CN112011615A - Gene fusion kit for human thyroid cancer and detection method - Google Patents

Abstract

The invention discloses a gene fusion kit and a detection method for human thyroid cancer in the technical field of molecular biology, wherein the kit adopts a multiple PCR capture technology and an NGS sequencing technology, is used for qualitatively detecting various variations of CCC6-RET, NCOA4-RET, PAX8/PPARG and ETV6-NTRK3 fusion genes in a fresh tissue sample with uncertain thyroid nodule biopsy cytology, determines a detection threshold value according to a molecular label, and can provide help for auxiliary diagnosis and treatment of thyroid cancer by detection of fusion sites of the kit and combination of clinical pathological analysis results.

Description

Gene fusion kit for human thyroid cancer and detection method

Technical Field

The invention relates to a gene fusion kit for human thyroid cancer and a detection method thereof, belonging to the technical field of molecular biology.

Background

Thyroid cancer is the most common tumor in human endocrine organs, accounting for about 4% of all human malignancies, and its incidence is steadily increasing in China and throughout the world in recent years. Thyroid cancer is classified into papillary carcinoma (PTC), follicular carcinoma (FTC), undifferentiated carcinoma (ATC), and medullary carcinoma (MTC) according to the pathological type. Among them, PTC and FTC are called Differentiated Thyroid Cancer (DTC), which accounts for about 85% to 90% of thyroid cancer. Differentiated Thyroid Cancer (DTC) usually develops in thyroid nodules. Such nodules occur commonly in the population, particularly with increasing age, with concomitant increases in incidence. However, most thyroid nodules are benign and it is clinically desirable to accurately and rapidly identify malignant nodules and remove them by surgery. Among diagnostic methods for thyroid nodules, cytological examination by ultrasound-guided Fine Needle Aspiration (FNA) is a common diagnostic method (hereinafter referred to as FNA cytological examination), and this method can reliably diagnose cancer or benign nodules in most cases. However, in about 25% of nodules, FNA cytological examination cannot exclude the presence of cancer, thereby hindering clinical management. Such remaining nodules, which cannot exclude cancer, fall within the class III, IV and V nodules defined by the Bethesda classification system with expected cancer risks of 5-15%, 20-30% and 50-75%, respectively. The uncertainty of cancer risk in these nodules has led many to diagnostic procedures, but these procedures can be avoided in many patients with benign nodules.

In the last decade, the rapid development of precise medicine has led to the gradual and deep understanding of thyroid cancer molecular targets and related signaling pathways through the research of predecessors. Thyroid cancer has been shown to be the result of the accumulation of a number of genetic alterations that are important diagnostic, prognostic and predictive biomarkers. Such as: the most common mutations in papillary carcinoma (PTC) are point mutations in the BRAF and RAS genes and RET/PTC recombination, all of which are capable of activating the Mitochondrial Activated Protein Kinase (MAPK) pathway to accelerate the onset and progression of cancer; follicular carcinoma (FTC) often contains RAS mutations or PAX8/PPAR γ rearrangements. Mutations involved in the PIK3CA/AKT signaling pathway (PIK3CA, PTEN mutation), TP53, AKT1, and CTNNB1 genes are common in advanced and undifferentiated cancers (ATC); myeloid cancer (MTC) often carries point mutations located in the RET and RAS genes. Based on the research results of the predecessors, the guidelines such as NCCN and ATA and the consensus of experts indicate that the gene detection can assist in diagnosing the benign and malignant thyroid nodules, and the gene detection-assisted identification is recommended for nodule patients who can not determine the benign and malignant thyroid nodules by FNA. Meanwhile, the gene detection can also assist the prognosis evaluation and medication guidance of thyroid cancer patients: genetic mutations such as BRAF in thyroid cancer may indicate risk of recurrence, aid in molecular typing and risk stratification of patients. A number of drugs have been FDA approved for thyroid cancer and genetic testing may suggest that patients benefit from targeted drugs.

