CN117106911A - Gene set for detecting schwannoma and multiplex PCR-high flux sequencing detection kit thereof - Google Patents
Gene set for detecting schwannoma and multiplex PCR-high flux sequencing detection kit thereof Download PDFInfo
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- 108090000623 proteins and genes Proteins 0.000 title claims abstract description 39
- 208000007538 neurilemmoma Diseases 0.000 title claims abstract description 31
- 206010039667 schwannoma Diseases 0.000 title claims abstract description 31
- 238000012163 sequencing technique Methods 0.000 title claims description 14
- 238000001514 detection method Methods 0.000 title abstract description 11
- 230000004907 flux Effects 0.000 title description 3
- 230000001717 pathogenic effect Effects 0.000 claims abstract description 12
- 238000007403 mPCR Methods 0.000 claims abstract description 10
- 108700026244 Open Reading Frames Proteins 0.000 claims abstract description 6
- 102000009512 Cyclin-Dependent Kinase Inhibitor p15 Human genes 0.000 claims abstract description 3
- 108010009356 Cyclin-Dependent Kinase Inhibitor p15 Proteins 0.000 claims abstract description 3
- 108010009392 Cyclin-Dependent Kinase Inhibitor p16 Proteins 0.000 claims abstract description 3
- 102100024458 Cyclin-dependent kinase inhibitor 2A Human genes 0.000 claims abstract description 3
- 108700039691 Genetic Promoter Regions Proteins 0.000 claims abstract description 3
- 101001038435 Homo sapiens Leucine-zipper-like transcriptional regulator 1 Proteins 0.000 claims abstract description 3
- 101000869796 Homo sapiens Microprocessor complex subunit DGCR8 Proteins 0.000 claims abstract description 3
- 101000702545 Homo sapiens Transcription activator BRG1 Proteins 0.000 claims abstract description 3
- 101000952936 Homo sapiens Ubiquinone biosynthesis monooxygenase COQ6, mitochondrial Proteins 0.000 claims abstract description 3
- 102100040274 Leucine-zipper-like transcriptional regulator 1 Human genes 0.000 claims abstract description 3
- 102100032459 Microprocessor complex subunit DGCR8 Human genes 0.000 claims abstract description 3
- 108700028341 SMARCB1 Proteins 0.000 claims abstract description 3
- 101150008214 SMARCB1 gene Proteins 0.000 claims abstract description 3
- 102100025746 SWI/SNF-related matrix-associated actin-dependent regulator of chromatin subfamily B member 1 Human genes 0.000 claims abstract description 3
- 102100031027 Transcription activator BRG1 Human genes 0.000 claims abstract description 3
- 102100037292 Ubiquinone biosynthesis monooxygenase COQ6, mitochondrial Human genes 0.000 claims abstract description 3
- 230000000694 effects Effects 0.000 claims abstract description 3
- 238000003752 polymerase chain reaction Methods 0.000 claims description 8
- 239000000203 mixture Substances 0.000 claims description 7
- 238000012408 PCR amplification Methods 0.000 claims description 2
- 238000011144 upstream manufacturing Methods 0.000 claims description 2
- 238000003759 clinical diagnosis Methods 0.000 abstract description 5
- 238000013461 design Methods 0.000 abstract description 5
- 238000012165 high-throughput sequencing Methods 0.000 abstract description 4
- 230000003321 amplification Effects 0.000 description 8
- 238000003199 nucleic acid amplification method Methods 0.000 description 8
- 238000000034 method Methods 0.000 description 6
- 108091093088 Amplicon Proteins 0.000 description 4
- 238000012986 modification Methods 0.000 description 3
- 230000004048 modification Effects 0.000 description 3
- 208000002761 neurofibromatosis 2 Diseases 0.000 description 3
- 208000022032 neurofibromatosis type 2 Diseases 0.000 description 3
- 108020004414 DNA Proteins 0.000 description 2
- 238000007400 DNA extraction Methods 0.000 description 2
- 102000007517 Neurofibromin 2 Human genes 0.000 description 2
- 108010085839 Neurofibromin 2 Proteins 0.000 description 2
- 238000004458 analytical method Methods 0.000 description 2
- 238000002474 experimental method Methods 0.000 description 2
- 239000007791 liquid phase Substances 0.000 description 2
- 210000005259 peripheral blood Anatomy 0.000 description 2
- 239000011886 peripheral blood Substances 0.000 description 2
- 102000004169 proteins and genes Human genes 0.000 description 2
- 238000007482 whole exome sequencing Methods 0.000 description 2
- 238000012070 whole genome sequencing analysis Methods 0.000 description 2
- 108091026890 Coding region Proteins 0.000 description 1
- KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 description 1
- 206010064571 Gene mutation Diseases 0.000 description 1
- 238000003766 bioinformatics method Methods 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
- 238000003748 differential diagnosis Methods 0.000 description 1
