CN116516009A - Amplification primer group for detecting pheochromocytoma and paraganglioma pathogenic genes and application thereof - Google Patents
Amplification primer group for detecting pheochromocytoma and paraganglioma pathogenic genes and application thereof Download PDFInfo
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Abstract
The application relates to the technical field of gene detection, and particularly provides an amplification primer group for detecting pheochromocytoma and paraganglioma pathogenic genes and application thereof, which are suitable for high-throughput sequencing (Next Generation Sequencing, NGS), and has the advantages of comprehensive mutation type detection, low requirement on sample initial quantity, high detection accuracy and flux, simplicity, convenience, rapidness and low cost.
Description
Technical Field
The application relates to the technical field of gene detection, and in particular provides an amplification primer group for detecting pathogenic genes of pheochromocytoma and paraganglioma and application thereof.
Background
Pheochromocytomas and paragangliomas (Pheochromocytoma and paraganglioma, PPGL) are tumors originating from neuroectodermal pheochromonas, mainly secreting catecholamines, and are classified into parasympathetic paragangliomas (including chemoreceptor tumors, carotid aneurysms, etc.) and sympathogenic paragangliomas (including retroperitoneal, pelvic and postmediastinal paragangliomas) depending on whether the tumor is from a sympathetic or parasympathetic nerve. Some patients can be endangered by serious heart, brain and kidney damage caused by long-term hypertension or crisis caused by sudden severe hypertension, but if diagnosis and treatment can be timely and early obtained, the patients are curable secondary hypertension.
At present, most detection methods for PPGL pathogenic gene mutation only comprise partial related genes, the detection range is not comprehensive enough, and the region with higher GC content or higher homology of partial pathogenic genes cannot be effectively amplified, or higher DNA initial quantity is required for detection, so that the amplification uniformity is influenced.
In view of this, the present application is presented.
Disclosure of Invention
In order to solve the technical problems, the application aims to provide a method or a system for detecting PPGL pathogenic genes, which has the advantages of comprehensive detection, low sample initial quantity requirement, high amplification efficiency, better accuracy and higher flux.
It is therefore a first object of the present application to provide a library construction method suitable for NGS for detecting pheochromocytoma and paraganglioma pathogenic genes;
a second object is to provide a method for detecting pheochromocytoma and paraganglioma pathogenic genes which can fully contain PPGL pathogenic genes;
a third object of the present application is to provide a primer set for detecting pathogenic genes of pheochromocytoma and paraganglioma, and a composition or kit thereof.
Specifically, the technical scheme adopted by the application is as follows:
the application firstly provides a library construction method for detecting pathogenic genes of pheochromocytoma and paraganglioma, which is applicable to NGS, and takes Ion torrent platform sequencing as an example, and is characterized by comprising the following steps:
1) Extracting sample DNA;
2) Library amplification: amplifying the extracted DNA;
3) And (3) joint connection: sequencing the amplified products and connecting the sequencing joints;
4) And (5) purifying the library.
Further, the amplification in 1) is performed for VHL, SDHA, SDHB, SDHC, SDHD, NF1, FH, MAX, TMEM127, RET, MET, KIF1B, SDHAF2, EPAS1, EGLN1, MDH2, KMT2D, TP, H3F3A, HRAS, IDH2, ATRX, BRAF, EGLN2, FGFR1, MERTK, CDKN2A, BAP1 and MEN1 genes.
Further, the amplification in 1) is performed for INDELs within SNV and 22bp of CDs region and variable cleavage region of VHL, SDHA, SDHB, SDHC, SDHD, NF1, FH, MAX, TMEM127, RET, MET, KIF1B, SDHAF2, EPAS1, EGLN1, MDH2, KMT2D, TP, H3F3A, HRAS, IDH2, ATRX, BRAF, EGLN2, FGFR1, MERTK, CDKN2A, BAP1 and MEN1 genes.