The mutation of the thyroid related genes such as BRAF, RAS, PIK3CA, PTEN, TP53, AKT1 and CTNNB1 is mostly point mutation or tiny indel, DNA is usually used as input clinically, a qPCR method is used for detection, and the DNA is relatively stable in clinical samples and large in extraction amount, so that the existing clinical test method is maintained without excessive optimization. For fusion assays, the current gold standard for clinical gene fusion assays is Fluorescence In Situ Hybridization (FISH). Although FISH sensitivity is high, there are: only one of the known fusions can be detected, with a large sample size and poor specificity. RNA nucleic acid directly reflects the splicing condition of exons of a fusion gene and a fusion partner gene, is the first choice material for clinical test of the fusion gene at present, is spliced by exons based on RNA level in companies at present, detects fusion by using an RT-PCR method, commonly uses ARMS-PCR and digital PCR, and has the advantages of high sensitivity and good specificity, but has small flux, and only can detect a plurality of known fusions at one time. If one needs to cover several fusion types common to thyroid cancer, a large amount of RNA nucleic acid is required, which is contrary to the micro-RNA amount of FNA tissue. With the rapid development of high throughput sequencing technology, the means for detecting fusion genes based on NGS technology is also gradually applied and accepted. At present, a detection kit based on a second-generation sequencing platform on the market is commonly established by a probe capture method based on DNA level, but the cost is high, and the operation process is complicated; the RNA level reverse transcription and multiple PCR technology combines the sensitivity and specificity of qPCR and the intuition of the NGS technical result, has simple and convenient operation and short time consumption, can detect all fusion types by only one sample, and has outstanding methodological advantages.

However, most of the existing technical solutions are to reverse RNA into cDNA and then construct a library with cDNA (patent numbers: CN 111088365A, CN 110241215A and CN109371139A), which not only increases the experimental operation process and increases the risk of pollution, but also has relatively less information for detecting fusion genes and limited clinical use. The fusion positive threshold of the prior art (patent No. CN 104894271A) is defined according to the reads value, and the method is extremely unstable and is easily interfered by various factors such as the sequencing data volume, methodology and the like. The molecular tag is obtained by attaching a random sequence to each nucleic acid fragment in the sample, so that each original sample has a unique tag. We can distinguish different original molecules by the respective tags of wild type and fused type to accurately quantify the fusion ratio in the original RNA. The method can stably and accurately trace back to the original fusion positive state, and solves the existing fusion detection problem.

Disclosure of Invention

The invention aims to provide a high-throughput sequencing detection kit which is sensitive, accurate and simple and convenient to operate and aims at thyroid cancer fusion gene detection so as to realize detection and auxiliary diagnosis of clinical thyroid cancer lesions.

The invention is realized by the following technical scheme:

a thyroid cancer gene fusion checkpoint comprising: the thyroid cancer gene fusion detection points are shown in the following table 1 as fusion genes and fusion types:

TABLE 1 fusion genes and fusion types

A gene fusion kit for human thyroid cancer is characterized in that: the kit also comprises an RNA extraction kit, RNA reverse transcriptase, a multiplex PCR amplification primer, PCR reaction liquid, purified magnetic beads, an NGS library building joint, a positive control and a negative control;

preferably, the kit performs PCR amplification on a target sequence through a specific primer, performs two rounds of PCR enrichment amplification on an amplification product after magnetic bead purification and enrichment, and finally obtains a sequencing library through magnetic bead purification;

preferably, the reverse transcription and first round PCR (amplification of the target region) reactions are performed in a single tube.

