CN110241215B - Primer and kit for detecting benign and malignant genetic variation of thyroid nodule - Google Patents
Primer and kit for detecting benign and malignant genetic variation of thyroid nodule Download PDFInfo
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Abstract
The invention discloses a primer and a kit for detecting benign and malignant genetic variation of thyroid nodules. The invention can simultaneously detect the variation of 15 sites of 6 genes and the variation of 3 fusion genes, wherein the variation comprises BRAF gene, KRAS gene, HRAS gene, NRAS gene, TERT gene, EIF1AX gene, RET/PTC1 fusion, RET/PTC3 fusion and PAX8/PPAR gamma fusion. The hot spot mutation and fusion variation of the genes are closely related to the benign and malignant nature of thyroid nodules. Therefore, the kit is used for detecting a sample of a patient, can assist a doctor in performing benign and malignant identification on thyroid nodules which cannot be diagnosed clearly in cytology, improves the accuracy of benign and malignant identification of the thyroid nodules, reduces the over-treatment of the thyroid nodule patients, saves medical resources for the country, and undoubtedly has very important practical significance and great economic benefit.
Description
Technical Field
The invention relates to the technical field of molecular biology, in particular to a primer and a kit for detecting benign and malignant related gene variation of thyroid nodules.
Background
Thyroid nodules are a thyroid disease commonly existing in people and are a pathological change caused by local abnormal growth of thyroid cells. In most cases, thyroid nodules are benign and can be treated conservatively, but 5% -10% are malignant and require early surgical treatment to obtain a good prognosis.
At present, the type-B ultrasonic is a preferred means for examining thyroid nodules, and the nature of the nodules can be preliminarily judged according to the imaging characteristics of the thyroid nodules, so that the detection rate of the thyroid nodules can reach 76%, in the detected thyroid nodules, the proportion of benign nodules such as nodular goiter and thyroid adenoma is about 85% -95%, and only 5% -15% of the nodules are malignant nodules, namely thyroid cancer, and the operation treatment of patients with each thyroid nodule cannot be carried out or cannot be carried out. Therefore, in order to further improve the detection rate of thyroid cancer, thyroid cells are obtained by a fine needle puncture (FNA) method, cytological examination is the current gold standard for assessing whether thyroid nodules are malignant tumors, and even then, 20% -30% of thyroid nodules cannot be clearly diagnosed in this way.
With the development of molecular biology techniques, molecular diagnosis has been receiving attention in the differential diagnosis of benign and malignant thyroid nodules. The clinical practice guidelines of the united states National Comprehensive Cancer Network (NCCN)2019 edition specify: (1) for suspected follicular and Hurthle cell carcinoma (diagnosed by infiltration of blood vessels or envelopes) which cannot be confirmed by fine needle puncture (FNA), molecular detection is helpful for judging whether the cancer is benign or malignant; (2) the method is characterized in that the nodules which cannot be diagnosed clearly in cytology have an important effect on auxiliary diagnosis of thyroid cancer by detecting the variation of BRAF, RAS, RET/PTC, PAX8/PPAR gamma and other oncogenes by utilizing a molecular diagnosis technology.
Therefore, the primers and the kit for detecting the benign and malignant genetic variation of the thyroid nodule are invented, the detection result can be used for assisting a doctor to identify the benign and malignant thyroid nodule which cannot be diagnosed clearly in cytology by detecting the benign and malignant genetic variation of a group of thyroid nodule, the accuracy of the benign and malignant thyroid nodule identification can be improved, the over-treatment of a thyroid nodule patient can be reduced, the medical resources can be saved for the country, various sequelae produced by unnecessary treatment can be reduced for the patient, and the primers and the kit have very important practical significance and huge economic benefit undoubtedly.
Disclosure of Invention
The invention mainly aims to provide a primer and a kit for detecting the benign and malignant related gene variation of thyroid nodules, which have high accuracy, good sensitivity and good specificity, and provide a good auxiliary effect for identifying the benign and malignant thyroid nodules.
