CN117070624A - Capture probe and kit for gene diagnosis and typing of pheochromocytoma and paraganglioma - Google Patents
Capture probe and kit for gene diagnosis and typing of pheochromocytoma and paraganglioma Download PDFInfo
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Abstract
The invention belongs to the field of biotechnology, and particularly relates to a capture probe and a kit for gene diagnosis and typing of pheochromocytoma and paraganglioma. The probes include combinations of RET probes, SDHD probes, HRAS probes, SDHB probes, NF1 probes, EPAS1 probes, VHL probes, FGFR1 probes. Compared with the prior art, the capture probe can be used for diagnosing and parting the gene of the pheochromocytoma and the paraganglioma, is convenient to use and simple to operate, has the advantages of rapidness, specificity, sensitivity, accuracy, practicability and the like, has diagnostic and parting values, and is convenient for mass production.
Description
Technical Field
The invention belongs to the field of biotechnology, and particularly relates to a capture probe and a kit for gene diagnosis and typing of pheochromocytoma and paraganglioma.
Background
Pheochromocytomas and paragangliomas (pheochromocytoma and paraganglioma, PPGL) are tumors originating in the adrenal medulla or the sympathetic or parasympathetic ganglions outside the adrenal gland, producing predominantly one or more catecholamines: epinephrine, norepinephrine, and dopamine. At present, the pathogenesis of PPGL is considered to be related to gene mutation, and the gene diagnosis of PPGL patients is not only beneficial to the parting of tumors, but also beneficial to the formulation of diagnosis and treatment schemes of PPGL patients.
At present, the clinical gene detection process for PPGL patients is relatively complex, firstly, a clinician determines one or more highly suspected known genes possibly existing in the patients through the information of clinical manifestations, family history and the like of the patients, and then the known genes are detected through methods such as first-generation sequencing, whole exon sequencing and the like, however, the detection period of the methods is longer, and the economic cost is higher. And for the first generation sequencing, there is a possibility that various known gene detections need to be performed multiple times. Therefore, the current methods such as the first generation sequencing, the whole exon sequencing and the like are not suitable for long-term clinical use and popularization.
At present, a method for accurately detecting genes of PPGL patients is required to have short detection period and small economic burden on patients clinically.
Disclosure of Invention
Based on the current demand for diagnosis and typing of pheochromocytoma and paraganglioma genes in the prior art, the invention provides a capture probe and a kit for diagnosis and typing of pheochromocytoma and paraganglioma genes.
The aim of the invention can be achieved by the following technical scheme:
the invention provides a capture probe for diagnosis and typing of pheochromocytoma and paraganglioma genes, which consists of a capture probe of Pheochromocytoma and Paraganglioma (PPGL) related genes, wherein the probe comprises a combination of RET probe, SDHD probe, HRAS probe, SDHB probe, NF1 probe, EPAS1 probe, VHL probe and FGFR1 probe.
In one embodiment of the invention, RET probes, SDHD probes, HRAS probes, SDHB probes, NF1 probes, EPAS1 probes, VHL probes, FGFR1 probes are mixed in the same mass ratio in capture probes for diagnosis and typing of pheochromocytoma and paraganglioma genes.
In one embodiment of the present invention, the sequences of RET probe, SDHD probe, HRAS probe, SDHB probe, NF1 probe, EPAS1 probe, VHL probe, FGFR1 probe are shown in Table 1, respectively, in the capture probes for diagnosis and typing of pheochromocytoma and paraganglioma genes.
In one embodiment of the invention, the capture probe is capable of covering all target regions of Pheochromocytoma and Paraganglioma (PPGL) associated genes; the capture probe can capture all exons and partial boundary areas of exons and introns of related genes of Pheochromocytoma and Paraganglioma (PPGL); the overlapping area exists between the capture probes, so that all target areas can be covered by more than two probes.
In one embodiment of the invention, the capture probes each have a biotin label.
In one embodiment of the invention, the capture probe is 80-120bp in length; the Tm value of the capture probe is 64-75 ℃.
The invention also provides a kit for gene diagnosis and typing of pheochromocytoma and paraganglioma, comprising the capture probe.
