CN112251515B - Primer and kit for detecting thyroid cancer targeted medication related gene variation - Google Patents
Primer and kit for detecting thyroid cancer targeted medication related gene variation Download PDFInfo
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Abstract
The invention discloses a primer and a kit for detecting thyroid cancer targeted medication related gene variation. 32 site variations and 6 fusion gene variations of 14 genes closely related to thyroid cancer targeted drug application are simultaneously detected, and the site variations and the 6 fusion gene variations comprise BRAF gene, RET gene, KRAS gene, HRAS gene, NRAS gene, FLT3 gene, KIT gene, TET2 gene, PDGFR-alpha gene, LRP1B gene, PIK3CA gene, PTEN gene, MAP2K1 gene, MAP2K2 gene and RET/CCDC6 fusion, RET/NCOA4 fusion, NTRK1/TPM3 fusion, NACC2/NTRK2 fusion, NTRK2/QKI fusion and NTRK3/ETV6 fusion. The invention can help doctors select proper target drugs for thyroid cancer patients, and improves the treatment effect of thyroid cancer.
Description
Technical Field
The invention relates to the technical field of molecular biology, in particular to a primer and a kit for detecting thyroid cancer targeted medication related gene variation.
Background
Thyroid cancer is a common malignancy of the endocrine system. According to the data of Chinese tumor registration center, the incidence of thyroid cancer of female in urban areas of China is in the 4 th place of all malignant tumors of female. Thyroid cancer is classified into Differentiated Thyroid Cancer (DTC), Medullary Thyroid Cancer (MTC), and undifferentiated thyroid cancer (ATC). Various types of thyroid cancer have different degrees of malignancy, and treatment modes and prognosis are also greatly different.
Targeted therapy is an important treatment modality for patients with thyroid cancer who are resistant to radioiodine or cannot be resected by surgery. Sorafenib (sorafenib) is a small molecule Tyrosine Kinase Inhibitor (TKI) that targets VEGFRs, RET/PTC and BRAF genes simultaneously. In 2013, the U.S. Food and Drug Administration (FDA) approved sorafenib for the treatment of local recurrence or metastasis, a progressive DTC of radioiodine resistance. Vandetanib (vandetanib) is an oral small molecule TKI, can act on multiple targets such as VEGFR-1, VEGFR-2 and RET genes and the like at the same time, and becomes the first symptomatic treatment or progressive MTC targeted drug approved by FDA in 2011. Cabozantinib (cabozantinib) is another oral small molecule multi-target therapeutic drug, can act on VEGFR-1, VEGFR-2, MET and RET genes at the same time, is approved by FDA as a second targeted drug for treating advanced metastatic MTC in 2012, and has great help for treating advanced, unresectable or metastatic MTC. In 2018, the targeted drug for NTRK1-3 gene fusion, larotinib (LOXO-101), the first broad-spectrum anti-cancer drug approved by the FDA for specific gene variation, spanning tumor sites, was approved for marketing. In addition, the targeted drug against RET gene fusion and mutation, seerpatinib (LOXO-292), is FDA approved for thyroid cancers that are progressive and distant metastatic and carry RET gene fusions or mutations. A plurality of thyroid cancer diagnosis and treatment guidelines and expert consensus indicate that: the targeted therapy is an important treatment mode of refractory or late-stage thyroid cancer, can relieve the symptoms of patients and prolong the life, and recommends gene detection and target point screening to guide the selection of targeted drugs.
Therefore, the primers and the kit for detecting the genetic variation related to the targeted drug use of the thyroid cancer are invented, and the detection result can be used for assisting a doctor to select a proper targeted drug for treating a thyroid cancer patient by detecting the genetic variation related to the targeted drug use of the thyroid cancer, so that the accuracy of the selection of the targeted drug for the thyroid cancer can be improved, the symptoms of the patient can be relieved, the life cycle of the patient can be prolonged, and the primers and the kit have extremely important clinical use value.
Disclosure of Invention
The invention mainly aims to provide a primer and a kit for detecting the genetic variation related to the targeted drug for thyroid cancer with high accuracy, and provide good auxiliary action for the selection of the targeted drug for thyroid cancer.