Further, a primer for detecting thyroid nodule benign and malignant related gene variation comprises the following amplification primer pairs: an amplification primer pair of BRAF gene with nucleotide sequences shown as SEQ ID NO.1 and SEQ ID NO.2, an amplification primer pair of KRAS gene with nucleotide sequences shown as SEQ ID NO.3, SEQ ID NO.4, SEQ ID NO.5 and SEQ ID NO.6, an amplification primer pair of HRAS gene with nucleotide sequences shown as SEQ ID NO.7, SEQ ID NO.8, SEQ ID NO.9 and SEQ ID NO.10, an amplification primer pair of NRAS gene with nucleotide sequences shown as SEQ ID NO.11, SEQ ID NO.12, SEQ ID NO.13 and SEQ ID NO.14, an amplification primer pair of TERT gene with nucleotide sequences shown as SEQ ID NO.15 and SEQ ID NO.16, an amplification primer pair of EIF1AX gene with nucleotide sequences shown as SEQ ID NO.17 and SEQ ID NO.18, SEQ ID NO.19 and SEQ ID NO.20, and a PTC primer pair fusion of the BRAF 1/REF 1 with nucleotide sequences shown as SEQ ID NO.21 and SEQ ID NO.22, the primer pair for amplifying RET/PTC3 fusion gene with nucleotide sequences shown as SEQ ID NO.23 and SEQ ID NO.24, the primer pair for amplifying PAX8/PPAR gamma fusion gene with nucleotides shown as SEQ ID NO.25, SEQ ID NO.26, SEQ ID NO.27, SEQ ID NO.28 and SEQ ID NO.29 and the primer pair for universal sequencing with nucleotide sequences shown as SEQ ID NO.30 and SEQ ID NO.31 can be used for specifically detecting the variation of 15 mutation sites on 6 genes of BRAF gene, HRAS gene, KRAS gene, NRAS gene, TERT gene and EIF1AX gene in a sample and 3 fusion variations of RET/PTC1 fusion, RET/PTC3 fusion and PAX 8/gamma fusion.
The 15 mutation sites are respectively shown in table 1:
TABLE 1 Gene name and mutation site Table
The 3 fusion genes are respectively shown in table 2:
TABLE 2 fusion Gene name and fusion location Table
Further, a kit for detecting thyroid nodule benign and malignant related gene variation comprises all the primers for detecting thyroid nodule benign and malignant related gene variation.
Further, a kit for detecting benign and malignant genetic variation of thyroid nodules comprises a reverse transcription reaction solution, a PCR reaction solution, 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 nodule benign and malignant related gene variation, wherein the reverse transcription buffer solution system comprises: RNA reverse transcriptase, 5 Xbuffer solution, DTT, dNTPs and RNase inhibitor.
The PCR reaction solution contains high-fidelity DNA polymerase, KCl, MgCl2, Tris-HCl and dNTPs.
The positive DNA quality control product is normal human genome DNA.
The positive RNA fusion quality control product is RET/PTC1 fusion RNA.
The negative RNA fusion quality control material is human non-fusion RNA.
Preferably, the storage temperature of the kit is-20 ℃.
Further, a kit for detecting benign and malignant related gene variation of thyroid nodule, wherein the detection method 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 the nucleotide sequence shown in SEQ ID NO. 1-20 as a primer to perform a first round of PCR amplification, and purifying an amplification product;
(3) performing second-round PCR amplification by using the amplification product in the step (2) as a template and using the nucleotide sequence shown in SEQ ID NO. 30-31 as a primer, 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 a first round of PCR amplification by using the cDNA in the step (5) as a template and using the nucleotide sequence shown in SEQ ID NO. 21-29 as a primer, and purifying an amplification product;
(7) taking the amplification product in the step (6) as a template, and using the sequence shown in SEQ ID No.: taking the nucleotide sequence shown by 30-31 as a primer, carrying out second-round PCR amplification, and purifying an amplification product to obtain an RNA 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. 1-20 is 10 mu M.