In one embodiment of the invention, the kit further comprises the following individual components individually packaged:
universal library construction system: fragmenting/terminal repairing enzyme, fragmenting/terminal repairing buffer solution, ligation reaction premix solution, linker, PCR Mix and purified magnetic beads;
liquid phase hybridization system: hybridization Mix, whs-PPGL-54gene Panel, human Cot, universal Blockers, hybridization Enhancer, streptavidin Binding Beads, binding Buffer, wash Buffer1, wash Buffer2, 2X KAPA HiFi HotStart ReadyMix, amplification Primers;
universal library construction kit:
liquid phase hybridization system:
the invention also provides application of the capture probe or the kit in preparation of products for detecting pheochromocytoma and paraganglioma genetic variation in a sample to be detected.
The invention also provides application of the capture probe or the kit in preparing a product for detecting or predicting whether a patient to be detected has pheochromocytoma and paraganglioma.
At present, the clinical gene detection process for PPGL patients is relatively complex, firstly, a clinician determines one or more highly suspected known genes possibly existing in the patients through the information of clinical manifestations, family history and the like of the patients, and then the known genes are detected through methods such as first-generation sequencing, whole exon sequencing and the like, however, the detection period of the methods is longer, and the economic cost is higher. And for the first generation sequencing, since all known genes are not included, there is also a possibility that detection of other known genes needs to be performed a plurality of times. Therefore, the current methods such as the first generation sequencing, the whole exon sequencing and the like are not suitable for long-term clinical use and popularization. At present, all reported known genes of PPGL are not collected and then detected, and a capture probe special for detecting all the known genes of PPGL is not provided.
Compared with the prior art, the invention has the following advantages:
the capture probe for diagnosis and typing of pheochromocytoma and paraganglioma genes can capture all exons and exon-intron part boundary areas of related genes of Pheochromocytoma and Paraganglioma (PPGL). Therefore, the capture probe can be used for diagnosing and parting the gene of the pheochromocytoma and the paraganglioma, is convenient to use and easy to operate, has the advantages of rapidness, specificity, sensitivity, accuracy, practicability and the like, has diagnostic and parting values, and is convenient for mass production.
Based on the capture probe for gene diagnosis and typing of pheochromocytoma and paraganglioma, the detection period of the PPGL patient is obviously shortened to 2-3 weeks, the economic burden of the patient is obviously reduced, the capture probe is more suitable for clinical gene diagnosis and typing of the PPGL, and certain auxiliary effect is also provided for follow-up and treatment scheme formulation of the PPGL patient.
By adopting the scheme of the invention, the tumor tissues (fresh or paraffin sections) of patients with pheochromocytoma and paraganglioma can be subjected to high-throughput sequencing, so that pathogenic gene mutations can be identified.
Drawings
FIG. 1 shows the result of electrophoresis in step 4.6 of example 1;
FIG. 2 shows the result of electrophoresis in step 5.17 of example 1;
FIG. 3 is a flow chart of the analysis in example 1.
Detailed Description
The terms "capture probe for diagnosis and typing of Pheochromocytoma and Paraganglioma Genes (PPGL)," capture probe ", or" gene capture probe "are used interchangeably to refer to capture probes for diagnosis and typing of Pheochromocytoma and Paraganglioma Genes (PPGL) as described in the present invention.
The invention will now be described in detail with reference to the drawings and specific examples.
Example 1
The embodiment provides a capture probe for diagnosis and typing of pheochromocytoma and paraganglioma genes, which consists of a capture probe of Pheochromocytoma and Paraganglioma (PPGL) related genes, wherein the probe comprises a combination of RET probe, SDHD probe, HRAS probe, SDHB probe, NF1 probe, EPAS1 probe, VHL probe and FGFR1 probe. The sequences of the probes are shown in Table 1.
TABLE 1 probe sequences
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Taking the first gene RET as an example, 39 probes were designed in total, so there are 39 long base sequences ending in RET in the text, chr10 preceding the first sequence represents the corresponding chromosome 10, and 43572683 and 43572803 correspond to the start and stop sites of the probes, respectively. The sequences of these probes were 100% matched to the coding sequences in the genome. Different genes, depending on the size of the gene and the number of exons, will have different numbers of probes designed. For example, for the gene SDHD, 6 probes were designed. And the rest and so on.
After the design of the capture probe is finished, the capture probe is delivered to Shanghai Wei Hansi biological medicine technology Co., ltd to finish the probe synthesis.
The embodiment also provides a capture sequencing technology, which comprises a capture probe obtained by preparation, a general library construction Kit, a liquid phase hybridization system, a sequencing device (Illumina Hi-Seq) and a paraffin extraction Kit FFPE DNA Kit (200) (OMEGA, D3399-02).