- 238000000605 extraction Methods 0.000 description 1
- 238000002360 preparation method Methods 0.000 description 1
- 238000012827 research and development Methods 0.000 description 1
- 238000012216 screening Methods 0.000 description 1
- 230000008685 targeting Effects 0.000 description 1
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- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
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- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/16—Primer sets for multiplex assays
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Abstract
The invention discloses a gene set for detecting schwannoma and a multiplex PCR-high throughput sequencing detection kit thereof, and particularly relates to the technical field of molecular biology. The gene set comprises a combination of one or more of the following genes: the protein coding region of DGCR8/COQ6/CDKN2A/CDKN2B/SMARCA4 and flanking alternative splicing regions; the protein coding region and flanking alternative splicing regions, most UTR, promoter region and intron regions of NF2/LZTR1/SMARCB1 may cause splicing effects; covers all the SNV/indels reported or likely to be pathogenic for the above genes. The gene set of the invention screens a database, designs aiming at pathogenic sites, realizes full coverage through specific multiplex PCR primers, has simple operation and controllable cost, and can meet the clinical diagnosis and treatment requirements of the schwannoma.
Description
Technical Field
The invention relates to the technical field of molecular biology, in particular to a gene set for detecting schwannoma and a multiplex PCR-high throughput sequencing detection kit thereof.
Background
The neurofibromatosis type 2 and the schwannoma pathogenic genes are completely different, but have overlapped phenotypes, and gene detection is a crucial means for differential diagnosis of the neurofibromatosis type 2 and schwannoma pathogenic genes, and the main current gene detection methods comprise whole genome sequencing, whole exome sequencing, targeted gene high-throughput sequencing and the like, wherein the whole genome sequencing and the whole exome sequencing respectively cover the whole genome sequence and the exome (coding region) sequence of all genes, the coverage range is wide, the detected genes are more, but the cost is higher, the price is high, a large number of unknown gene loci are difficult to read, the targeted gene capturing sequencing is specific to specific regions of specific genes, the target is clear, the related gene mutation loci can be obtained quickly, and the cost is low, so that the method is suitable for clinical practice. The capturing technical route comprises liquid phase capturing, multiplex PCR amplification and the like, the liquid phase capturing cost is high, the experiment is complex, and the method is suitable for the condition of large target area, the multiplex PCR amplification has the characteristics of simple and rapid experiment and controllable cost, and the design and system adjustment of the multiplex primer are difficult.
Disclosure of Invention
Therefore, the invention provides a gene set for detecting schwannoma and a multiplex PCR-high throughput sequencing detection kit thereof, so as to solve the problem that the design and system adjustment of the existing multiplex primer are difficult. The invention aims to provide a multiplex PCR-high flux detection kit for detecting genes related to schwannoma, which is used for assisting clinical diagnosis.
In order to achieve the above object, the present invention provides the following technical solutions:
according to a first aspect of the present invention there is provided a gene set for detecting a schwannoma comprising a combination of one or more of the following genes: the protein coding region of DGCR8/COQ6/CDKN2A/CDKN2B/SMARCA4 and flanking alternative splicing regions; the protein coding region and flanking alternative splicing regions, most UTR, promoter region and intron regions of NF2/LZTR1/SMARCB1 may cause splicing effects; covers all the SNV/indels reported or likely to be pathogenic for the above genes.
According to a second aspect of the present invention, there is provided a primer composition for detecting a gene of a schwannoma, the composition comprising a primer of a gene set related to the schwannoma of the first round plus a sequence of 20 bp.
Furthermore, the upstream F sequence of the primer is added with ACACGACGCTCTTCCGATCT (shown as SEQ ID NO.1 of the sequence table), and the downstream R sequence of the primer is added with GACGTGTGCTCTTCCGATCT (shown as SEQ ID NO.2 of the sequence table).