Further, a primer for detecting thyroid cancer targeted drug related gene variation comprises the following amplification primer pairs: an amplification primer pair of a BRAF gene with a nucleotide sequence shown as SEQ ID NO.1-4, an amplification primer pair of a RET gene with a nucleotide sequence shown as SEQ ID NO.5-10, an amplification primer pair of a KRAS gene with a nucleotide sequence shown as SEQ ID NO.11-14, an amplification primer pair of a HRAS gene with a nucleotide sequence shown as SEQ ID NO.15-18, an amplification primer pair of an NRAS gene with a nucleotide sequence shown as SEQ ID NO.19-22, an amplification primer pair of a FLT3 gene with a nucleotide sequence shown as SEQ ID NO.23-24, an amplification primer pair of a KIT gene with a nucleotide sequence shown as SEQ ID NO.25-26, an amplification primer pair of a TET2 gene with a nucleotide sequence shown as SEQ ID NO.27-28, an amplification primer pair of a PDGFR-alpha gene with a nucleotide sequence shown as SEQ ID NO.29-30, an amplification primer pair of an LRP1B gene with a nucleotide sequence shown as SEQ ID NO.31-32, an amplification primer pair of PIK3CA gene having a nucleotide sequence shown in SEQ ID NO.33-34, an amplification primer pair of PTEN gene having a nucleotide sequence shown in SEQ ID NO.35-36, an amplification primer pair of MAP2K1 gene having a nucleotide sequence shown in SEQ ID NO.37-42, an amplification primer pair of MAP2K2 gene having a nucleotide sequence shown in SEQ ID NO.43-48, an amplification primer pair of NCOA4/RET fusion having a nucleotide sequence shown in SEQ ID NO.49-50, an amplification primer pair of CCDC6/RET fusion having a nucleotide sequence shown in SEQ ID NO.51-52, an amplification primer pair of NTRK1/TPM3 fusion having a nucleotide sequence shown in SEQ ID NO.53-56, an amplification primer pair of NACC2/NTRK2 fusion having a nucleotide sequence shown in SEQ ID NO.57-58, an amplification primer pair of QRK 2 fusion having a nucleotide sequence shown in SEQ ID NO.59-60, an amplification primer pair of ETV6/NTRK3 fusion with the nucleotide sequence shown in SEQ ID NO. 61-64.
The 32 DNA mutation sites of the 14 genes are respectively shown in Table 1:
TABLE 1 Gene mutation sites
The 6 gene fusion types are respectively shown in table 2:
TABLE 2 Gene fusion types
TABLE 3 primer sequence Listing
Further, a kit for detecting the gene variation related to the targeted drug administration of the thyroid cancer comprises all the primers for detecting the gene variation related to the targeted drug administration of the thyroid cancer.
Further, a kit for detecting thyroid cancer targeted drug related gene variation, which comprises a reverse transcription reagent, a library establishing reagent, a positive DNA quality control product, a positive RNA fusion quality control product, a negative RNA fusion quality control product and nuclease-free water.
Further, a kit for detecting thyroid cancer targeted drug related gene variation, wherein the Reverse transcription reagent comprises RNA Reverse Transcriptase, 5 Xbuffer, 0.1M DTT, dNTPs, RNase Inhibitor and random primer, and the commercial SuperScript III Reverse Transcriptase (Thermo Fisher) and RNaseOUT (biocompatible Nuclear Inhibitor) can be selected.
Further, the kit for detecting the gene variation related to the targeted drug administration of the thyroid cancer comprises DNA polymerase, KCl, MgCl2, Tris-HCl, dNTPs, universal primers and a joint, and can select a second-generation sequencing rapid DNA library construction kit (Illumina) sold in the market.
The positive DNA quality control product is normal human genome DNA.
The positive RNA fusion quality control product is human RET/CCDC6 fusion RNA.
The negative RNA fusion quality control material is human non-fusion RNA.
Preferably, the storage temperature of the kit is-20 ℃.
Further, the sample used is thyroid cancer solid tissue of the test subject.
Preferably, the sample used may be one or more of fresh surgical and/or punctured tissue, frozen surgical and/or punctured tissue, Formalin Fixed Paraffin Embedded (FFPE) tissue samples. More preferably, the test sample is fresh surgical tissue and/or punctured tissue.