The concentration of the RNA specific primer mixing pool shown by the nucleotide sequence SEQ ID NO. 21-29 is 10 mu M.
The concentration of the universal primer mixing pool shown by the nucleotide sequence SEQ ID NO. 30-31 is 10 mu M.
The magnetic bead used for purifying the PCR amplification product is a Beckman Agencour AMPure XP magnetic bead.
The invention has the following beneficial effects:
(1) the invention can simultaneously detect 15 mutation sites on 6 genes of BRAF gene, KRAS gene, HRAS gene, NRAS gene, TERT gene and EIF1AX gene and RET/PTC1, RET/PTC3 and PAX8/PPAR gamma 3 fusion genes, and the gene mutations and fusion variations are related to the benign and malignant of thyroid gland, so the detection result can be used for assisting doctors to identify the benign and malignant of thyroid nodules which cannot be clearly diagnosed in cytology, and the accuracy of the benign and malignant identification of the thyroid nodules can be improved.
(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.
Figure 5 is a depth map of sequencing of 10 different clinical sample-constructed libraries.
FIG. 6 shows the sequencing depth of 5 libraries constructed from the same clinical specimen by the same experimenter.
FIG. 7 shows the sequencing depth of 5 libraries constructed by different experimenters on the same clinical specimen.
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 1a primer for detecting benign and malignant genetic variation of thyroid nodule.
The thyroid nodule benign and malignant related gene and the site related to the embodiment are selected from a COSMIC (catalytic of genetic Mutations in Cancer) database, mutation hotspot primer design is carried out according to the related gene sequence, and the design range comprises mutation hotspots in the thyroid nodule benign and malignant related gene.
As shown in Table 3, 16 pairs of specific primers are designed for the hot spot mutation of the benign and malignant related genes of the thyroid nodule in the embodiment, the amplification target region size of each pair of specific primers is 180-200bp, and the amplification product size is 220-280bp, so that the kit has the advantages of wide coverage range, more detection sites, balanced GC content, stable product structure, few dimer structure and the like. In particular, the method comprises the following steps of,
TABLE 3 primer sequence Listing
Wherein i5 and i7 are 6 base sequences, respectively, and the index numbers are different, and the 6 base sequences are also different, so that different samples can be distinguished.
In this example, the 5' end of each amplification primer pair is connected with a sequence fragment with a length of 13bp which is completely complementary with the universal primer. The sequences of the two primers of the universal primer pair are shown as SEQ ID NO.30 and SEQ ID NO. 31.
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.
Example 2a kit for detecting benign and malignant genetic variation of thyroid nodule.
The kit for detecting benign and malignant related gene variation of thyroid nodules in the embodiment mainly comprises:
(1) PCR specific primers: the method is used for amplifying a plurality of target regions on a target gene of a sample to be detected, the amplification range at least covers a hot spot mutation region of the target gene, and the sequences are shown as SEQ ID NO.1 and SEQ ID NO.29 in Table 3. Preferably, the multiple amplification primer pairs are mixed together to form a primer pool;
(2) the general primer is as follows: the method is used for amplifying the amplification product of the specific primer amplification target region again in the library construction process, marking the sequencing libraries of different samples to be detected and further distinguishing different samples, and the sequences are shown as SEQ ID NO.30 and SEQ ID NO.31 in the table 3;
(3) reverse transcription buffer: comprises RNA reverse transcriptase, 5 Xbuffer solution, DTT, dNTPs and RNase inhibitor;
(4) PCR reaction solution: comprises high-fidelity DNA polymerase, KCl, MgCl2, Tris-HCl and dNTPs;
(5) positive DNA quality control: normal human genomic DNA;
(6) positive RNA fusion quality control: human RET/PTC1 fusion RNA;
(7) negative RNA fusion quality control: human non-fused RNA.