Further, the amplification primer sequence is shown as SEQ ID NO. 1-650.
Further, the amplification conditions were as follows:
further, the library amplification step of 2) is specifically as follows:
Pre-Mix was prepared for a total of 2 pool, 5ul per reaction, with the following formulation method:
split charging is carried out on the prepared Pre-Mix, genomic DNA is added, and a reaction system is prepared as follows:
covering the tube cover, gently swirling, mixing, and centrifuging briefly, wherein bubbles are generated (if bubbles are generated, the tube wall is flicked, and the bubbles are broken and then separated instantaneously); the prepared reaction system is put into a PCR instrument, and the following procedure is started for amplification:
further, the 3) joint connection step is specifically as follows:
putting all products of pool1 into pool2 correspondingly, adding 1 μl FuPa Reagent into each PCR product, covering with a cover, mixing, and centrifuging instantaneously to prevent bubbles (if bubbles are generated, flicking the tube wall, breaking the bubbles and then instantaneously separating); the mixed reactants are put into a PCR instrument, and the following procedure is started for reaction:
barcode adapter Mix is formulated according to the following table
Configuring the sample to establish a corresponding relation between the sample name and the Barcode adapter, and preparing a joint connection system according to the following table:
adding 1 μl of DNA (deoxyribonucleic acid) Ligase into the reaction system, covering with a cover, mixing, and performing instantaneous centrifugation to prevent bubbles (if bubbles are generated, flicking the tube wall to break the bubbles, and then instantaneously separating); the prepared reaction system is put into a PCR instrument, and the following procedure is started for reaction:
the application also provides a detection method of pheochromocytoma and paraganglioma, comprising any one of the library construction methods described above, and further comprising the steps of sequencing and belief analysis by NGS.
The application also provides a primer group for detecting the pathogenic genes of pheochromocytoma and paraganglioma, wherein the primer in 1) is a primer for amplifying VHL, SDHA, SDHB, SDHC, SDHD, NF1, FH, MAX, TMEM127, RET, MET, KIF1B, SDHAF2, EPAS1, EGLN1, MDH2, KMT2D, TP53, H3F3A, HRAS, IDH2, ATRX, BRAF, EGLN2, FGFR1, MERTK, CDKN2A, BAP1 and MEN1 genes.
Further, the primer is a primer for amplifying INDEL within 22bp of SNV of CDs region and variable shearing region of VHL, SDHA, SDHB, SDHC, SDHD, NF1, FH, MAX, TMEM127, RET, MET, KIF1B, SDHAF, EPAS1, EGLN1, MDH2, KMT2D, TP53, H3F3A, HRAS, IDH2, ATRX, BRAF, EGLN2, FGFR1, MERTK, CDKN2A, BAP1 and MEN1 genes.
Further, the primer sequence is shown as SEQ ID NO. 1-650.
The application also provides a composition comprising any one of the primer sets.
The application also provides a kit for detecting pathogenic genes of pheochromocytoma and paraganglioma, which comprises any one of the primer groups.
The application also provides application of any one of the primer groups or the composition in preparation of a construction kit or a pathogenic gene detection kit for detecting pheochromocytoma and paraganglioma library.
Compared with the prior art, the application has at least the following advantages:
1) According to the invention, 29 genes of VHL, SDHA, SDHB, SDHC, SDHD, NF1, FH, MAX, TMEM127, RET, MET, KIF1B, SDHAF, EPAS1, EGLN1, MDH2, KMT2D, TP53, H3F3A, HRAS, IDH2, ATRX, BRAF, EGLN2, FGFR1, MERTK, CDKN2A, BAP and MEN1 are determined as detection target pathogenic genes through screening, three levels of basically, expanding and synthesizing are covered, and the CDs region of the pathogenic genes and the INDEL within SNV and 22bp of the variable shearing region are further detected, so that the method can be effectively used for detecting pheochromocytoma and paraganglioma diseases through high-throughput gene sequencing, and has the advantages of comprehensive detection, high accuracy and the like.