Further, a detection method for detecting thyroid cancer targeted drug-related gene variation, which comprises the following steps:
(1) extracting DNA from a sample to be detected, and detecting the concentration and purity of the extracted DNA;
(2) taking the DNA extracted in the step (1) as a template, and taking nucleotide sequences shown in SEQ ID numbers 1-48 as primers to perform first round PCR amplification, and purifying an amplification product;
(3) taking the amplification product in the step (2) as a template, performing second round PCR amplification by using a universal primer and a joint in a library building reagent, and purifying the amplification product to obtain a DNA sequencing library;
(4) extracting purified RNA from a sample to be detected, and detecting the concentration and purity of the extracted RNA;
(5) taking the RNA extracted in the step (4) as a template, generating corresponding cDNA through reverse transcription, and purifying the cDNA;
(6) performing first round of PCR amplification by using the cDNA in the step (5) as a template and nucleotide sequences shown in SEQ ID numbers 49-64 as primers, and purifying an amplification product;
(7) taking the amplification product in the step (6) as a template, performing second round PCR amplification by using a universal primer and a joint in a library building reagent, and purifying the amplification product to obtain a cDNA sequencing library;
(8) performing quality inspection on the sequencing libraries in the steps (3) and (7), and then sequencing by using a second-generation sequencer;
(9) the sequencing results were analyzed and annotated with bioinformatic software.
Preferably, the first and second liquid crystal materials are,
the concentration of the DNA specific primer mixing pool shown by the nucleotide sequence SEQ ID NO.1-48 is 2 mu M.
The concentration of the RNA specific primer mixing pool shown by the nucleotide sequence SEQ ID NO.49-64 is 6 mu M.
The magnetic beads used for purifying the amplification products are Beckman Agencour AMPure XP magnetic beads.
The invention has the following beneficial effects:
(1) the KIT can simultaneously detect 32 mutation sites on 14 genes of BRAF gene, RET gene, KRAS gene, HRAS gene, NRAS gene, FLT3 gene, KIT gene, TET2 gene, PDGFR-alpha gene, LRP1B gene, PIK3CA gene, PTEN gene, MAP2K1 gene and MAP2K2 gene and NCOA4/RET fusion, CCDC6/RET fusion, NTRK1/TPM3 fusion, NACC2/NTRK2 fusion, QKI/NTRK2 fusion and ETV6/NTRK3 fusion, and the gene mutations and fusion variations are related to thyroid cancer targeted drug selection, so the detection result can be used for assisting a doctor to select a proper targeted drug for a thyroid cancer patient and improve the thyroid cancer treatment effect.
(2) The invention is based on a high-throughput sequencing technology, and the primer combination, the kit and the detection method thereof have the characteristics of high detection sensitivity, good specificity and good repeatability.
Drawings
FIG. 1 is a schematic diagram of the construction of the library of the kit.
FIG. 2 is a detection flow chart of the kit.
FIG. 3 is a DNA library fragment distribution diagram constructed using the kit.
FIG. 4 is a diagram showing a cDNA library fragment distribution constructed using the kit.
Fig. 5 is a depth map of BRAF gene T1799A site sequencing for 10 different clinical sample construction libraries.
Fig. 6 shows the sequencing depth of site T1799A of BRAF gene in 5 times of library construction by the same experimenter for the same clinical sample.
Fig. 7 shows the sequencing depth of site T1799A of BRAF gene in 5 times of library construction by different experimenters on the same clinical sample.
Detailed Description
The present invention may be embodied in many different forms and should not be limited to the embodiments set forth herein, which will be apparent to those of ordinary skill in the art of the present invention and may be embodied with several non-intended modifications and additions without departing from the spirit and scope thereof as hereinafter claimed.
Example 1 a primer for detecting thyroid cancer targeted drug related gene variation.
The thyroid cancer targeted drug related gene and site related to the embodiment are selected from the national and foreign thyroid cancer guideline/norm/expert consensus such as NCCN clinical practice guideline for oncology, thyroid cancer diagnosis and treatment norm and the like, and a COSMIC (catalytic of viral Mutations in cancer) database, and the targeted drug related mutation site primer design is performed according to the related gene sequence, and the design range includes drug selection related mutation in the thyroid cancer targeted drug related gene.
As shown in Table 3, 32 pairs of specific amplification primers are designed for the drug selection related mutation of the thyroid cancer targeted drug related gene in the embodiment, and the amplification target region of each pair of primers is 180-200bp in size, so that the method has the advantages of wide coverage, more detection sites, balanced GC content, stable product structure, less dimer structure and the like.
The specific primer of the embodiment shows good specificity, stability and uniformity during multiplex PCR amplification, and can ensure the amplification efficiency of the PCR amplification specificity at the same time.
