CN113999906A - Method for detecting free DNA multi-site mutation of lung tumor plasma - Google Patents
Method for detecting free DNA multi-site mutation of lung tumor plasma Download PDFInfo
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Abstract
The invention discloses a method for detecting free DNA multi-site mutation in lung tumor plasma, which is characterized in that specific primers, specific PNA probes and a capturing technology are adopted to improve ctDNA detection and specificity, so that trace ctDNA can be detected, and primers, probes and a detection method for detecting gene variation in peripheral blood samples of patients with lung space occupying lesion can be used. The method has the advantages of high sensitivity, strong specificity, good repeatability, simple operation, rapid detection, safety and the like, and has an auxiliary effect on the diagnosis of the lung cancer.
Description
Technical Field
The invention relates to a DNA multi-site mutation detection method, in particular to a method for detecting free DNA multi-site mutation in plasma of a free lung tumor.
Background
With the development of the second-generation sequencing technology, the liquid biopsy is a novel diagnostic technology, and has been rapidly developed in recent years due to the characteristics of non-invasiveness, real-time dynamics and the like, and is becoming a new hotspot in the accurate tumor medical field. Among them, genetic testing of body fluid samples represented by plasma is attracting attention, and this method obtains free nucleic acids or cells from peripheral blood, searches for tumor information, and provides a basis for early diagnosis and the like. The incidence and mortality of lung cancer in China are the first of all cancer species, and early diagnosis is urgent.
The technical difficulties existing in the accurate sequencing of ctDNA and the realization of early tumor screening at present comprise effective extraction and identification of ctDNA, background noise subtraction, interpretation of medical significance and the like, the selection of a gene panel is very critical to overcome the difficulties, and proper genes and sites can play a role of markers, reduce the requirement on the extraction amount of ctDNA, reduce the difficulty in background noise subtraction, improve the purpose and accuracy of result interpretation, and play a decisive role in the detection result of ctDNA. The existing ctDNA detection method mainly has the defects of low sensitivity and poor specificity.
Disclosure of Invention
The invention aims to provide a method for detecting a circulating tumor gene mutation signal from a clinical peripheral blood sample.
The invention relates to a method for detecting free DNA multi-site mutation of lung tumor plasma, which adopts specific primers
Forward:
5’AGGCGAAGATCGGAAGAGCACACGTCTGAACTCCAGTCAC3’
Reverse:
5’ACACTCTTTCCCTACACGACGCTCTTCCGATCTTCGCCT3’
The specific RNA probe and the capturing technology improve the sensitivity and specificity of ctDNA detection, thereby being capable of detecting trace (0.1%) ctDNA.
b. The invention provides a specific primer and a specific probe required by specific ctDNA detection, and a corresponding ctDNA detection program.
1, set of specific genes (gene-panel)
Specific DNA primer set (primer-panel)
3, set of specific RNA probes (probe-panel)
5’GTTCTGGAAGATCTTGAACCCTCTTCTGGAAAGGGGTACCTATTATTACTTTATGGGGCAGCAGCCTGGAAAAGTACTTGGGGACCAAAGAAGGCCAAGCTTGCCTGCCCTGCATTTTAT3’
5’CAAAGGAGCAGGGAAGAAGGAATCATCGAGGCATGGGGGTCCACACTGCAATGTTTTTGTGGAACATGGTGAGTGCTTTTCAAAATTTCTGCTCATGGTTTTCCTCATGCATTCATCTTA3’
5’CCCGACGTGCTGGCGCGGGAAAATGTTGGAGATCTGCCTGAAGCTGGTGGGCTGCAAATCCAAGAAGGGGCTGTCCTCGTCCTCCAGCTGTTATCTGGAAGGTAAGCCCGGGCCGCACGG3’
… … Total 3179 probes
The method for detecting the free DNA multi-site mutation of the lung tumor plasma comprises the following steps:
firstly, preparing a pre-library by fragmenting, adding a linker, PCR (polymerase chain reaction) enrichment and the like on the gDNA of normal white blood cells, the gDNA of paraneoplastic normal cells and the gDNA of tumor cells extracted from an FFPE (fringe field plasma enhanced plasma) sample of lung tumor;
hybridizing an RNA probe with a specific sequence with a pre-library so as to specifically capture partial exon and intron regions in 68 genes from a human genome;
enriching the DNA fragments captured by the probe by a magnetic bead method, and carrying out quantification, quality control and sequence determination on the captured library;
fourthly, judging whether variation from tumor exists in 68 target genes by adopting bioinformatics software;
preparing a pre-library by adding a linker to a standard ctDNA and cfDNA extracted from a lung tumor plasma sample, PCR enrichment and the like;
sixthly, hybridizing an RNA probe with a specific sequence with the pre-library so as to specifically capture partial exon and intron regions in 68 genes from the human genome;
seventhly, enriching the DNA fragments captured by the probe by a magnetic bead method, and carrying out quantification, quality control and sequence determination on the captured library;
and the biological informatics software is adopted to judge whether the 68 target genes have the variation from the tumor.