Universal library construction kit:
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liquid phase hybridization system:
the biological materials used in this example were all from the secondary mountain hospital at the university of double denier, and all samples were informed consent and ethical evidence provided by the tissues signed by the patient himself.
The embodiment also provides an application of the capture probe for detecting the gene of Pheochromocytoma and Paraganglioma (PPGL) based on a high-throughput sequencing technology, and the experimental process is as follows:
(1) And (5) sample collection. Paraffin embedded tissue (FFPE) samples, 10% neutral formalin fixed surgical resection samples, were sampled according to pathology practice specifications, and 22 FFPE samples were collected for testing.
When paraffin sections were prepared, 5 serial sections were cut, 1 of which was HE stained to confirm the content of tumor cells.
DNA is easily affected by fixation, and DNA of a sample immersed in formalin for a long period of time (1 week or more) is fragmented, and mutation cannot be detected. The fixing time of the biopsy material is generally 24 hours, and for a biopsy sample such as a puncture, the fixing time is preferably controlled to be 6 to 24 hours.
(2) Extraction of human genome DNA. The DNA extraction procedure is described with reference to the Paraffin extraction Kit FFPE DNA Kit (200) (OMEGA, D3399-02) extraction Kit.
(3) And (5) DNA quality control. Performing quality inspection on the human genome DNA obtained in the step (1), detecting the integrity of the DNA by agarose electrophoresis, taking 100ng of genome DNA for electrophoresis detection, wherein the main band of the DNA is more than 1kb, and the DNA is not completely degraded; detecting the concentration of DNA by Qubit3.0, wherein the concentration of DNA is more than or equal to 10 ng/. Mu.L, and the total amount of DNA is more than or equal to 500ng; onedrop detected DNA purity, OD260/280 was 1.6-2.0.
The concentration measurement results are shown in table 2 below.
TABLE 2 concentration detection results
| Sample numbering | Concentration of | 260/280 | 260/230 | Volume of |
| PPGL-S01 | 54.42 | 1.76 | 1.91 | 50 |
| PPGL-S02 | 133.03 | 1.87 | 1.96 | 50 |
| PPGL-S03 | 316.45 | 1.87 | 2.07 | 50 |
| PPGL-S04 | 165.44 | 1.83 | 2.07 | 50 |
| PPGL-S05 | 38.71 | 1.86 | 1.83 | 50 |
| PPGL-S06 | 81.16 | 1.85 | 2.06 | 50 |
| PPGL-S07 | 81.78 | 1.88 | 2.06 | 50 |
| PPGL-S08 | 94.87 | 1.92 | 2.09 | 50 |
| PPGL-S09 | 331.4 | 1.83 | 1.51 | 50 |
| PPGL-S10 | 177.01 | 1.94 | 1.22 | 50 |
| PPGL-S11 | 101.69 | 1.9 | 0.9 | 50 |
| PPGL-S12 | 60.23 | 1.93 | 0.63 | 50 |
| PPGL-S13 | 116.52 | 1.95 | 0.91 | 50 |
| PPGL-S14 | 81.19 | 1.78 | 1.39 | 50 |
| PPGL-S15 | 87.99 | 1.9 | 1.45 | 50 |
| PPGL-S16 | 143.16 | 1.78 | 1.64 | 50 |
| PPGL-S17 | 269.48 | 1.93 | 2.11 | 50 |
| PPGL-S18 | 106.26 | 1.84 | 2.04 | 50 |
| PPGL-S19 | 111.06 | 1.84 | 2.02 | 50 |
| PPGL-S20 | 165.93 | 1.89 | 2.04 | 50 |
| PPGL-S21 | 253.43 | 1.83 | 2.13 | 50 |
| PPGL-S22 | 68.06 | 1.85 | 2.02 | 50 |
(4) And (5) DNA library establishment. And (3) taking 200ng of gDNA obtained in the step (1) for operations such as fragmentation/end repair, addition of A, connection joint, magnetic bead purification, genome library enrichment and the like, so as to obtain an enriched DNA library.
4.1 fragmentation/end repair with A the reaction system was formulated as follows
The fragmentation/end repair reaction system is placed on a thermal cycler for reaction, and the specific reaction procedure is as follows: 37℃for 7min,65℃for 30min,4℃hold (thermal lid 105 ℃).