The kit for detecting thyroid cancer targeted drug-related gene variation in the embodiment mainly comprises:
(1) an amplification primer: is used for amplifying a plurality of target areas on the target gene of the sample to be detected and fusing the genes. Amplifying a plurality of target regions on a target gene of a sample to be detected, wherein the amplification range at least covers a hot spot mutation region of the target gene, and the primer sequence is shown as SEQ ID NO.1-48 in Table 3; amplifying gene fusion of a sample to be detected, wherein the sequence of a primer is shown as SEQ ID NO.49-64 in the table 3;
(2) reverse transcription reagent: RNA reverse transcriptase, 5 Xbuffer solution, 0.1M DTT, dNTPs, RNase inhibitor and random primer;
(3) a library establishing reagent: DNA polymerase, KCl, MgCl2, Tris-HCl, dNTPs, universal primers and a joint;
(4) positive DNA quality control: normal human genomic DNA;
(5) positive RNA fusion quality control: human RET/CCDC6 fusion RNA;
(6) negative RNA fusion quality control: human non-fused RNA.
Preferably, the storage temperature of the kit is-20 ℃.
The detection method of the embodiment comprises the following steps:
(1) sample DNA and RNA extraction: according to the instruction, the ALPrep DNA/RNA Mini Kit is used for extracting DNA and RNA respectively, the NanoDrop is used for measuring the concentration and the purity, and according to the concentration result of the measured nucleic acid sample, the nucleic acid sample is diluted to 10-50 ng/. mu.L by using nuclease-free water to be used as the initial concentration of the nucleic acid for amplification and library building.
(2) Construction of a DNA library:
(2a) amplification of the target region of DNA:
the first round of PCR amplification was performed according to the following reaction system and amplification conditions:
| reagent | Volume of |
| DNA polymerase mixture | 12.5μL |
| Amplification primer mixing pool | 4μL |
| DNA(50-100ng) | 2μL |
| Nuclease-free water | Make up to 25. mu.L |
Setting a DNA multiplex PCR amplification program:
(2b) first round PCR product purification:
the first round of PCR products were purified using AMPure XP Beads purification kit:
1) transferring 25ul of the amplified product to a new centrifuge tube with the volume of 1.5mL, adding 22.5 mu L of AMPure XP Beads, blowing and uniformly mixing by using a pipette, and standing for 5 minutes at room temperature;
2) placing the 1.5ml centrifuge tube filled with the supernatant on a magnetic frame, standing for 5 minutes, after the solution is clarified, sucking 50ul of the supernatant out and discarding, sucking out the tube bottom residues with a 10ul gun head, keeping the magnetic beads, taking down the centrifuge tube, adding 50ul of YF Buffer B, blowing and uniformly mixing with a gun, and incubating at room temperature for 5 minutes;
3) placing the centrifugal tube on a magnetic frame, standing for 5 minutes, carefully absorbing the supernatant after the solution is clarified, and remaining the centrifugal tube containing the magnetic beads on the magnetic frame;
4) adding 180 mu L of 80% ethanol into the centrifuge tube, standing for 30 seconds, and carefully sucking the supernatant by using a pipette;
5) repeating the steps once;
6) standing for 10 minutes at room temperature until the ethanol is completely volatilized;
7) adding 24 mu L of nuclease-free water into a centrifuge tube, blowing and uniformly mixing by a pipette, fully suspending magnetic beads, and standing for 5 minutes at room temperature;
8) the centrifuge tube was placed on a magnetic rack, allowed to stand for 5 minutes, and after the solution was clarified, 13.5. mu.L of the supernatant was carefully pipetted into a new 0.2mL PCR tube for a second round of PCR amplification.
(2c) Second round PCR amplification of DNA:
the second round of PCR amplification was performed according to the following reaction system and amplification conditions:
| reagent | Volume of |
| DNA polymerase mixture | 14.5μL |
| Universal primer | 1μL |
| Joint | 1μL |
| First round PCR purified product | 13.5μL |
| Nuclease-free water | Make up to 30. mu.L |
Different sample libraries will require the use of different adaptor-numbered primers.
Setting a DNA multiplex PCR amplification program:
(2d) DNA second round PCR product recovery
The second round PCR products were purified using AMPure XP cultures purification kit:
1) uniformly mixing the 30 mu L PCR amplification product in a vortex mode and centrifuging, transferring the supernatant into a new 1.5mL centrifuge tube, adding 27 mu L AMPure XP Beads, uniformly mixing by blowing with a pipette, and standing for 5 minutes at room temperature;
2) placing the 1.5ml centrifuge tube filled with the supernatant on a magnetic frame, standing for 5 minutes, after the solution is clarified, sucking 50ul of the supernatant out and discarding, sucking out the tube bottom residues with a 10ul gun head, keeping the magnetic beads, taking down the centrifuge tube, adding 50ul of YF Buffer B, blowing and uniformly mixing with a gun, and incubating at room temperature for 5 minutes;
3) placing the centrifugal tube on a magnetic frame, standing for 5 minutes, carefully absorbing the supernatant after the solution is clarified, and remaining the centrifugal tube containing the magnetic beads on the magnetic frame;
4) adding 180 mu L of 80% ethanol into the centrifuge tube, standing for 30 seconds, and carefully sucking the supernatant by using a pipette;
5) repeating the steps once;
6) standing for 10 minutes at room temperature until the ethanol is completely volatilized;
7) adding 24 mu L of nuclease-free water into a centrifuge tube, blowing and uniformly mixing by a pipette, fully suspending magnetic beads, and standing for 5 minutes at room temperature;
8) placing the centrifuge tube on a magnetic frame, standing for 5 minutes, carefully sucking 20 mu L of supernatant into a new 1.5mL centrifuge tube after the solution is clarified, wherein the purified product is the constructed library, and performing quality inspection (figure 3) through a post-machine sequencing.