Preferably, the storage temperature of the kit is-20 ℃.
Example 3a method for detecting a kit for detecting benign and malignant genetic variation of thyroid nodule.
The detection method of the embodiment comprises the following steps:
(1) sample DNA and RNA extraction: DNA and RNA were extracted using the AllPrep DNA/RNA Mini Kit, the concentration and purity were measured using NanoDrop, and the nucleic acid samples were diluted to 10-50 ng/. mu.L using nuclease-free water as the initial concentration of nucleic acid for amplification and pooling, according to the results of the concentration of nucleic acid samples measured, with reference to the Kit instructions.
(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:
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, according to kit instructions:
1) uniformly mixing the 25 mu L of PCR amplification product by vortex, centrifuging, transferring the supernatant into a new 1.5mL centrifuge tube, adding 17.5 mu L of AMPure XP Beads, uniformly blowing 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, carefully sucking the supernatant after the solution is clarified, transferring the supernatant into another new 1.5mL centrifuge tube, adding 15 mu L of AMPure XP Beads, blowing and uniformly mixing by using a pipettor, and standing for 5 minutes at room temperature;
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 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 12 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, 10. 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:
different sample libraries require the use of different index-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 according to kit instructions:
1) mixing the 50 mu L PCR amplification products evenly by vortex and centrifuging, transferring the supernatant into a new 1.5mL centrifuge tube, adding 27.5 mu L AMPure XP Beads, blowing and mixing evenly 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, carefully sucking the supernatant and transferring the supernatant to a new 1.5mL centrifuge tube, adding 15 mu L of AMPure XP Beads, blowing and uniformly mixing by using a pipettor, and standing for 5 minutes at room temperature;
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 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 22 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) and (3) 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 sequencing on the centrifuge tube after quality inspection.
(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:
setting an amplification program:
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:
setting a multiplex PCR amplification program:
(3c) first round PCR product purification of cDNA:
and (3) purifying the first round of cDNA PCR amplification products by using an AMPure XP Beads purification kit according to the kit specification:
1) uniformly mixing the 25 mu L of PCR amplification product by vortex, centrifuging, transferring the supernatant into a new 1.5mL centrifuge tube, adding 25 mu L of AMPure XP Beads, uniformly blowing 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, carefully absorbing the supernatant, and still keeping the centrifuge tube containing the magnetic beads on the magnetic frame;
3) adding 200 μ L of 80% ethanol into the centrifuge tube, standing for 30 s, and carefully sucking off the supernatant with a pipette;
4) repeating the steps once;
5) standing for 10 minutes at room temperature until the ethanol is completely volatilized;
6) adding 12 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;
7) the centrifuge tube was placed on a magnetic rack, allowed to stand for 5 minutes, and after the solution was clarified, 10. 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:
different sample libraries require the use of different index-numbered primers.
Setting a DNA multiplex PCR amplification program:
(3e) second round cDNA PCR product recovery
The second round PCR products were purified using AMPure XP cultures purification kit according to kit instructions:
1) vortexing, uniformly mixing and centrifuging the 50 mu L of amplification product, transferring the supernatant into a new 1.5mL centrifuge tube, adding 40 mu L of AMPure XP Beads, blowing and uniformly mixing by using a pipettor, 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, carefully absorbing the supernatant, and remaining the centrifuge tube containing the magnetic beads on the magnetic frame;
3) adding 200 μ L of 80% ethanol into the centrifuge tube, standing for 30 s, and carefully sucking off the supernatant with a pipette;
4) repeating the steps once;
5) standing at room temperature for 10 minutes to completely volatilize ethanol;
6) adding 22 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;
7) and (3) 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 sequencing on the centrifuge tube after quality inspection.
(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 validation of a kit for detecting benign and malignant thyroid nodule associated gene variation.
