing Reaction, wherein the sequencing Reaction program is as follows: 37 ℃/10 min; 96 ℃/1 min; at 96 deg.C/10 s, at 50 deg.C/5 s, at 60 deg.C/2 min for 35 cycles; 60 ℃/2 min; 25 ℃/1 min; wherein, the sequence of the M13R sequencing primer is as follows:
Seq ID NO.4：CAGGAAACAGCTATGACC；
4. adding 16 mu L of sodium acetate-ethanol mixture into a sequencing product, performing vortex oscillation, standing in a dark place, performing centrifugal separation at 4 ℃, discarding the upper layer liquid, adding 70 mu L of precooled 70% ethanol, performing vortex oscillation, performing centrifugal separation at 4 ℃, discarding the upper layer liquid, volatilizing the ethanol at room temperature, adding formamide for dissolving to obtain a purified sequencing product, completing sequencing on the purified sequencing product on a high-throughput sequencer (Illumina), and performing sequencing experiment operation according to an operation instruction provided by a manufacturer.
The blood samples of myeloproliferative tumor patients were analyzed by the present invention and the common Sanger sequencing method, and the results are shown in FIG. 1:
as can be seen from FIG. 1, compared with the common Sanger sequencing method which can only detect more than 20% of mutation ratio, the mutation ratio as low as 0.5% can be detected after the specific probe matched with the wild type sequence is added, wherein the left side is a detection result peak diagram of the common Sanger sequencing method for detecting different mutation ratios, and the right side is a detection result peak diagram of the high-sensitivity Sanger sequencing method for detecting different mutation ratios.
Finally, it should be noted that the above-mentioned description is only a preferred embodiment of the present invention, and those skilled in the art can make various similar representations without departing from the spirit and scope of the present invention.
Claims (10)
1. A specific probe for MPL gene mutation detection, characterized in that: consists of a nucleotide chain comprising two segments;
a first fragment, all or a part of which has a recognition sequence for molecular recognition of a target nucleic acid;
a second fragment including, but not limited to, C3 Spacer, C6 Spacer, C12 Spacer, Spacer 9, or Spacer 18;
the sequence of the first fragment is
Seq ID NO.1：TGCAGGAAACTGCCACCTCAGCAGCAGCAG。
2. The specific probe for MPL gene mutation detection according to claim 1, wherein: the first and second fragments are joined 3-3'.
3. A method for detecting MPL gene mutation, comprising the steps of:
s1, system configuration: preparing an amplification system by using a genome extracted from a blood sample as a template, a PCR amplification premix, the specific probe, the MPL upstream primer and the MPL downstream primer according to claim 1;
s2, performing multiple PCR amplification, and purifying an amplification product;
s3, adding an M13R sequencing primer into the purified product serving as a template to perform sequencing reaction;
and S4, purifying the sequencing product and then carrying out sequencing analysis.
4. The method of claim 5 for detecting mutations in MPL gene, wherein the mutation is detected by the method
The sequence of the MPL upstream primer is as follows:
Seq ID NO.2：CAGGAAACAGCTATGACCGGCCGAAGTCTGACCCTTTT；
the sequence of the MPL downstream primer is as follows:
Seq ID NO.3：GTACCTGTAGTGTGCAGGAAACT；
the sequence of the M13R sequencing primer is as follows:
Seq ID NO.4：CAGGAAACAGCTATGACC。
5. the method of claim 5, wherein the PCR amplification mix of S1 comprises Taq DNA polymerase and a fluorescent dye.
6. The method according to claim 5, wherein the amplification process of S2 comprises: 95 ℃/10 min; 40 cycles of 95 ℃/15s, 60 ℃/20s, 72 ℃/30 s; 72 ℃/5 min.
7. The method according to claim 5, wherein the purification system of S2 is: enzymatic purification reaction of PCR amplification product was carried out by taking 6.0. mu.L of PCR amplification product, 1.0. mu.L of exonuclease ExoI, 0.2. mu.L of calf intestinal alkaline phosphatase, and 2.8. mu.L of purified water.
8. The method according to claim 5, wherein the purification process of S2 comprises: 37 ℃/60 min; 80 ℃/15 min; 25 ℃/1 min.
9. The method according to claim 5, wherein the sequencing reaction of S3 is performed by: 37 ℃/10 min; 96 ℃/1 min; at 96 deg.C/10 s, at 50 deg.C/5 s, at 60 deg.C/2 min, for 35 cycles; 60 ℃/2 min; 25 ℃/1 min.
10. The method according to claim 5, wherein the purification conditions of S4 are as follows: adding 16 mu L of sodium acetate-ethanol mixture into a sequencing product, performing vortex oscillation, standing in the dark, performing centrifugal separation at 4 ℃, removing upper layer liquid, adding 70 mu L of precooled 70% ethanol, performing vortex oscillation, performing centrifugal separation at 4 ℃, removing upper layer liquid, and adding formamide for dissolution.
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Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
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| WO2009072084A2 (en) * | 2007-12-07 | 2009-06-11 | Universita' Degli Studi Di Firenze | Mutational analysis of chronic myeloproliferative disorders |
| CN102925559A (en) * | 2012-09-29 | 2013-02-13 | 童永清 | Kit for quantitatively detecting W515 site mutation of MPL genes |
| CN103757100A (en) * | 2013-12-20 | 2014-04-30 | 郑州艾迪康医学检验所（普通合伙） | Method and primer for detecting mutation type of 515th locus of MPL (myeloproliferative leukemia) gene |
| CN110396517A (en) * | 2019-05-21 | 2019-11-01 | 珠海圣美生物诊断技术有限公司 | It is a kind of that type oligonucleotide probe and its application being quenched for expanding the non-of anomaly target fragment |
| CN110938693A (en) * | 2019-12-04 | 2020-03-31 | 阅尔基因技术（苏州）有限公司 | Primer group, kit and method for detecting BRAF gene mutation |
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- 2022-02-11 CN CN202210128436.3A patent/CN114525339A/en active Pending
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
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| WO2009072084A2 (en) * | 2007-12-07 | 2009-06-11 | Universita' Degli Studi Di Firenze | Mutational analysis of chronic myeloproliferative disorders |
| CN102925559A (en) * | 2012-09-29 | 2013-02-13 | 童永清 | Kit for quantitatively detecting W515 site mutation of MPL genes |
| CN103757100A (en) * | 2013-12-20 | 2014-04-30 | 郑州艾迪康医学检验所（普通合伙） | Method and primer for detecting mutation type of 515th locus of MPL (myeloproliferative leukemia) gene |
| CN110396517A (en) * | 2019-05-21 | 2019-11-01 | 珠海圣美生物诊断技术有限公司 | It is a kind of that type oligonucleotide probe and its application being quenched for expanding the non-of anomaly target fragment |
| CN110938693A (en) * | 2019-12-04 | 2020-03-31 | 阅尔基因技术（苏州）有限公司 | Primer group, kit and method for detecting BRAF gene mutation |
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| Title |
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| 汤琴等: "慢性骨髓增殖性肿瘤患者钙网蛋白基因突变及其临床意义", 中国实验血液学杂志, vol. 25, no. 1, pages 151 - 156 * |
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