Further, the primers further comprise a second round of primers:
F：
AATGATACGGCGACCACCGAGATCTACACNNNNNNNNACACTCTTTCCCTACACGACGCTCTTCCGATCT; as shown in a sequence table SEQ ID NO. 3;
R：
CAAGCAGAAGACGGCATACGAGATNNNNNNNNGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT; as shown in a sequence table SEQ ID NO. 4;
wherein NNN represents a sequencing index sequence.
According to a third aspect of the present invention, there is provided a kit for detecting a schwannoma, comprising the primer composition for detecting schwannoma gene as described above.
Further, multiplex PCR was performed with two pool.
Further, the PCR amplification system is characterized in that:
further, the first round of PCR amplification procedure was: first round PCR amplification procedure: 95 ℃ for 2min; (98 ℃,20s;65 ℃,1min;72 ℃,30 s). Times.16 cycles;72 ℃ for 3min;4 ℃ and infinity;
second round PCR amplification procedure: 95 ℃ for 2min; (98 ℃,20s;65 ℃,1min;72 ℃,30 s). Times.10 cycles;72 ℃ for 3min;4 ℃ and infinity.
The invention has the following advantages:
the invention detects the gene related to the schwannoma by using a multiplex PCR targeting capture technology and a high-throughput second-generation sequencing technology. The method comprises the steps of simultaneously amplifying a plurality of target areas on genome DNA by utilizing a multiplex PCR technology to obtain amplicons, adding second generation sequencing joints to two sides of the amplicons in a PCR mode to obtain an amplicon library, carrying out second generation sequencing to obtain sequence information of the target areas, and carrying out SNV/Indel identification. Under the premise of guaranteeing the amplification uniformity, the method carries out rapid targeted linear amplification on hundreds of areas, and then carries out parallel detection on a large number of samples and data depth analysis based on a bioinformatics method by using a domestic autonomous research and development sequencing platform (GenoLab). Based on the multi-parameter primer design, the capturing efficiency is greatly improved, the omission ratio is reduced, the cost is reduced, the clinical detection rate is improved, and the method is suitable for clinical diagnosis and treatment of the schwannoma.
The gene set of the kit is obtained by screening a database, designing a related gene protein coding exon region, currently known schwannoma pathogenic sites and potential alternative splicing pathogenic sites, realizing full coverage through specific multiplex PCR primers, and has the advantages of simple operation and controllable cost, can meet the clinical diagnosis and treatment requirements of schwannoma, and can discover new pathogenic sites in the region.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below. It will be apparent to those of ordinary skill in the art that the drawings in the following description are exemplary only and that other implementations can be obtained from the extensions of the drawings provided without inventive effort.
The structures, proportions, sizes, etc. shown in the present specification are shown only for the purposes of illustration and description, and are not intended to limit the scope of the invention, which is defined by the claims, so that any structural modifications, changes in proportions, or adjustments of sizes, which do not affect the efficacy or the achievement of the present invention, should fall within the ambit of the technical disclosure.
FIG. 1 is a diagram showing the length distribution of a primer amplification product according to example 1 of the present invention.
Detailed Description
Other advantages and advantages of the present invention will become apparent to those skilled in the art from the following detailed description, which, by way of illustration, is to be read in connection with certain specific embodiments, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
Example 1
Multiplex PCR-high-throughput detection kit for detecting genes related to schwannoma:
1. preparation of templates
1) Sample type: genomic DNA extracted from human tissue/peripheral blood
2) Sample collection: peripheral blood collected by 2ml EDTA tube, soybean-sized tissue
3) Genome extraction: DNA extraction Using the DNA extraction kit of the root of the Tian
2. The primer and the sequence thereof are shown in the table 1, wherein the primer with the subscript 1 adds 20bp sequence, the F sequence adds ACACGACGCTCTTCCGATCT and the R sequence adds GACGTGTGCTCTTCCGATCT, respectively, as shown in the sequence table SEQ ID NO.1 and the sequence thereof, the specific sequence of the primer in the first round is shown in the table 1, wherein pool1 represents a first pool, pool2 represents a second pool, and the multiplex PCR is performed by dividing into two pools:
TABLE 1
The amplification primer profile is shown in FIG. 1.