The kit provided by the invention is used for detecting 10 thyroid cancer samples provided by the head and neck surgery of the affiliated tumor hospital of Shanghai Compound denier university, and the detection method is carried out according to the steps of the embodiment 3. The detection results of 10 samples show that the kit of the invention has good detection depth for different samples, such as BRAF gene T1799A site, and the detection depths in 10 samples are respectively: 9834.93, 5915.42, 4201.23, 3713.57, 6742.76, 11361.92, 5752.67, 27824.62, 8728.12 and 33962.28 (figure 5), the lowest sequencing depth 3713.57 and the highest sequencing depth 33962.28 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 benign and malignant genetic variation of thyroid nodules.
The kit provided by the invention is used for 5 detections of thyroid cancer samples of the same 1 case provided by the head and neck surgery of the subsidiary tumor hospital of Shanghai Compound Dane university by the same technician in a laboratory, the detection method is carried out according to the steps of example 3, in the 5 detections, the sequencing depths of the T1799A site of the BRAF gene are 37824.62, 35531.44, 39144.74, 38655.30 and 34960.28 (figure 6), the mutation of the T1799A site of the BRAF gene can be stably detected, the detected mutation frequencies are 40.09%, 40.2%, 39.9%, 39.2% and 40.3%, 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 benign and malignant genetic variation of thyroid nodule.
The kit provided by the invention is used for detecting the same thyroid cancer samples 1 provided by the head and neck surgery of the subsidiary tumor hospital of Shanghai Compound Dane university by 5 different laboratory personnel according to the steps of example 3, taking the detection result of the BRAF gene T1799A site as an example, the sequencing depths of the site are 41420.42, 23812.79, 47981.92, 47522.67 and 36519.78 (figure 7), the mutation of the BRAF gene T1799A site can be stably detected, and the detected mutation frequencies are 27.11%, 26.65%, 27.34%, 26.92% and 26.25% respectively. 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.
Example 7 the performance of the kit of the invention was tested by retrospective experiments on 50 clinical samples.
By using the kit, thyroid samples of 50 patients provided by head and neck surgery of subsidiary tumor hospital of Shanghai Compound denier university are detected, and the detection method is performed according to the steps of example 3.
Specifically, the 50 samples are extracted and purified through DNA and RNA, a library is constructed, a sample library is sequenced through a NovaSeq high-throughput sequencing platform of Illumina company, the sequencing read length is PE150, the average sequencing depth of a sequencing result is required to be larger than 1000 and reaches 95%, and unqualified samples are subjected to resequencing.
Analyzing the sequencing result, judging whether the thyroid nodule of each patient is benign or malignant, comparing the judging result with the pathological detection result of the patient, and comparing the difference between the results. Specifically, as shown in table 4:
TABLE 4 comparison table of test results of patient sample kit and clinical pathological results of patient
For the 50 clinical thyroid nodule patients, the detection result of the kit is compared with the clinical actual pathological detection result, and the statistical method is applied to calculate the following results: the sensitivity of the kit reaches 81.8%, the specificity reaches 92.9%, the positive predictive value reaches 90%, the negative predictive value reaches 86.7%, and the accuracy reaches 88%, so that the kit can become a very important reference basis for a clinician to identify the benign and malignant thyroid nodules.