A detection method for a human thyroid cancer gene fusion kit comprises the following steps:

1) extraction of RNA: extracting RNA in the punctured tissue sample by using an RNA extraction kit;

2) reverse transcription and amplification of specific fragments: reverse transcription and the first round of PCR (amplification of a specific fragment of interest) reactions are performed in a single tube. Taking RNA as a template and a fused 3' end gene reverse primer with 6 random bases as a reverse transcription specific primer to obtain cDNA; amplifying the cDNA by using a specific multiplex PCR primer and an amplification reagent to obtain a specific target fragment;

3) and (3) purifying a product: adding quantitative magnetic beads into the PCR amplification product obtained in the step (2) for purification, and removing RNA and residual amplification primers;

4) connecting a sequencing joint: adding a specific label and a joint reagent in the NGS library establishing reagent into the purified product obtained in the step (3), and performing PCR amplification to obtain a library fragment;

5) library detection: quantifying the library prepared in the step (4) by using a Qubit, performing quality inspection by using an Agilent Bioanalyzer 2100 or an instrument with the same function, and detecting the concentration of the library and the fragment size of the library to ensure that the final library fragment is in the range of 220-250 bp;

6) and (3) machine sequencing: cyclizing the library qualified in the step (5), and performing high-throughput sequencing by using a Huada MGI sequencing platform;

7) and (4) analyzing the data result by using bioinformatics analysis software.

The invention has the beneficial effects that:

the kit adopts a multiple PCR capture technology, respectively designs primers aiming at CCC6-RET, NCOA4-RET, PAX8-PPARG and ETV6-NTRK3, specifically screens the low expression housekeeping gene TBP and HMBS in the thyroid gland, and expresses the housekeeping gene LMNA. The kit RNA is built by using gene specific primers with molecular labels, reverse transcription and amplification reaction of a target region are completed in one tube, so that the detection accuracy is greatly improved, and the extra tube opening/pipetting risk is reduced.

Drawings

FIG. 1 CCDC6-RET library construction principle.

FIG. 2 TBP amplification efficiency curves.

Figure 3 TBP dissolution profile.

FIG. 4 HMBS amplification efficiency curves.

Fig. 5 HMBS dissolution profile.

FIG. 6 LMNA amplification efficiency curves.

FIG. 7 LMNA dissolution curves.

FIG. 8 fragments of the library.

Detailed Description

The technical means adopted by the present invention and the effects thereof are further explained below with reference to the drawings and the examples, and the technical solutions of the present invention are further explained by the specific embodiments, but the present invention is not limited within the scope of the examples.

Material

1. Cell RNA extraction kit (centrifugation column method) produced by Rui Jing JING organism, having a product number of RJ 001T-A;

PCR reaction solution, 2 XKAPA 2G Fast Multiplex Mix, cat # KK 5802;

vazyme reverse transcriptase, cat # R201-02;

4. purified magnetic beads Beackman XP beads, cat # A63881;

a Qsep-100 analyzer;

6. huada sequencer MGI-200; MGI-2000

The materials used in the following examples are not limited to those listed above, and other similar materials may be substituted, and those skilled in the art should understand that the materials and equipment used are conventional and the equipment is not specifically defined, or the equipment is recommended by the manufacturer.

Example 1 primer sequences for amplifying mutant sites of thyroid cancer-associated genes were synthesized by Shanghai.

The RNA primer design principle of the present invention is described in detail below by taking CCDC6-RET as an example. As shown in figure 1, the 5 'end CCDC6 gene exon 1 is fused with 3' RET gene exon 12 by using the position of the break point as a boundary, so that the upstream primer is designed on the CCDC6 gene exon 1, and the downstream primer is designed on the RET gene exon 12; the RET wild type upstream primer is designed on the No. 11 exon, and the downstream is consistent with the fusion gene downstream primer.