2) The invention solves the problem that partial gene areas cannot be effectively amplified due to high GC content or high homology by optimizing and matching the primer sequences and the reaction system, and ensures the effectiveness of an ultra-high-weight amplification system, improves the amplification efficiency and has better data uniformity on the premise of low initial quantity of 20ng DNA.
3) The invention provides an overall solution for refractory hypertension molecular diagnosis and differential diagnosis, which has the advantages of simple operation, high flux, comprehensive detection, high accuracy and the like.
Drawings
In order to more clearly illustrate the embodiments of the present application or the technical solutions in the prior art, the drawings that are needed in the description of the embodiments or the prior art will be briefly described below, and it is obvious that the drawings in the following description are some embodiments of the present application, and other drawings may be obtained according to these drawings without inventive effort for a person skilled in the art.
FIG. 1, amplification efficiencies of different GC content regions under conventional amplification conditions;
FIG. 2, amplification efficiency of different GC content regions in an optimized amplification primer set application;
FIG. 3, results of detection of pathogenic mutation sites and one generation of sequencing validation of the present application.
Detailed Description
The following description of the embodiments of the present application will be made apparent and fully in view of the accompanying drawings, in which some, but not all embodiments of the invention are shown. All other embodiments, which can be made by one of ordinary skill in the art without undue burden from the present disclosure, are within the scope of the present disclosure.
The following terms or definitions are provided solely to aid in the understanding of the present application. These definitions should not be construed to have a scope less than understood by those skilled in the art.
Unless defined otherwise hereinafter, all technical and scientific terms used in the detailed description of the present application are intended to be the same as commonly understood by one of ordinary skill in the art. While the following terms are believed to be well understood by those skilled in the art, the following definitions are set forth to better explain the present application.
As used in this application, the terms "comprising," "including," "having," "containing," or "involving" are inclusive or open-ended and do not exclude additional unrecited elements or method steps. The term "consisting of …" is considered to be a preferred embodiment of the term "comprising". If a certain group is defined below to contain at least a certain number of embodiments, this should also be understood to disclose a group that preferably consists of only these embodiments.
The indefinite or definite article "a" or "an" when used in reference to a singular noun includes a plural of that noun.
The terms "about", "substantially" in this application refer to a range of accuracy that one of ordinary skill in the art would understand yet still guarantee the technical effect of the features in question. The term generally means a deviation of + -10%, preferably + -5%, from the indicated value.
Furthermore, the terms first, second, third, (a), (b), (c), and the like in the description and in the claims, are used for distinguishing between similar elements and not necessarily for describing a sequential or chronological order. It is to be understood that the terms so used are interchangeable under appropriate circumstances and that the embodiments described herein are capable of operation in other sequences than described or illustrated herein.
Experimental example 1 establishment of detection Gene
In the embodiment, 20 common genes VHL, SDHA, SDHB, SDHC, SDHD, NF1, FH, MAX, TMEM127, RET, MET, KIF1B, SDHAF2, EGLN1, MDH2, KMT2D, MERTK, CDKN2A, BAP1 and MEN1 are selected as gene sets for detection, but analysis finds that the detection results are incomplete easily due to the common genes, and 9 genes of EPAS1, TP53, H3F3A, HRAS, IDH2, ATRX, BRAF, EGLN2 and FGFR1 are expanded for detection through screening and exploration, so that the detection genes cover three levels basically, expanded and integrated, the detection integrity is enriched, and the detection results are more comprehensive.