(3) constructing a cDNA library:
(3a) preparation of cDNA by reverse transcription:
reverse transcription of RNA was performed according to the following reaction system and amplification conditions:
| reagent | Volume of | |
| Random primer | 2μL | |
| Total RNA | 2μL | |
| dNTPs | 4μL | |
| Nuclease- | 5μL | |
| 5 Xbuffer | 4μL | |
| 0.1M DTT | 1μL | |
| RNase inhibitors | 1μL | |
| RNA reverse transcriptase | 1μL |
Setting an amplification program:
| temperature of | Time | Number of cycles |
| 25 ˚ |
5 |
1 |
| 50 ˚ |
60 |
1 |
| 70 ˚C | 15 |
1 |
The obtained cDNA was used as a template for amplification reaction.
(3b) Amplification of the cDNA target region:
PCR amplification was performed according to the following reaction system and amplification conditions:
| reagent | Volume of |
| DNA polymerase mixture | 12.5μL |
| Amplification primer pool | 2μL |
| cDNA template | 2μL |
| Nuclease-free water | Make up to 25. mu.L |
Setting a multiplex PCR amplification program:
(3c) first round PCR product purification of cDNA:
and (3) purifying the first round PCR amplification product of the cDNA by using an AMPure XP Beads purification kit:
1) adding water to the 25 mu L PCR amplification product to 50 mu L, uniformly mixing by vortex, centrifuging, transferring the supernatant into a new 1.5mL centrifuge tube, adding 30 mu L AMPure XP Beads, uniformly mixing by blowing with a pipette, and standing for 5 minutes at room temperature;
2) placing the 1.5ml centrifuge tube with the supernatant on a magnetic frame, standing for 5 minutes, after the solution is clarified, sucking out all the supernatant in the tube into a new tube, adding 35ul of magnetic beads, blowing and uniformly mixing by using a gun, and incubating for 5min at room temperature;
3) placing the centrifuge tube on a magnetic frame, opening the cover, standing at room temperature for 5min, sucking out the supernatant, discarding, and keeping the magnetic beads;
4) adding 200 μ L of 80% ethanol into the centrifuge tube, standing for 30 s, and carefully sucking off the supernatant with a pipette;
5) repeating the steps once;
6) standing for 10 minutes at room temperature until the ethanol is completely volatilized;
7) adding 20 mu L of nuclease-free water into a centrifuge tube, blowing and uniformly mixing by a pipette, fully suspending magnetic beads, and standing for 5 minutes at room temperature;
8) the centrifuge tube was placed on a magnetic rack, allowed to stand for 5 minutes, and after the solution was clarified, 17. mu.L of the supernatant was carefully pipetted into a new 0.2mL PCR tube for a second round of PCR amplification.
(3d) Second round of cDNA PCR amplification:
the second round of PCR amplification was performed according to the following reaction system and amplification conditions:
| reagent | Volume of |
| DNA polymerase mixture | 25μL |
| Universal primer | 1μL |
| Joint | 1μL |
| First round PCR purified product | 17μL |
| Nuclease-free water | Make up to 50. mu.L |
Different sample libraries will require the use of different adaptor-numbered primers.