(1) RNA specific primers whose primer sequences are shown in Table 2:

TABLE 2 fusion genes and their primer sequences

Gene	Primer and method for producing the same
		CCDC6-E1-F	5’-GAACGACATGGCTACGATCCGACTTCAGGAGGAGAACCGCGAC-3’
CCDC6-E2-F	5’-GAACGACATGGCTACGATCCGACTTAAAGAAGAAGAATTCCTCACTAATGAG-3’
		NCOA4-E8-F	5’-GAACGACATGGCTACGATCCGACTTTCCTTACATACCCAGCACCGA-3’
NCOA4-E7-F	5’-GAACGACATGGCTACGATCCGACTTTCCATGCCAGAGCAGGTAAAA-3’
		RET-E12-R	5’-TCCTAAGACCGCTTGGCCTCCGACTTNNNNNNCAAGAACCAAGTTCTTCCGAGG-3’
PAX8-E10-F	5’-GAACGACATGGCTACGATCCGACTTCAGGGCAGCTATGCCTCCT-3’
		PAX8-E9-F	5’-GAACGACATGGCTACGATCCGACTTGCCTTTCCCCATGCTGC-3’
PAX8-E8-F	5’-GAACGACATGGCTACGATCCGACTTGCACTACCCAGAGGCCTATGC-3’
		PAX8-E7-F	5’-GAACGACATGGCTACGATCCGACTTGCAACCTCTCGACTCACCAGAC-3’
PPARG-E2-R	5’-TCCTAAGACCGCTTGGCCTCCGACTTNNNNNNCCAAAGTTGGTGGGCCAGA-3’
		ETV6-E5-F	5’-GAACGACATGGCTACGATCCGACTTATGGTCTCTGTCTCCCCGC-3’
ETV6-E4-F	5’-GAACGACATGGCTACGATCCGACTTCTGGAAACTCTATACACACACAGCC-3’
		NTRK3-E15-R	5’-TCCTAAGACCGCTTGGCCTCCGACTTNNNNNNGTGAGTTGATGGGACTAGATGATCTC-3’
NTRK3-E14-R	5’-TCCTAAGACCGCTTGGCCTCCGACTTNNNNNNGTGGGCTGGCTGAGTCCTC-3’

Wild type gene and primer sequence thereof

P represents phosphorylated nnnnnnnn represents a 6-base molecular tag the reaction solution contains the following primers: mu.L of each 10. mu. mol/L primer was 0.4. mu.L.

(2) Primer of housekeeping gene

The method is a database construction process through housekeeping gene quality control database construction. Screening low-expression genes TBP and HMBS respectively in thyroid cancer by consulting documents, expressing genes LMNA, designing primers for adjacent exons of each gene in a cross-intron manner, and requiring the length of an intron to be more than 1000bp for preventing DNA from polluting; designing multiple pairs of primers for each gene, verifying the amplification efficiency and the melting curve of each pair of primers by QPCR, and finally screening out the primers with the amplification efficiency (Eff%) ranging from 95% to 115% and only a single melting curve (FIGS. 2-6), wherein the primer lists are shown in Table 3:

TABLE 3 housekeeping genes and their primer sequences

Gene	Primer (5 '-3')
		TBP-F	5’-GAACGACATGGCTACGATCCGACTTGGGAGCTGTGATGTGAAGTTTCC-3’
TBP-R	5’-TCCTAAGACCGCTTGGCCTCCGACTTNNNNNNGGAGAACAATTCTGGGTTTGATCAT-3’
		HMBS-F	5’-GAACGACATGGCTACGATCCGACTTGCAGAGAAAGTTCCCGCATC-3’
HMBS-R	5’-TCCTAAGACCGCTTGGCCTCCGACTTNNNNNNCACTGAACTCCTGCTGCTCG-3’
		LMNA-F	5’-GAACGACATGGCTACGATCCGACTTCTCAGTGAGAAGCGCACGCT-3’
LMNA-R	5’-TCCTAAGACCGCTTGGCCTCCGACTTNNNNNNGCAGCATCTCATCCTGAAGTTG-3’

Example 2: pretreatment of samples and nucleic acid extraction

The qualified professional samples with a needle, the sample comprising greater than 1mg of the punctured tissue sample. After completion of the biopsy, the tissue was completely infiltrated in RNA storage solution as soon as possible. Samples were stored at-20 ℃ prior to sample shipment.

RNA in the sample was extracted using a tissue cell RNA extraction kit (centrifugal column method) by referring to the kit instructions.