Experimental example 2 exploration and optimization of targeting region and primer sequences
This example was based on the genes VHL, SDHA, SDHB, SDHC, SDHD, NF, FH, MAX, TMEM127, RET, MET, KIF1B, SDHAF2, EPAS1, EGLN1, MDH2, KMT2D, TP53, H3F3A, HRAS, IDH2, ATRX, BRAF, EGLN2, FGFR1, MERTK, CDKN2A, BAP1 and MEN1 determined in example 1, and primer amplification was performed with the CDs region and variable sheared region of these target genes, and by analysis, the present application selected amplification assays for SNV and INDELs of length within 22bp for these genes, thereby obtaining more favorable pathogenic mutation information.
This example designs amplification primers for the above regions and optimizes the final primer targeting region and primer sequence, etc. The spread is for reasons of coverage only.
The method comprises the following steps: table 1 shows the coverage of the target region before optimization, and the example adds the primers SEQ ID NO.53-54 to ensure that the exon1 region of the EGLN1 gene reaches 100% coverage; the primers of SEQ ID NO.293-294 are added to ensure that the exo 1 region of the MDH2 gene reaches 100% coverage; the SEQ ID NO.257-258 primer is added, so that the exon10 region of the KMT2D gene reaches 100% coverage; the SEQ ID NO.193-194 primer is added, so that the exon42 region of the KMT2D gene reaches 100% coverage; the primers of SEQ ID NO.635-636 were optimized to achieve 100% coverage of the exon4 region of the SDHD gene (see in particular Table 3).
In addition, the present application also adjusts the base sequence of the primer sequence to the problem of poor coverage due to poor amplification of the original primer. Table 2 shows the original primer sequences, and the primers SEQ ID NO.563-564 are obtained by optimizing and adjusting part of the original sequences, so that the exon1 region of the SDHA gene reaches 100% amplification coverage; optimizing to obtain SEQ ID NO.199-200 primer, so that the exon39 region of the KMT2D gene reaches 100% amplification coverage; optimizing to obtain SEQ ID NO.23-24 primer to ensure that the exon6 region of the BAP1 gene reaches 100% amplification coverage; optimizing to obtain a primer of SEQ ID NO.159-160, so that the exon45 region of the KIF1B gene reaches 100% amplification coverage; optimizing to obtain SEQ ID NO.283-284 primer to make the exon4 region of MAX gene reach 100% amplification coverage; the primers SEQ ID NO.531-532 are obtained by optimization, so that the exon5 region of RET gene reaches 100% amplification coverage, and the sequence before primer optimization is shown in Table 2. After the above optimization, the target area reached 100% coverage, see in particular table 3.
TABLE 1 target area coverage Condition before primer optimization
TABLE 2 primer sequences before partial optimization
TABLE 3 target area coverage after primer optimization
Through the optimization, the application finally determines the primer sequence as follows:
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example 3 optimization of amplification System
In this example, 1 clinical sample diagnosed positive was selected, the amplification primer set was used to amplify the target region and sequenced by NGS, the coverage of the amplified region in the sequencing result was analyzed, and the amplification efficiency of the amplification primer set and the rationality of the test method were evaluated. The operation flow is as follows:
1) Extracting sample DNA according to a conventional process;
2) Amplifying the DNA obtained in the step one according to a conventional test flow, and sequencing by using NGS, wherein the test amplification conditions are as follows:
3) Counting data coverage information to obtain a target area coverage result;
4) The reason for the uncovered area was analyzed and an improvement scheme was specified.
Target area coverage results: the partial genes of the target region are not completely covered, and the high GC region cannot be amplified due to the high GC content of the partial region, so that the target region is not completely covered, and the amplification efficiency is shown in the figure 1.
The application further adjusts the assay procedure, improving amplification conditions to increase coverage.