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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gctcttccga tctcctgggg ctgggcctg 29
<210>9
<211>29
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>9
gctcttccga tctgacgcag ccggcctgg 29
<210>10
<211>33
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>10
gctcttccga tctggtccct gagccctgtc ctc 33
<210>11
<211>37
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>11
gctcttccga tctccgacaa gtgagagaca ggatcag 37
<210>12
<211>36
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>12
gctcttccga tctgctcgcc aattaaccct gattac 36
<210>13
<211>41
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>13
gctcttccga tctatccttt cagagaaaat aatgctccta g 41
<210>14
<211>32
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>14
gctcttccga tcttccctcc ctgccccctt ac 32
<210>15
<211>30
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>15
gctcttccga tctcagggag cgcacggctc 30
<210>16
<211>30
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>16
gctcttccga tctactgggg acccgggcac 30
<210>17
<211>40
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>17
gctcttccga tcttctccat ggataataac aaaaattgga 40
<210>18
<211>46
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>18
gctcttccga tctgagtaaa ttacagtgct gacttatgag tatttc 46
<210>19
<211>42
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>19
gctcttccga tctaagaaag gatgttattt taaaaagcgt aa 42
<210>20
<211>47
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>20
gctcttccga tctcatactg ttttacagat aattaatgtc atttacc 47
<210>21
<211>34
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>21
gctcttccga tctgcattgt catctcgccg ttcc 34
<210>22
<211>36
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>22
gctcttccga tctctgctct gcctttcaga tggaag 36
<210>23
<211>38
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>23
gctcttccga tctaagtcag catccggtat tgtagctg 38
<210>24
<211>36
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>24
gctcttccga tctgtgtacc ctgctctgcc tttcag 36
<210>25
<211>32
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>25
gctcttccga tctcatgcct gcgatggcag ag 32
<210>26
<211>38
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>26
gctcttccga tctgtaggtc tggtgagtcg agaggttg 38
<210>27
<211>37
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>27
ctcttccgat ctaaggcgga gctagataaa gaggaag 37
<210>28
<211>33
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>28
gctcttccga tctcgaggtg gtgctggctg aag 33
<210>29
<211>38
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>29
gctcttccga tcttggatct gttcttgtga atggaatg 38
<210>30
<211>62
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>30
aatgatacgg cgaccaccga gatctacaca cactctttcc ctacacgacg ctcttccgat 60
ct 62
<210>31
<211>58
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>31
caagcagaag acggcatacg agatgtgact ggagttcaga cgtgtgctct tccgatct 58
Claims (4)
1. A primer group for detecting thyroid nodule benign and malignant related gene variation is characterized by consisting of nucleotide sequences shown in SEQ ID NO. 1-31, and being used for specifically detecting variation of 15 sites of 6 genes including BRAF gene, KRAS gene, HRAS gene, NRAS gene, TERT gene and EIF1AX gene in a sample and 3 fusion variations including RET/PTC1 fusion, RET/PTC3 fusion and PAX8/PPAR gamma fusion.
2. A kit for detecting thyroid nodule benign and malignant related gene variation, comprising the primer set for detecting thyroid nodule benign and malignant related gene variation according to claim 1.
3. The kit of claim 2, further comprising a reverse transcription reaction solution, a PCR reaction solution, a positive DNA quality control substance, a positive RNA fusion quality control substance, a negative RNA fusion quality control substance, and nuclease-free water.
4. The kit according to claim 3, wherein the reverse transcription reaction solution comprises RNA reverse transcriptase, 5 Xbuffer, DTT, dNTPs, RNase inhibitor; the PCR reaction solution contains high-fidelity DNA polymerase, KCl, MgCl2, Tris-HCl and dNTPs; the positive DNA quality control substance is normal human genome DNA, the positive RNA fusion quality control substance is human RET/PTC1 fusion RNA, and the negative RNA fusion quality control substance is human non-fusion RNA.
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| CN201910592171.0A CN110241215B (en) | 2019-07-03 | 2019-07-03 | Primer and kit for detecting benign and malignant genetic variation of thyroid nodule |
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| 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 |
| CN111349694A (en) * | 2020-04-14 | 2020-06-30 | 深圳市新合生物医疗科技有限公司 | Detection method and kit for thyroid cancer related gene fusion mutation |
| CN111748628B (en) * | 2020-07-14 | 2022-04-05 | 润安医学科技(苏州)有限公司 | Primer and kit for detecting thyroid cancer prognosis related gene variation |
| CN116987791B (en) * | 2023-09-22 | 2023-12-22 | 润安医学科技(苏州)有限公司 | Application of plasma markers in identification of benign and malignant thyroid nodule |
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