1. Mechanical tissue sample homogenization:

the sample is placed in a suitable glass tube or a centrifuge tube, 350. mu.L of Buffer RL and DTT mixed lysate (100:1) is added, a probe is inserted into the lysate, and the sample is homogenized intermittently at a high speed for 15-20 seconds each time until the sample is completely homogenized.

2. Extraction of nucleic acids after homogenization

Approximately 350. mu.L of the homogenized sample was centrifuged at 14000rpm for 3min and the supernatant was transferred to a centrifuge tube and kept ready for use.

2.1 Total RNA extraction

2.1.1 adding 70% ethanol with equal volume to the supernatant after centrifugation, and sucking and beating for 3-5 times by using a pipette gun.

2.1.2 transfer the mixture to HiPure RNA Mini Columns I, which are loaded into collection tubes, and centrifuge at 12000rpm for 1 min.

2.1.3 discard waste, put the column back into the collection tube, add 350. mu.L Buffer RW1 to the column, centrifuge at 12000rpm for 1 min.

2.1.4 discard the waste liquid and put the column back into the collection tube. 50U DNase I enzyme was added to the center of the membrane of the column. Standing at room temperature for 15 min.

2.1.5 Add 500. mu.L Buffer RW1 to the column, let stand for 3min, centrifuge at 12000rpm for 1 min.

2.1.6 discard the waste and put the column back into the collection tube. Add 500. mu.L Buffer RW2 to the column and centrifuge at 12000rpm for 1 min.

2.1.7 discard the waste and put the column back into the collection tube. Add 500. mu.L of Buffer RW2 to the column. Centrifuge at 12000rpm for 1 min.

2.1.8 discard the waste liquid and return the column to the collection tube. The column was emptied at 14000rpm for 5 min.

2.1.9 the RNA columns were packed into 1.5mL centrifuge tubes. Add 15-30. mu.L RNase Free Water to the center of the membrane of the column. Standing at room temperature for 1 min. Centrifuge at 12000rpm for 1 min. RNA samples were stored at-80 ℃.

Example 3: construction of the library

The library establishing process of the kit is as follows:

(1) RNA reverse transcription and first round PCR amplification (amplification of target region)

The following systems were prepared using the reverse transcriptase of Vazyme and its KAPA multi-reconstitution library mix, respectively:

the PCR instrument is set with the following conditions for reaction:

(2) first round PCR product purification

1) After the programmed reaction is finished, centrifuging the sample for a short time, adding non-nucleic acid water into the sample to make up for 20 ul, transferring the sample to XP magnetic beads containing 14 ul (0.7X), mixing uniformly by vortex, and incubating for 2min at room temperature;

2) placing the sample on a magnetic frame, after the solution is clarified, transferring the supernatant into 10 microliter (0.5X) XP magnetic beads, uniformly mixing by vortex, and incubating for 5min at room temperature;

3) placing the sample on a magnetic frame, and removing the supernatant after the solution is clarified;

4) adding 200 mul of freshly prepared 80% ethanol into the sample, rotating the sample on a magnetic frame for one circle, and after 30s, removing the ethanol; repeating the step 1 time and 2 times in total;

5) centrifuging the sample, discarding residual liquid on a magnetic frame, uncovering and drying the magnetic beads for 1-2min until the magnetic beads do not reflect light;

6) adding 9 μ l of nucleic-Free Water into the magnetic beads, mixing by vortex, and centrifuging for a short time;

7) transferring the sample with the magnetic beads into a new PCR tube, and directly carrying out second round amplification without removing the magnetic beads;

(3) second round of amplification

Performing a second round of amplification on the purified product, using sequencing adapter primers and index primers, and formulating the reaction on a clean bench according to the following system:

different sample libraries using different index-numbered primers

The PCR instrument is set with the following conditions for reaction:

(4) second round PCR product purification

1) Transferring 20 μ l of the sample with beads to 20 μ l (1 ×) XP beads, vortexing, and incubating at room temperature for 5 min;

2) placing the sample on a magnetic frame, and removing the supernatant after the solution is clarified;

3) adding 200 mul of freshly prepared 80% ethanol into the sample, rotating the sample on a magnetic frame for one circle, and after 30s, removing the ethanol; repeating the step 1 time and 2 times in total;

4) centrifuging the sample tube, discarding residual liquid on a magnetic frame, uncovering and drying the magnetic beads for 1-3min until the magnetic beads do not reflect light;

5) adding 15 μ l of nucleic-Free Water into the magnetic beads, vortexing, and incubating at room temperature for 5 min;

6) placing the sample tube on a magnetic frame, after clarification, transferring the supernatant to a new clearly marked 1.5ml EP tube, wherein the purified product is a constructed library;

(5) detection of the library: the constructed library is accurately quantified by using the Qubit, quality inspection is carried out by using Qsep-100, and the fragment size of the library is detected to ensure that the library fragment is in the range of 220-250bp (figure 8).

(6) And (3) machine sequencing: performing high-throughput sequencing on the library qualified in the step (5) by using a Huada sequencer after cyclization by using a Huada cyclization kit;

(7) and (4) performing data result analysis by using bioinformatics analysis software. According to Q30, firstly, the sequencing quality of the base is evaluated, and simultaneously, the random base sequence at the front end of the sequencing sequence is cut and combined into the sequence ID; duplicate determinations were made based on the random tag sequences added to the ID positions, and sequences aligned to the same position and having compatible tags were considered to be derived from the same starting RNA template. Firstly, comparing reads to a reference genome, screening split reads (reads containing two gene fusion breakpoints) meeting corresponding parameters and trans-intron region 3' end gene wild-type reads, and calculating the ratio of the split reads to the sum of the split reads and the wild-type reads so as to obtain the fusion proportion of RNA in the original state.

Example 4 Performance verification

1. And (3) sensitivity analysis: the fusion ratio of 1% detectable in RNA samples was determined by using a standard of 5% positive cells, and performing gradient dilution of 2%, 1%, and 0.5%.

2. And (3) repeatability experiment: different initial amounts of RNA were added for each reaction, and high-throughput sequencing was repeated 10 times, with consistent results for 10 tests, and a compliance rate of 100%.

3. Clinical sample validation

30 clinical thyroid gland puncture tissue samples of Shanghai Ruiki hospital are collected, the kit is compared with a digital PCR detection result, the consistency of the detection results of the kit and the digital PCR detection result is 100%, and specific results are shown in Table 4. But the kit can simultaneously and rapidly detect the fusion of multiple genes, saves the detection cost and improves the detection efficiency, thereby having good clinical application value

TABLE 430 comparison of different methodologies for tissue sample puncture

The embodiments show that the kit can be used for simultaneously detecting multiple genes and multiple sites related to thyroid cancer, has the characteristics of high sensitivity and good specificity, and can meet the existing clinical requirements.

The kit adopts a multiple PCR capture technology and an NGS sequencing technology, is used for qualitatively detecting various variations of CCC6-RET, NCOA4-RET, PAX8/PPARG and ETV6-NTRK3 fusion genes in a fresh tissue sample with uncertain thyroid nodule puncture biopsy cytology, and provides help for the auxiliary diagnosis and treatment of thyroid cancer by detecting fusion sites and combining clinical pathological analysis results.

The kit is a one-round PCR amplification by designing a specific primer to carry out reverse transcription and amplification on a target sequence, carrying out two-round PCR enrichment amplification on an amplification product after purification and enrichment of magnetic beads, and finally obtaining a sequencing library through purification of the magnetic beads.

The kit can more accurately and stably detect RNA analysis fusion by designing a molecular label and screening out a specific housekeeping gene.