Compared with the prior art, the invention has the beneficial effects that: .
The invention discloses a two-wheeled power-assisted trolley.
Has the advantages that: primers and probes for detecting gene variation in peripheral blood samples of patients with lung space occupying lesions, and a detection method. The method can realize the ctDNA detection with the mutation frequency of 0.1% in the standard ctDNA, improves the sensitivity and specificity of the detection compared with other methods, has the advantages of good repeatability, simple operation, quick detection, safety and the like, and has an auxiliary effect on the diagnosis of the lung cancer.
Drawings
FIG. 1 is a schematic diagram of the distribution uniformity of molecular tags (shown by taking sequence analysis of gene BRAF as an example);
FIG. 2 is a schematic diagram showing the detection of a ctDNA standard (purchased from Horizon, containing 6 sites) with a mutation frequency of 0.1%;
FIG. 3 is a schematic diagram showing that positive detection rates obviously superior to tumor protein markers are obtained in 100 clinical peripheral blood samples of early lung cancer (stage I-II).
Detailed Description
1. Extracting cfDNA and WBC-gDNA, wherein the WBC-gDNA needs to be broken by ultrasonic to about 170 bp;
2. adding A tail, purifying the product, connecting the product with a joint, purifying and sorting DNA fragments with the length of less than 300bp, and introducing barcode;
3. tube PCR was performed under the same conditions as in NEB multiplex PCR (NEB, M0284 s). 95 ℃ for 1min, 60 ℃ for 60s, 68 ℃ for 90s, and 4 ℃ infinite. More than 2 cycles; after the PCR product is purified, performing second round PCR under the same reaction conditions as NEB multiplex PCR for 30 cycles;
4. the length of the fragment is detected by gel electrophoresis of the product, and the theoretical product is about less than 200 bp.
5. And finishing the library building.
Optimizing experimental conditions:
1. ensuring the efficiency of ligation
2. The random molecular tag barcode introduced at the adapter was ensured to attach to the DNA template (number of random bases was optimized). As shown in fig. 1
P5 primer (+) has a competitive relationship with P7-targetgene-3R and P7-targetgene-3F, so P5 primer (+) should be higher in concentration (ratio needs to be optimized) than the other two primers.
4. The number of cycles of two rounds of PCR need to be optimized.
And a ctDNA detection process is formed by using a specific DNA primer, a specific RNA probe, a primer of an internal reference gene and a probe, and detection is carried out after specific capture.
Embodiment I
Fragmenting a standard gDNA, adding a linker, performing PCR enrichment and the like to prepare a pre-library; then carrying out the subsequent steps (1) - (3)
Embodiment II
Fragmenting tissue DNA extracted from an FFPE sample, adding a linker, performing PCR enrichment and the like to prepare a pre-library;
then carrying out the subsequent steps (1) - (3)
Embodiment III
Preparing a pre-library by adding a linker to a standard cfDNA, enriching by PCR and the like;
then carrying out the subsequent steps (1) - (3)
Embodiment IV
Preparing a pre-library by adding a linker to cfDNA extracted from a lung tumor plasma sample, PCR enrichment and the like; as shown in fig. 2.
Then carrying out the subsequent steps (1) - (3)
The following steps:
(1) hybridizing the pre-libraries obtained in schemes I-IV with RNA probes having specific sequences, respectively, to specifically capture a portion of exon and intron regions in 70 genes from the human genome;
(2) enriching DNA fragments captured by the probe by a magnetic bead method, and carrying out quantification, quality control and sequence determination on a captured library;
(3) bioinformatics software was used to determine whether there was a tumor-derived variation in the 70 target genes. As shown in fig. 3.
The reliability of the method of the invention was verified by testing standards of different known mutation abundances, as well as FFPE and plasma cfDNA.
The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.