3. The amplification system is shown in Table 2.
TABLE 2
4. Amplification procedure
1) First round PCR amplification procedure: 95 ℃ for 2min; (98 ℃,20s;65 ℃,1min;72 ℃,30 s). Times.16 cycles;72 ℃ for 3min;4 ℃ and infinity;
2) Second round PCR amplification procedure: 95 ℃ for 2min; (98 ℃,20s;65 ℃,1min;72 ℃,30 s). Times.10 cycles;72 ℃ for 3min; at the temperature of 4 ℃ and infinity,
the second round of primers are:
F：
AATGATACGGCGACCACCGAGATCTACACNNNNNNNNACACTCTTTCCCTACACGACGCTCTTCCGATCT, as shown in SEQ ID NO.3 of the sequence Listing;
R：
CAAGCAGAAGACGGCATACGAGATNNNNNNNNGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT, as shown in SEQ ID NO.4 of the sequence Listing;
NNN represents a sequencing index sequence;
3) Double-ended sequencing using PE150 chips;
5. sequencing analysis results
Genome alignment was performed on the sequencing data using BWA, and coverage of the predetermined coverage area was counted, and the results showed that coverage of more than 500X for 99% or more of the area could be achieved at an overall sequencing average depth of more than 800X, and depth information of each amplicon is shown in table 3.
TABLE 3 Table 3
Therefore, the kit provided by the invention screens the database, designs the coding exon region of the related gene protein, the currently known schwannoma pathogenic sites and the potential alternative splicing pathogenic sites, realizes full coverage through specific multiplex PCR primers, has the advantages of simplicity in operation and controllable cost, can meet the clinical diagnosis and treatment requirements of schwannoma, and can discover new pathogenic sites in the region.
While the invention has been described in detail in the foregoing general description and specific examples, it will be apparent to those skilled in the art that modifications and improvements can be made thereto. Accordingly, such modifications or improvements may be made without departing from the spirit of the invention and are intended to be within the scope of the invention as claimed.
Claims (8)
1. A gene set for detecting a schwannoma, comprising a combination of one or more of the following genes: the protein coding region of DGCR8/COQ6/CDKN2A/CDKN2B/SMARCA4 and flanking alternative splicing regions; the protein coding region and flanking alternative splicing regions, most UTR, promoter region and intron regions of NF2/LZTR1/SMARCB1 may cause splicing effects; covers all the SNV/indels reported or likely to be pathogenic for the above genes.
2. A primer composition for detecting a schwannoma gene, which is characterized by comprising a primer of a gene set related to schwannoma in the first round plus a sequence of 20 bp.
3. The primer composition for detecting the schwannoma gene according to claim 2, wherein the upstream F sequence of the primer is added with ACACGACGCTCTTCCGATCT as shown in a sequence table SEQ ID NO.1, and the downstream R sequence of the primer is added with GACGTGTGCTCTTCCGATCT as shown in a sequence table SEQ ID NO. 2.
4. The primer composition for detecting a gene of a schwannoma according to claim 2, wherein the primer further comprises a second round primer:
F：
AATGATACGGCGACCACCGAGATCTACACNNNNNNNNACACTCTTTCCC TACACGACGCTCTTCCGATCT; as shown in a sequence table SEQ ID NO. 3;
R：
CAAGCAGAAGACGGCATACGAGATNNNNNNNNGTGACTGGAGTTCAGA CGTGTGCTCTTCCGATCT; as shown in a sequence table SEQ ID NO. 4;
wherein NNN represents a sequencing index sequence.
5. A test kit for detecting a schwannoma, comprising the primer composition for detecting a schwannoma gene according to any one of claims 2 to 4.
6. The kit for detecting a schwannoma according to claim 5, comprising two pool for multiplex PCR.
7. The kit for detecting a schwannoma according to claim 6, wherein the PCR amplification system is:
8. the kit for detecting a schwannoma of claim 7, wherein the first round of PCR amplification procedure is: first round PCR amplification procedure: 95 ℃ for 2min; (98 ℃,20s;65 ℃,1min;72 ℃,30 s). Times.16 cycles;72 ℃ for 3min;4 ℃ and infinity;
second round PCR amplification procedure: 95 ℃ for 2min; (98 ℃,20s;65 ℃,1min;72 ℃,30 s). Times.10 cycles;72 ℃ for 3min;4 ℃ and infinity.
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