Setting a cDNA multiplex PCR amplification program:
(3e) second round cDNA PCR product recovery
The second round PCR products were purified using AMPure XP cultures purification kit:
1) vortexing, uniformly mixing and centrifuging the 50 mu L of amplification product, transferring the supernatant into a new 1.5mL centrifuge tube, adding 27.5 mu L of AMPure XP Beads, blowing and uniformly mixing by using a pipette, and standing for 5 minutes at room temperature;
2) placing the 1.5ml centrifuge tube with the supernatant on a magnetic frame, standing for 5 minutes, after the solution is clarified, sucking out all the supernatant in the tube into a new tube, adding 15ul of magnetic beads, blowing and uniformly mixing by using a gun, and incubating for 5min at room temperature;
3) placing the centrifuge tube on a magnetic frame, opening the cover, standing at room temperature for 5min, sucking out the supernatant, discarding, and keeping the magnetic beads;
4) adding 200 μ L of 80% ethanol into the centrifuge tube, standing for 30 s, and carefully sucking off the supernatant with a pipette;
5) repeating the steps once;
6) standing at room temperature for 10 minutes to completely volatilize ethanol;
7) adding 20 mu L of nuclease-free water into a centrifuge tube, blowing and uniformly mixing by a pipette, fully suspending magnetic beads, and standing for 5 minutes at room temperature;
8) placing the centrifuge tube on a magnetic frame, standing for 5 minutes, carefully sucking 18 mu L of supernatant into a new 1.5mL centrifuge tube after the solution is clarified, wherein the purified product is the constructed library, and performing quality inspection (figure 4) through a post-machine sequencing.
(4) And (3) machine sequencing: and (3) accurately quantifying the constructed library, diluting the library to a proper concentration, and performing on-machine sequencing by referring to an official operating instruction of an Illumina company.
(5) And (3) letter generation analysis: and analyzing and annotating the sequencing result by using biological information software.
Example 4 sequencing depth performance verification of a kit for detecting thyroid cancer targeted drug-related genetic variation.
By using the kit, 10 thyroid cancer samples were detected, and the detection method was performed according to the procedure in example 3. The detection results of 10 samples show that the detection depth of the kit of the invention is good for different samples, for example, the detection depth of the site T1799A of the BRAF gene in 10 samples is respectively: 3935.15, 3179.52, 4102.31, 4851.93, 3910.39, 5010.91, 4651.79, 5710.71, 3726.91 and 4910.81 (figure 5), the lowest sequencing depth 3179.52 and the highest sequencing depth 5710.71 all reach the performance index that the lowest sequencing depth set by the kit is more than 1000, and the accuracy of the sequencing result is ensured.
Example 5 a repetitive experiment of a kit for detecting thyroid cancer targeted drug-related gene variation.
The kit of the invention is used for carrying out 5 times of detection on thyroid cancer samples of the same example 1 by the same technical personnel in a laboratory, the detection method is carried out according to the steps of the example 3, in the 5 times of detection, the sequencing depths of the BRAF gene T1799A site are taken as examples, the sequencing depths are respectively 3821.31, 3590.97, 3901.21, 3619.96 and 3489.13 (figure 6), the mutation of the BRAF gene T1799A site can be stably detected, the detected mutation frequencies are respectively 30.12%, 31.96%, 29.61%, 31.87% and 30.91%, and the detection conditions of other sites are similar. Therefore, the kit provided by the invention has good detection repeatability for the same sample.
Example 6 a reproducibility experiment of a kit for detecting gene variation related to targeted drug administration for thyroid cancer.
The kit provided by the invention is used for detecting the same 1 sample of thyroid cancer by 5 different experimenters in a laboratory according to the steps of the embodiment 3, taking the detection result of the BRAF gene T1799A as an example, the sequencing depths of the positions are 4001.34, 3898.97, 3711.91, 3529.13 and 3619.18 (figure 7), the mutation of the BRAF gene T1799A position can be stably detected, and the detected mutation frequencies are 23.90%, 25.89%, 23.42%, 27.13% and 24.19%. The results show that the detection conditions of other sites are similar. Therefore, the detection results of different experiment operators using the kit are good for the same sample. The kit has good detection reproducibility.
Finally, the above-mentioned embodiments are only preferred embodiments of the present invention, and do not limit the technical solutions of the present invention. The present invention is not intended to be limited to the embodiments shown herein but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.