In addition to the high-throughput multiplex PCR capture technology of the present invention, the fusion detection can be performed by digital PCR or ARMS-PCR, but the number of loci detected by the two is small. Therefore, this method is currently most suitable when multiple gene multiple sites are required for simultaneous detection.

The kit adopts a multiple PCR capture technology, respectively designs primers aiming at CCC6-RET, NCOA4-RET, PAX8-PPARG and ETV6-NTRK3, specifically screens the low expression housekeeping gene TBP and HMBS in the thyroid gland, and expresses the housekeeping gene LMNA.

The kit RNA is built by using gene specific primers with molecular labels, reverse transcription and amplification reaction of a target region are completed in one tube, so that the detection accuracy is greatly improved, and the extra tube opening/pipetting risk is reduced.

The above embodiments are only used to illustrate the technical solution of the present invention, and not to limit it. The basic principles and the main features of the invention have been described above with specific embodiments, on the basis of which some modifications or alterations can be made without departing from the essence of the corresponding technical solution.

Sequence listing

<110> Shanghai Rui JING Biotechnology GmbH

<120> gene fusion kit for human thyroid cancer and detection method

<160> 24

<170> SIPOSequenceListing 1.0

<210> 1

<211> 43

<212> DNA

<213> Unknown (Unknown)

<400> 1

gaacgacatg gctacgatcc gacttcagga ggagaaccgc gac 43

<210> 2

<211> 52

<212> DNA

<213> Unknown (Unknown)

<400> 2

gaacgacatg gctacgatcc gacttaaaga agaagaattc ctcactaatg ag 52

<210> 3

<211> 46

<212> DNA

<213> Unknown (Unknown)

<400> 3

gaacgacatg gctacgatcc gactttcctt acatacccag caccga 46

<210> 4

<211> 46

<212> DNA

<213> Unknown (Unknown)

<400> 4

gaacgacatg gctacgatcc gactttccat gccagagcag gtaaaa 46

<210> 5

<211> 48

<212> DNA

<213> Unknown (Unknown)

<400> 5

tcctaagacc gcttggcctc cgacttcaag aaccaagttc ttccgagg 48

<210> 6

<211> 44

<212> DNA

<213> Unknown (Unknown)

<400> 6

gaacgacatg gctacgatcc gacttcaggg cagctatgcc tcct 44

<210> 7

<211> 42

<212> DNA

<213> Unknown (Unknown)

<400> 7

gaacgacatg gctacgatcc gacttgcctt tccccatgct gc 42

<210> 8

<211> 46

<212> DNA

<213> Unknown (Unknown)

<400> 8

gaacgacatg gctacgatcc gacttgcact acccagaggc ctatgc 46

<210> 9

<211> 47

<212> DNA

<213> Unknown (Unknown)

<400> 9

gaacgacatg gctacgatcc gacttgcaac ctctcgactc accagac 47

<210> 10

<211> 45

<212> DNA

<213> Unknown (Unknown)

<400> 10

tcctaagacc gcttggcctc cgacttccaa agttggtggg ccaga 45

<210> 11

<211> 44

<212> DNA

<213> Unknown (Unknown)

<400> 11

gaacgacatg gctacgatcc gacttatggt ctctgtctcc ccgc 44

<210> 12

<211> 50

<212> DNA

<213> Unknown (Unknown)

<400> 12

gaacgacatg gctacgatcc gacttctgga aactctatac acacacagcc 50

<210> 13

<211> 52

<212> DNA

<213> Unknown (Unknown)

<400> 13

tcctaagacc gcttggcctc cgacttgtga gttgatggga ctagatgatc tc 52

<210> 14

<211> 45

<212> DNA

<213> Unknown (Unknown)

<400> 14

tcctaagacc gcttggcctc cgacttgtgg gctggctgag tcctc 45

<210> 15

<211> 46

<212> DNA

<213> Unknown (Unknown)

<400> 15

gaacgacatg gctacgatcc gactttcagc tactcctctt ccggtg 46

<210> 16

<211> 48

<212> DNA

<213> Unknown (Unknown)