4.2 connecting Joint, a connecting reaction system was prepared according to the following table
| Reagent name | Volume/. Mu.L |
| Fragmentation/end repair reaction system | 50 |
| Joint | 5 |
| Connection reaction premix | 25 |
| Total volume of | 75 |
The connection reaction system is placed on a thermal cycler for reaction, and the specific reaction procedure is as follows: 15min at 20℃and hold (close thermal lid) at 4 ℃.
4.3 purification of magnetic beads, recovery of ligation reaction product using Twist Binding and Purification Beads Kit purification.
Vortex oscillating DNA purifying magnetic beads, and fully and uniformly mixing;
adding 0.8 times system magnetic beads (60 mu L) into the connection system, mixing uniformly by vortex, and standing at room temperature for 5min;
centrifuging briefly, placing the reaction tube on a magnetic rack, standing for 2min, and removing supernatant after the solution is clear;
adding 200 mu L of 80% ethanol, standing for 30s, and sucking and discarding the supernatant; the reaction mixture was allowed to stand at room temperature for 4min (no reflection state was observed on the surface of the beads) after two operations.
17 μl of water was added, the tube cap was closed, vortexed and mixed well and allowed to stand at room temperature for 2min.
Transferring 15 μl of supernatant into a clean single tube of 0.2ml thin-walled PCR tube or plate hole of 96 Kong Rexun-ring plate without breaking up magnetic bead precipitate
4.5 enrichment of genomic library an enrichment reaction system was formulated as follows
The fragmentation/end repair reaction system is placed on a thermal cycler for reaction, and the specific reaction procedure is as follows: 98℃for 45s, (98℃for 15s,60℃for 30s,72℃for 30 s) 4cycles,72℃for 1min,4℃hold (thermal lid 105 ℃).
4.6, quality inspection, namely after the enrichment reaction of the genome library is finished, taking out a PCR tube, sucking 2 mu L of reaction liquid into a new PCR tube with a mark, transferring the new PCR tube to an electrophoresis chamber, adding 2 mu L of 2 xLoding Buffer into each tube, blowing and uniformly mixing, spotting, and carrying out 110V electrophoresis; the electrophoresis result is shown in figure 1
4.7 purification of magnetic beads, recovery of ligation reaction product using Twist Binding and Purification Beads Kit purification.
Vortex oscillating DNA purifying magnetic beads, and fully and uniformly mixing;
adding 1-time system magnetic beads (50 mu L) into the connection system, mixing uniformly by vortex, and standing at room temperature for 5min;
centrifuging briefly, placing the reaction tube on a magnetic rack, standing for 2min, and removing supernatant after the solution is clear;
adding 200 mu L of 80% ethanol, standing for 30s, and sucking and discarding the supernatant; the reaction mixture was allowed to stand at room temperature for 4min (no reflection state was observed on the surface of the beads) after two operations.
Adding 30 mu L of water, covering a tube cover, vortex uniformly mixing, and standing at room temperature for 2min.
The supernatant was allowed to stand for 30s and 27. Mu.L of supernatant was aspirated into a new labeled 1.5mL centrifuge tube. The Qubit is used for measuring the concentration, writing the concentration into the pipe wall and filling the experimental record table.
(5) Hybridization capture: and (3) carrying out liquid phase hybridization capture on the enriched DNA library obtained in the step (4) and the capture probe prepared in the step (1), and carrying out PCR enrichment and magnetic bead purification on hybridization products to obtain capture library DNA.
5.1 transferring the library to be mixed, whs-PPGL-54gene Panel, twist Universal Blockers and Blocker Solution from-20 ℃ to a refrigerator at 4 ℃ for thawing, reversing, mixing uniformly and centrifuging briefly;
calculating a mixed sample and a mixed sample amount according to the data amount, the number of samples, the library concentration and the barcode, wherein the mixed sample amount is calculated by referring to the following table: (the total mixing amount may be more than 1500ng, but not more than 4000 ng).