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<213> Artificial Sequence (Artificial Sequence)
<400> 7
gctcttccga tctctgacga ctcgtgctat ttttcctca 39
<210> 8
<211> 38
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 8
gctcttccga tctccttggg aagcctagga aagatacc 38
<210> 9
<211> 37
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 9
gctcttccga tctacagcac tcctctggtt actgaaa 37
<210> 10
<211> 36
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 10
gctcttccga tctttataca tgtagtgggg ccacga 36
<210> 11
<211> 35
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 11
gctcttccga tctggtcctg caccagtaat atgca 35
<210> 12
<211> 34
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 12
gctcttccga tctcagtcat tttcagcagg cctt 34
<210> 13
<211> 47
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 13
gctcttccga tctaattact ccttaatgtc agcttattat attcaat 47
<210> 14
<211> 41
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 14
gctcttccga tctgagaaac acagtctgga ttattacagt g 41
<210> 15
<211> 36
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 15
gctcttccga tctatagtgg ggtcgtattc gtccac 36
<210> 16
<211> 37
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 16
gctcttccga tctgccatct gaagggcaaa cccacag 37
<210> 17
<211> 33
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 17
gctcttccga tctcggtgcg catgtactgg tcc 33
<210> 18
<211> 37
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 18
gctcttccga tctgggactc ccctcctcta gaggaag 37
<210> 19
<211> 38
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 19
gctcttccga tctatccgac aagtgagaga caggatca 38
<210> 20
<211> 38
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 20
gctcttccga tctcaccagc aagaacctgt tggaaacc 38
<210> 21
<211> 33
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 21
gctcttccga tctcggtgcg catgtactgg tcc 33
<210> 22
<211> 38
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 22
gctcttccga tcttcaagcc catttctgcc tatctggt 38
<210> 23
<211> 33
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 23
gctcttccga tctaatgcac cacagtgagt gca 33
<210> 24
<211> 37
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 24
gctcttccga tctgccaggt ctctgtgaac acactgt 37
<210> 25
<211> 39
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 25
gctcttccga tcttggtttt cttttctcct ccaacctaa 39
<210> 26
<211> 37
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 26
gctcttccga tctacccatt ctctgcttga cagtcct 37
<210> 27
<211> 38
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 27
gctcttccga tctgagggca acagactaag tccattcc 38
<210> 28
<211> 36
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 28
gctcttccga tctaagccag aatagtcgtg tgagtc 36
<210> 29
<211> 37
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 29
gctcttccga tctgctacag atggcttgat cctgagt 37
<210> 30
<211> 38
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 30
gctcttccga tctttgcttc tagagattcg gtgcctgt 38
<210> 31
<211> 37
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 31
gctcttccga tctgcttgtg ctgctcacat acttggc 37
<210> 32
<211> 37
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 32
gctcttccga tcttctatgg agtcacaggt aagtgct 37
<210> 33
<211> 35
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 33
gctcttccga tctcatctgt gaatccagag gggaa 35
<210> 34
<211> 37
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 34
gctcttccga tcttctatgg agtcacaggt aagtgct 37
<210> 35
<211> 36
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 35
gctcttccga tctagaccat aacccaccac agctag 36
<210> 36
<211> 37
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 36
gctcttccga tcttgttttt cctttcctct tcctgga 37
<210> 37
<211> 35
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 37
gctcttccga tctgtattga cttgtgctcc ccact 35
<210> 38
<211> 38
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 38
gctcttccga tcttggccag aaaggtgagt ttgccttg 38
<210> 39
<211> 36
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 39
gctcttccga tctgatcata agggagctgc aggttc 36
<210> 40
<211> 35
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 40
gctcttccga tctaatcttt ggtctgggag gtgac 35
<210> 41
<211> 36
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 41
gctcttccga tcttggtctg tgtggaatgc tgatcc 36
<210> 42
<211> 36
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 42
gctcttccga tcttgcttga gcttcttgta cggtca 36
<210> 43
<211> 36
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 43
gctcttccga tctccagctc tgagatcctt tcgaag 36
<210> 44
<211> 37
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 44
gctcttccga tctacacaga ctggattcct gcacttg 37
<210> 45
<211> 35
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 45
gctcttccga tctaaatgct gatctccccg tcact 35
<210> 46
<211> 37
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 46
gctcttccga tctgttccca acagtggtca agaccaa 37
<210> 47
<211> 35
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 47
gctcttccga tctacatggg ggtgagagct gaggg 35
<210> 48
<211> 35
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 48
gctcttccga tctatgttgg agggcttcac atctg 35
<210> 49
<211> 36
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 49
gctcttccga tctcagtggt tatcaagctc cttaca 36
<210> 50
<211> 32
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 50