<400> 16

tcctaagacc gcttggcctc cgacttcaag aaccaagttc ttccgagg 48

<210> 17

<211> 49

<212> DNA

<213> Unknown (Unknown)

<400> 17

gaacgacatg gctacgatcc gacttggaga ttctcctatt gacccagaa 49

<210> 18

<211> 45

<212> DNA

<213> Unknown (Unknown)

<400> 18

tcctaagacc gcttggcctc cgacttccaa agttggtggg ccaga 45

<210> 19

<211> 46

<212> DNA

<213> Unknown (Unknown)

<400> 19

gaacgacatg gctacgatcc gacttactcg catccctgtc attgag 46

<210> 20

<211> 52

<212> DNA

<213> Unknown (Unknown)

<400> 20

tcctaagacc gcttggcctc cgacttgtga gttgatggga ctagatgatc tc 52

<210> 21

<211> 49

<212> DNA

<213> Unknown (Unknown)

<400> 21

gaacgacatg gctacgatcc gacttggttc tcttcgtcat gatcaacaa 49

<210> 22

<211> 45

<212> DNA

<213> Unknown (Unknown)

<400> 22

tcctaagacc gcttggcctc cgacttgtgg gctggctgag tcctc 45

<210> 23

<211> 25

<212> DNA

<213> Unknown (Unknown)

<400> 23

gaacgacatg gctacgatcc gactt 25

<210> 24

<211> 43

<212> DNA

<213> Unknown (Unknown)

<400> 24

tgtgagccaa ggagttgttg tcttcctaag accgcttggc ctc 43

Claims (5)

1. A thyroid cancer gene fusion checkpoint comprising: the thyroid cancer gene fusion detection points are shown in the fusion genes and the fusion types in table 1.

2. A gene fusion kit for human thyroid cancer is characterized in that: the kit comprises an RNA extraction kit, RNA reverse transcriptase, a multiplex PCR amplification primer, PCR reaction liquid, purified magnetic beads, an NGS library building joint, positive control and negative control.

3. The gene fusion kit for human thyroid cancer according to claim 2, wherein: the kit performs PCR amplification on a target sequence through a specific primer with a molecular label, performs two rounds of PCR enrichment amplification on an amplification product after magnetic bead purification and enrichment, and finally obtains a sequencing library through magnetic bead purification.

4. The gene fusion kit for human thyroid cancer according to claim 2, wherein: the RNA reverse transcription and first round PCR (amplification of the target region) reactions were completed in a single tube.

5. A detection method for a human thyroid cancer gene fusion kit is characterized by comprising the following steps: the detection process comprises the following steps:

1) extraction of RNA: extracting RNA in the punctured tissue sample by using an RNA extraction kit;

2) reverse transcription and amplification of specific fragments: reverse transcription and a first round of PCR (amplification of a specific target fragment) reaction are completed in a single tube, RNA is taken as a template, a fused 3' end gene reverse primer with 6 random bases is taken as a reverse transcription specific primer, and cDNA is obtained; amplifying the cDNA by using a specific multiplex PCR primer and an amplification reagent to obtain a specific target fragment;

3) and (3) purifying a product: adding quantitative magnetic beads into the PCR amplification product obtained in the step (2) for purification, and removing RNA and residual amplification primers;

4) connecting a sequencing joint: adding a specific label and a joint reagent in the NGS library establishing reagent into the purified product obtained in the step (3), and performing PCR amplification to obtain a library fragment;

5) library detection: quantifying the library prepared in the step (4) by using a Qubit, performing quality inspection by using an Agilient Bioanalyzer 2100 or an instrument with the same function, and detecting the concentration of the library and the fragment size of the library to ensure that the final library fragment is in the range of 220-250 bp;

6) and (3) machine sequencing: cyclizing the library qualified in the step (5), and performing high-throughput sequencing by using a Huada MGI sequencing platform;

and (4) analyzing the data result by using bioinformatics analysis software.

Images