| Library quantity | Mix amount of each library | Total mixing amount |
| 1 | 500ng | 500ng |
| 2 | 500ng | 1000ng |
| 3 | 500ng | 1500ng |
| 4 | 375ng | 1500ng |
| 8 | 187.5ng | 1500ng |
| 30 | 133.3 | 4000ng |
Mixing samples according to the calculation result to prepare a mixed library Mix;
5.2, transferring the Mix library Mix centrifuge tube to a vacuum concentrator, drying at a rotating speed of 3000rpm and a temperature of less than or equal to 40 ℃ (32 ℃ in default), and controlling the drying time within 1h as much as possible;
5.3 preheating Hybridization Mix at 65 ℃ for 10min until completely dissolved;
5.4 preparing prehybridization liquid in a 0.2mL centrifuge tube, and gently blowing and uniformly mixing;
| reagent name | Volume/. Mu.L |
| Hybridization Mix | 20 |
| whs-PPGL-54gene Panel | 4 |
| Water and its preparation method | 4 |
| Total | 28 |
The prehybridization solution was placed in a PCR apparatus at 95℃for 2 minutes (thermal lid 105 ℃) and then immediately cooled on ice for 5 minutes, and finally equilibrated to room temperature for 5 minutes;
5.5 the following table reagents were added to the dry mix library and blow mixed. Transfer to a 0.2ml centrifuge tube;
| reagent name | Volume/. Mu.L |
| Dried Indexed Library Pool | - |
| Human Cot | 5 |
| Universal Blockers | 2 |
| H 2 O | 5 |
| Total | 12 |
5.6 the 0.2mL centrifuge tube was placed in a 95℃PCR apparatus (105℃heat lid) and heated for 5 minutes, the library was removed from the PCR apparatus and cooled to room temperature (no more than 5 minutes, which may be cooled on ice).
5.7, carefully blowing and mixing by a pipette, performing instantaneous centrifugation, and adding 28 mu L of prehybridization liquid prepared in the step 5.4;
5.8 30. Mu. L Hybridization Enhancer was added per hybridization reaction. The air bubbles are not generated by light blowing. Instantaneous centrifugation to ensure that all reaction solution is at the bottom of the tube;
5.9 the hybridization reaction tube was placed in a PCR apparatus at 70℃and hybridized for 16h (hot cap 85 ℃).
5.10 aspirate 100. Mu. L Streptavidin Binding Beads into a new 0.5mL centrifuge tube; placing the centrifuge tube in a magnetic rack, standing for 1min, and discarding the supernatant after clarification;
adding 200 mu L of Binding Buffer into a centrifuge tube, carrying out brief vortex mixing, carrying out brief centrifugation, placing in a magnetic rack, standing for 1min, and discarding the supernatant after clarification; this step was repeated twice, for a total of 3 washes;
200. Mu.L of Binding Buffer was added to the centrifuge tube, and the mixture was vortexed and mixed well for use.
5.11 after hybridization, transferring all the liquid in the hybridized PCR tube into a centrifuge tube in the step 5.10; placing the centrifuge tube on a mixing instrument, clicking the start, and slowly turning over for 30min;
5.12, taking down the centrifuge tube after capturing, centrifuging briefly, placing the centrifuge tube in a magnetic rack, standing for 1min, and discarding the supernatant after clarifying;
5.13 adding 200 mu L of Wash Buffer1 into the centrifuge tube, blowing and mixing uniformly, performing instantaneous centrifugation to ensure that the liquid is at the bottom of the tube, transferring all the liquid in the tube into a new marked 0.5mL centrifuge tube, placing the tube into a magnetic rack, standing for 1min, and discarding the supernatant after clarification;
5.14 adding 200 mu L of Wash Buffer2 preheated to 48 ℃ into a centrifuge tube, blowing and mixing uniformly, centrifuging briefly, placing into a 48 ℃ PCR instrument, and incubating for 5min; taking out the centrifuge tube after incubation, placing the centrifuge tube in a magnetic rack, standing for 1min, and discarding the supernatant after clarification; this step was repeated twice and a total of 3 washes was performed
5.15, taking down the centrifuge tube, centrifuging briefly, placing the centrifuge tube on a magnetic rack, and discarding the supernatant; 22.5 mu L of Nuclease-Free Water is added into the centrifuge tube, mixed evenly by vortex and placed on an ice box for standby.
5.16 PCR enrichment of hybridization products the hybridization product PCR System was formulated as follows
The hybridization product PCR system is placed on a thermal cycler for reaction, and the specific reaction procedure is as follows: 98℃45s, (98℃15s,60℃30s,72℃30 s) 13cycles,72℃1min,4℃hold (thermal cover 105 ℃)
And 5.17 quality inspection. After the PCR enrichment of the hybridization product is finished, taking out a PCR tube, sucking 2 mu L of reaction liquid into a new PCR tube with a mark, transferring the new PCR tube to an electrophoresis chamber, adding 2 mu L of 2 xLoding Buffer into each tube, blowing and uniformly mixing, spotting, and carrying out 110V electrophoresis; the result of electrophoresis is shown in figure 2.