And (3) selecting the sample, amplifying the target region by using the amplification primer set, sequencing by NGS, analyzing the coverage of the amplified region in the sequencing result, and evaluating the amplification efficiency of the amplification primer set and the rationality of the test method. The operation flow is as follows:
1) Extracting sample DNA according to a conventional process;
2) The DNA obtained in step one was amplified according to the test procedure of the present invention and sequenced using NGS. From the above results, it is clear that the amplification efficiency of the high GC content region is low, and the template is difficult to open because of the high energy required for melting due to the formation of three pairs of hydrogen bonds between G, C, and the main difficulty faced in the amplification of the high GC content template is that the stable secondary structure is easily formed in the template to prevent the DNA polymerase from advancing on the template, and a plurality of non-specific primer renaturation sites may exist on the template, resulting in non-specific amplification, even in many times no target gene is amplified. To solve this problem, the present application attempts to improve by: the cycle number of the denaturation reaction is increased, so that the denaturation reaction can be fully carried out, and the high GC content area can be thoroughly separated; setting the temperature difference between annealing and extension procedures, fully combining the primer and the template, and then performing the extension reaction of the sequence, so that the target sequence can be effectively extended. The specific conditions after improvement are:
3) Counting data coverage information to obtain a target area coverage result;
4) And comparing the coverage results, and analyzing the feasibility of the scheme.
As shown in FIG. 2, comparing FIG. 1, the present application solves the coverage problem of the target region under the condition of adjusting the amplification conditions, and effectively solves the problem of failure in amplification of the high GC region.
Example 4 establishment of the inventive method System
Optimized by the above examples, the amplification primer set of the gene detection method for determining the final pheochromocytoma and paraganglioma diseases of the application and the method thereof are as follows:
1. library construction and primer amplification sequencing
1. Sample preparation: extracting DNA;
2. library amplification: pre-Mix was prepared for a total of 2 pool, 5ul per reaction, with the following formulation method:
split charging is carried out on the prepared Pre-Mix, genomic DNA is added, and a reaction system is prepared as follows:
covering the tube cover, gently swirling, mixing, and centrifuging briefly, wherein bubbles are generated (if bubbles are generated, the tube wall is flicked, and the bubbles are broken and then separated instantaneously); the prepared reaction system is put into a PCR instrument, and the following procedure is started for amplification:
3. digestion of primer, enzyme digestion and ligation to adaptor
Putting all products of pool1 into pool2 correspondingly, adding 1 μl FuPa Reagent into each PCR product, covering with a cover, mixing, and centrifuging instantaneously to prevent bubbles (if bubbles are generated, flicking the tube wall, breaking the bubbles and then instantaneously separating); the mixed reactants are put into a PCR instrument, and the following procedure is started for reaction:
barcode adapter Mix (pollution prevention) is formulated according to the following table
The configuration magnet establishes a corresponding relation between a sample name and a Barcode adapter, and a joint connection system is prepared according to the following table:
adding 1 μl of DNA (deoxyribonucleic acid) Ligase into the reaction system, covering with a cover, mixing, and performing instantaneous centrifugation to prevent bubbles (if bubbles are generated, flicking the tube wall to break the bubbles, and then instantaneously separating); the prepared reaction system is put into a PCR instrument, and the following procedure is started for reaction:
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4. library purification:
fresh 70% ethanol was prepared in an amount of 300ul per purification step for each sample; incubating Agencourt AMPure XP Beads in advance for half an hour at room temperature, and fully and uniformly mixing when in use; adding 22.5 mu l Agencourt AMPure XP Beads into the product obtained in the step 3, blowing and sucking the mixture, standing the mixture at room temperature for 5min, then transferring the Tube to DynaMag-2magnet, standing the Tube for more than 5min until the Tube is clear, and removing the supernatant; 150 μl of freshly prepared 70% ethanol was added to the centrifuge tube and after 2 weeks of rotation on a magnetic rack, the supernatant was carefully removed; repeating the steps, removing the supernatant, and air-drying the magnetic beads (the back surfaces of the magnetic beads are provided with a crack); adding 50 μl of low TE, blowing and sucking, mixing, standing at room temperature for 2min, centrifuging, standing in DynaMag-2Magnet until the solution is completely clear (> 3 min), transferring the supernatant into a new centrifuge tube, and completing the warehouse building.