gctcttccga tctaccctgc tctgcctttc ag 32
<210> 51
<211> 33
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 51
gctcttccga tctaggagga gaaccgcgac ctg 33
<210> 52
<211> 31
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 52
gctcttccga tctgccgttg ccttgaccac t 31
<210> 53
<211> 35
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 53
gctcttccga tctgtcaaca acggcaacta cacgc 35
<210> 54
<211> 35
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 54
gctcttccga tctctcattc aggtcaagca gggtc 35
<210> 55
<211> 31
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 55
gctcttccga tctcggtgga gaagaaggac g 31
<210> 56
<211> 31
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 56
gctcttccga tcttcagcaa actcagcacg g 31
<210> 57
<211> 31
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 57
gctcttccga tctactggca tccgctcgtc c 31
<210> 58
<211> 35
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 58
gctcttccga tctttacttc tgttcgtggt gtccc 35
<210> 59
<211> 31
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 59
gctcttccga tcttcctcac ccaactgctg c 31
<210> 60
<211> 34
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 60
gctcttccga tcttgacagg gatcttggtc attc 34
<210> 61
<211> 33
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 61
gctcttccga tctggaaggg aagcccatca acc 33
<210> 62
<211> 33
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 62
gctcttccga tctttgtagc actcggccag gaa 33
<210> 63
<211> 34
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 63
gctcttccga tctttttcac cattcttcca ccct 34
<210> 64
<211> 31
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 64
gctcttccga tctggcatcc agtgacgagg g 31
Claims (5)
1. A primer group for detecting thyroid cancer targeted drug related gene variation is characterized by consisting of nucleic acids shown in SEQ ID NO 1-64, and being used for specifically amplifying BRAF gene, RET gene, KRAS gene, HRAS gene, NRAS gene, FLT3 gene, KIT gene, TET2 gene, PDGFR-alpha gene, LRP1B gene, PIK3CA gene, PTEN gene, MAP2K1 gene and MAP2K2 gene variation of 32 sites in 14 genes in a sample, RET/NCOA4 fusion, RET/CCDC6 fusion, NTRK 1/3 fusion, NACC2/NTRK2 fusion, NTRK2/QKI fusion and NTRK3/ETV6 fusion of the 6 fusion variations.
2. A kit for detecting thyroid cancer targeted drug related gene variation, which comprises the primer set for detecting thyroid cancer targeted drug related gene variation according to claim 1.
3. The kit of claim 2, further comprising a reverse transcription reagent, a library construction reagent, a positive DNA quality control, a positive RNA fusion quality control, a negative RNA fusion quality control, and nuclease-free water.
4. The kit of claim 3, wherein the reverse transcription reagent comprises RNA reverse transcriptase, 5 x buffer, 0.1M DTT, dNTPs, RNase inhibitor, random primer, and the banking reagent comprises DNA polymerase, KCl, MgCl2The positive DNA quality control material is normal human genome DNA, the positive RNA fusion quality control material is human RET/CCDC6 fusion RNA, and the negative RNA fusion quality control material is human non-fusion RNA.
5. The kit of claim 2, wherein the solid thyroid cancer tissue is selected from the group consisting of fresh surgical tissue, fresh punctured tissue, frozen surgical tissue, frozen punctured tissue, and formalin-fixed paraffin-embedded (FFPE) tissue.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202011538550.0A CN112251515B (en) | 2020-12-23 | 2020-12-23 | Primer and kit for detecting thyroid cancer targeted medication related gene variation |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202011538550.0A CN112251515B (en) | 2020-12-23 | 2020-12-23 | Primer and kit for detecting thyroid cancer targeted medication related gene variation |
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| CN112251515A CN112251515A (en) | 2021-01-22 |
| CN112251515B true CN112251515B (en) | 2021-03-12 |
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| Application Number | Title | Priority Date | Filing Date |
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| CN202011538550.0A Active CN112251515B (en) | 2020-12-23 | 2020-12-23 | Primer and kit for detecting thyroid cancer targeted medication related gene variation |
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| CN113403397B (en) * | 2021-07-15 | 2024-03-08 | 西安医臻生物医药科技有限公司 | Primer set and kit for detecting melanoma targeted drug-related gene mutation |
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| CN109371139A (en) * | 2018-12-29 | 2019-02-22 | 杭州迪安医学检验中心有限公司 | A kind of primer and its application being used to detect the variation of thyroid cancer pathogenic related gene based on high throughput sequencing technologies |
| CN110013555B (en) * | 2019-04-18 | 2021-07-23 | 山东大学齐鲁医院 | Application of LRP11 as target in preparation of product for treating cervical cancer |
| CN110904235A (en) * | 2019-12-20 | 2020-03-24 | 深圳市新合生物医疗科技有限公司 | Gene panel for detecting tumor targeted drug related gene mutation, method, application and kit |
| CN111088365B (en) * | 2020-03-23 | 2020-06-19 | 上海润安医学科技有限公司 | Primer and kit for detecting thyroid cancer RET gene mutation and fusion |
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