(6) High throughput sequencing. The captured library DNA obtained in (5) was subjected to high throughput sequencing using Illumina Hi-Seq.
(7) And (5) data analysis. The sequencing raw data was quality controlled using fasstqc software (FASTQC v 0.11.8). After removal of low mass reads, sequences were aligned to the human reference genome (GRCh 37/hg 19) using BWA (Burrows-Wheeler Aligner 0.7.17-r 1194-dirty) software. After deduplication and base calibration of the sequences, SNP and Indel variation was analyzed using GATK software (v4.1.0.0). Finally, the detected variations were annotated by Annovar software (2020-06-08:00:46:07-0400). Genetic diagnosis and typing of pheochromocytoma and paraganglioma were performed on the high throughput sequencing data of (6).
The analysis flow is shown in figure 3.
The key data quality control results are shown in table 3.
TABLE 3 quality control results for critical data
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The data analysis results are shown in table 4.
TABLE 4 data analysis results
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Note that: the samples are not listed in the table as the samples did not detect Pathogenic/potential Pathogenic (Pathogenic/pathogenic_pathogenic) mutation sites.
The previous description of the embodiments is provided to facilitate a person of ordinary skill in the art in order to make and use the present invention. It will be apparent to those skilled in the art that various modifications can be readily made to these embodiments and the generic principles described herein may be applied to other embodiments without the use of the inventive faculty. Therefore, the present invention is not limited to the above-described embodiments, and those skilled in the art, based on the present disclosure, should make improvements and modifications without departing from the scope of the present invention.
Claims (10)
1. A capture probe for diagnosis and typing of pheochromocytoma and paraganglioma genes, which consists of a capture probe of pheochromocytoma and paraganglioma related genes, characterized in that the probe comprises a combination of RET probe, SDHD probe, HRAS probe, SDHB probe, NF1 probe, EPAS1 probe, VHL probe, FGFR1 probe.
2. The capture probe for diagnosis and typing of pheochromocytoma and paraganglioma genes according to claim 1, wherein RET probe, SDHD probe, HRAS probe, SDHB probe, NF1 probe, EPAS1 probe, VHL probe, FGFR1 probe are mixed in the same mass ratio.
3. A capture probe for diagnosis and typing of pheochromocytoma and paraganglioma genes according to claim 1, wherein the capture probe covers all target areas of pheochromocytoma and paraganglioma related genes; the capture probe can capture all exons and partial junction areas of exons and introns of related genes of pheochromocytoma and paraganglioma; the overlapping area exists between the capture probes, so that all target areas can be covered by more than two probes.
4. A capture probe for diagnosis and typing of pheochromocytoma and paraganglioma genes according to claim 1, wherein each of the capture probes has a biotin label.
5. A capture probe for diagnosis and typing of pheochromocytoma and paraganglioma genes according to claim 1, wherein the capture probe is 80-120bp in length; the Tm value of the capture probe is 64-75 ℃.
6. A capture probe for gene diagnosis and typing of pheochromocytoma and paraganglioma according to claim 1,
the sequences of RET probe, SDHD probe, HRAS probe, SDHB probe, NF1 probe, EPAS1 probe, VHL probe, FGFR1 probe are as follows:
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7. kit for gene diagnosis and typing of pheochromocytomas and paragangliomas, characterized in that it comprises a capture probe according to any one of claims 1 to 6.
8. The kit for diagnosis and typing of pheochromocytoma and paraganglioma genes according to claim 7, further comprising the following individual components packaged separately:
universal library construction system: fragmenting/terminal repairing enzyme, fragmenting/terminal repairing buffer solution, ligation reaction premix solution, linker, PCR Mix and purified magnetic beads;
liquid phase hybridization system: hybridization Mix, whs-PPGL-54gene Panel, human Cot, universal Blockers, hybridization Enhancer, streptavidin Binding Beads, binding Buffer, wash Buffer1, wash Buffer2, 2X KAPA HiFi HotStart ReadyMix, amplification Primers.
9. Use of the capture probe of claim 1 or the kit of claim 7 for the preparation of a product for detecting pheochromocytoma and paraganglioma genetic variations in a sample to be tested.
10. Use of the capture probe of claim 1 or the kit of claim 7 for the preparation of a product for detecting or predicting whether a patient to be tested has pheochromocytoma and paraganglioma.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202311010584.6A CN117070624A (en) | 2023-08-11 | 2023-08-11 | Capture probe and kit for gene diagnosis and typing of pheochromocytoma and paraganglioma |
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