5. Dilution and quantification (quality control) of DNA library
Library quantification was performed using Real-Time PCR (kit selection Ion Library TaqMan Quantitation Kit, instrument VII 7); firstly, diluting the standard substance according to the sequence of S1, S2 and S3, and diluting the original standard substance S0-68pM and 10 times of the original standard substance S0-68pM to 6.8pM,0.68pM and 0.068pM; the library of each sample was diluted 100-fold by 5ul and single sample quantification was performed; the reagents for qPCR were prepared according to the number of samples, in a total volume of 10ul, according to the following table:
PCR was performed under the following conditions:
2. sequencing and data analysis:
1. after library preparation and qualification, the NGS method was used to sequence, and the sequencing steps were performed according to existing protocols.
2. According to ClinVar, 1000G, HGMD, clinGen, dbVar, gnomAD etc. databases, vep is used
v96 annotates detected SNPs and INDELs, analyzing the pathogenicity of mutations against pheochromocytoma and paraganglioma diseases.
3. According to guidelines such as ACMG, the mutation is manually interpreted, pathogenic mutation is screened out, and a detection report is provided according to a pathogenicity result.
Example 5 advantage in starting amount of DNA
In PPGL-associated pathogenic genes, the GC content of 13 total regions is greater than 70%. These regions are affected in amplification efficiency, so that conventional methods require amplification using a larger initial amount of DNA in order to obtain efficient amplification. According to the invention, the optimized amplification conditions are used, and all the regions can be effectively amplified only by using the same initial quantity (20 ng) of DNA as other regions, so that the high initial quantity of DNA is not required to be amplified, the original sample is saved, the influence of the high initial quantity on the amplification quality of other regions can be avoided, the amplification result is more uniform, the application is convenient, and the amplification effect is good. Amplification was performed using a starting amount of 20ng DNA, the high GC content region was fully covered, and the statistics of the high GC content region and its coverage are shown in Table 4.
TABLE 4 Gene regions with GC content of more than 70%
| Gene | Exon | GC | Coverage(Depth>＝4X) |
| SDHB | exon1 | 71.83％ | 100％ |
| FH | exon1 | 70.23％ | 100％ |
| TMEM127 | exon2 | 72.43％ | 100％ |
| MERTK | exon1 | 76.67％ | 100％ |
| VHL | exon1 | 71.98％ | 100％ |
| SDHA | exon1 | 79.03％ | 100％ |
| MDH2 | exon1 | 75.38％ | 100％ |
| BRAF | exon1 | 77.37％ | 100％ |
| CDKN2A | exon2 | 72.22％ | 100％ |
| CDKN2A | exon1 | 73.15％ | 100％ |
| RET | exon4 | 71.78％ | 100％ |
| HRAS | exon5 | 72.88％ | 100％ |
| NF1 | exon1 | 71.19％ | 100％ |
Example 6 verification of clinical sample detection and first generation sequencing
In this example, 1 patient sample was selected for clinical diagnosis of paraganglioma (heart, mediastinum, post-operation) and pheochromocytoma (bi-adrenal) recurrence after metastatic operation, target region amplification was performed using the amplification primer set and amplification system determined in example 3, and sequencing was performed by NGS, analyzing gene mutation and performing site annotation, interpreting the annotation results and determining the site of pathogenic mutation. The operation flow is as follows:
1) Extracting sample DNA according to a conventional process;
2) Amplifying the DNA obtained in the step one according to the test flow, and sequencing and mutation detection by using NGS;
3) Performing site annotation on the mutation result, reading the annotation result, and searching the mutation site of the pathogenic gene;
4) Further carrying out first-generation sequencing verification on the pathogenic mutation site.
Pathogenic mutation results: the pathogenic gene mutation of the patient is detected to be SDHD c.278_280del p.Tyr93del, the detection result is consistent with clinical symptoms, and the specific pathogenic mutation information is shown in the attached table 5.