Sequence listing
<110> Shenzhen Si Ji Yiyun science and technology Limited
<120> method for detecting free DNA multi-site mutation in lung tumor plasma
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<212> PRT
<213> Human lung tumor (Human)
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Cys Thr Thr Thr Ala Thr Gly Gly Gly Gly Cys Ala Gly Cys Ala Gly
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Cys Cys Thr Gly Gly Ala Ala Ala Ala Gly Thr Ala Cys Thr Thr Gly
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<211> 120
<212> PRT
<213> Human lung tumor (Human)
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Ala Thr Gly Gly Gly Gly Gly Thr Cys Cys Ala Cys Ala Cys Thr Gly
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Cys Ala Ala Thr Gly Thr Thr Thr Thr Thr Gly Thr Gly Gly Ala Ala
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Cys Ala Thr Gly Gly Thr Gly Ala Gly Thr Gly Cys Thr Thr Thr Thr
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Gly Gly Gly Cys Thr Gly Cys Ala Ala Ala Thr Cys Cys Ala Ala Gly
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Claims (3)
1. A method for detecting free DNA multi-site mutation in lung tumor plasma adopts specific primers, specific RNA probes and a capture technology to improve ctDNA detection and specificity, so that trace ctDNA can be detected, and the method comprises the following specific steps:
firstly, preparing a pre-library by fragmenting, adding a linker, PCR (polymerase chain reaction) enrichment and the like on the gDNA of normal white blood cells, the gDNA of paraneoplastic normal cells and the gDNA of tumor cells extracted from an FFPE (fringe field plasma enhanced plasma) sample of lung tumor;
hybridizing an RNA probe with a specific sequence with a pre-library so as to specifically capture partial exon and intron regions in 68 genes from a human genome;
enriching the DNA fragments captured by the probe by a magnetic bead method, and carrying out quantification, quality control and sequence determination on the captured library;
fourthly, judging whether variation from tumor exists in 68 target genes by adopting bioinformatics software;
preparing a pre-library by adding a linker to a standard ctDNA and cfDNA extracted from a lung tumor plasma sample, PCR enrichment and the like;
sixthly, hybridizing an RNA probe with a specific sequence with the pre-library so as to specifically capture partial exon and intron regions in 68 genes from the human genome;
seventhly, enriching the DNA fragments captured by the probe by a magnetic bead method, and carrying out quantification, quality control and sequence determination on the captured library;
and the biological informatics software is adopted to judge whether the 68 target genes have the variation from the tumor.
2. The method for detecting lung tumor plasma free DNA multi-site mutation according to claim 1, wherein the specific primers are:
Forward:5’AGGCGAAGATCGGAAGAGCACACGTCTGAACTCCAGTCAC 3’
Reverse:5’ACACTCTTTCCCTACACGACGCTCTTCCGATCTTCGCCT 3’。
3. the method for detecting lung tumor plasma free DNA multi-site mutation according to claim 1, wherein the RNA probe sequence is:
5’GTTCTGGAAGATCTTGAACCCTCTTCTGGAAAGGGGTACCTATTATTACTTTATGGGGCAGCAGCCTGGAAAAGTACTTGGGGACCAAAGAAGGCCAAGCTTGCCTGCCCTGCATTTTAT 3’
5’CAAAGGAGCAGGGAAGAAGGAATCATCGAGGCATGGGGGTCCACACTGCAATGTTTTTGTGGAACATGGTGAGTGCTTTTCAAAATTTCTGCTCATGGTTTTCCTCATGCATTCATCTTA3’
5’CCCGACGTGCTGGCGCGGGAAAATGTTGGAGATCTGCCTGAAGCTGGTGGGCTGCAAATCCAAGAAGGGGCTGTCCTCGTCCTCCAGCTGTTATCTGGAAGGTAAGCCCGGGCCGCACGG3’。
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107619867A (en) * | 2017-10-18 | 2018-01-23 | 广州漫瑞生物信息技术有限公司 | For detecting the combined sequence and probe of lung cancer several genes mutation type simultaneously |
| CN110452981A (en) * | 2017-06-07 | 2019-11-15 | 深圳市海普洛斯生物科技有限公司 | The kit of early screening of lung cancer based on peripheral blood |
| WO2021108620A1 (en) * | 2019-11-25 | 2021-06-03 | The Johns Hopkins University | Methods and compositions for analyses of cancer |
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- 2021-10-08 CN CN202111169647.3A patent/CN113999906A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110452981A (en) * | 2017-06-07 | 2019-11-15 | 深圳市海普洛斯生物科技有限公司 | The kit of early screening of lung cancer based on peripheral blood |
| CN107619867A (en) * | 2017-10-18 | 2018-01-23 | 广州漫瑞生物信息技术有限公司 | For detecting the combined sequence and probe of lung cancer several genes mutation type simultaneously |
| WO2021108620A1 (en) * | 2019-11-25 | 2021-06-03 | The Johns Hopkins University | Methods and compositions for analyses of cancer |
Non-Patent Citations (3)
| Title |
|---|
| NICK BEIJE等: "Somatic mutation detection using various targeted detection assays in paired samples of circulating tumor DNA, primary tumor and metastases from patients undergoing resection of colorectal liver metastases", MOL ONCOL ., vol. 10, no. 10, pages 1575 - 1584, XP029836517, DOI: 10.1016/j.molonc.2016.10.001 * |
| YANG LIU等: "NGS-based accurate and efficient detection of circulating cell-free mitochondrial DNA in cancer patients", MOL THER NUCLEIC ACIDS, vol. 23, pages 658 * |
| 吕爽等: "高通量基因测序技术检测外周血循环肿瘤DNA基因突变在非小细胞肺癌中的应用", 现代肿瘤医学, vol. 27, no. 13, pages 2291 - 2295 * |
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