TABLE 5 clinical diagnostic positive sample test result information
A first generation sequencing verification result is shown in figure 3, and is consistent with the detection result of the invention. The above results demonstrate the accuracy and effectiveness of the present application in clinical testing.
The foregoing descriptions of specific exemplary embodiments of the present application are presented for purposes of illustration and description. It is not intended to be exhaustive or to limit the application to the precise form disclosed, and obviously many modifications and variations are possible in light of the above teaching. The exemplary embodiments were chosen and described in order to explain the specific principles of the present application and its practical application to thereby enable one skilled in the art to make and utilize the present application in various exemplary embodiments and with various modifications as are suited to the particular use contemplated. The scope of the application is intended to be defined by the claims and the equivalents thereof.
Claims (10)
1. A library construction method for NGS for detecting pheochromocytoma and paraganglioma causative genes, comprising the steps of:
1) Extracting sample DNA;
2) Library amplification: amplifying the extracted DNA;
3) And (3) joint connection: sequencing adaptor connection is carried out on the amplified products;
4) And (5) purifying the library.
2. The library construction method according to claim 1, wherein the amplification in 1) is for VHL, SDHA, SDHB, SDHC, SDHD, NF, FH, MAX, TMEM127, RET, MET, KIF1B, SDHAF2, EPAS1, EGLN1, MDH2, KMT2D, TP53, H3F3A, HRAS, IDH2, ATRX, BRAF, EGLN2, FGFR1, MERTK, CDKN2A, BAP1 and MEN1 genes;
preferably, the amplification is performed on INDELs within 22bp of the SNV and 22bp of the CDs region and variable cleavage region of VHL, SDHA, SDHB, SDHC, SDHD, NF1, FH, MAX, TMEM127, RET, MET, KIF1B, SDHAF, EPAS1, EGLN1, MDH2, KMT2D, TP53, H3F3A, HRAS, IDH2, ATRX, BRAF, EGLN2, FGFR1, MERTK, CDKN2A, BAP1 and MEN1 genes.
3. The method of library construction according to claim 2, wherein the amplification primer sequences are shown in SEQ ID NOS.1-650.
4. A library construction method according to any one of claims 1-3, wherein the amplification conditions are as follows:
5. a method of detecting pheochromocytoma and paraganglioma comprising the library construction method of any one of claims 1-4, and further comprising the steps of sequencing and belief analysis by NGS.
6. A primer set for detecting pheochromocytoma and paraganglioma causative genes, characterized in that the primer is a primer for amplifying VHL, SDHA, SDHB, SDHC, SDHD, NF1, FH, MAX, TMEM127, RET, MET, KIF1B, SDHAF2, EPAS1, EGLN1, MDH2, KMT2D, TP53, H3F3A, HRAS, IDH2, ATRX, BRAF, EGLN2, FGFR1, MERTK, CDKN2A, BAP1 and MEN1 genes;
preferably, the primer is a primer for amplification of the CDs region of VHL, SDHA, SDHB, SDHC, SDHD, NF, FH, MAX, TMEM127, RET, MET, KIF1B, SDHAF, EPAS1, EGLN1, MDH2, KMT2D, TP53, H3F3A, HRAS, IDH2, ATRX, BRAF, EGLN2, FGFR1, MERTK, CDKN2A, BAP1 and MEN1 genes and the SNV of the variable cleavage region and the INDEL within 22 bp.
7. The primer set of claim 6, wherein the primer has a sequence as shown in SEQ ID NO. 1-650.
8. A composition comprising the primer set of any one of claims 6 to 7.
9. A kit for detecting a disease causing gene of pheochromocytoma and paraganglioma, comprising the primer set of any one of claims 6 to 7.
10. Use of a primer set according to any one of claims 6 to 7 or a composition according to claim 8 for the preparation of a kit for detecting pheochromocytoma and paraganglioma pathogenic genes.
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