CN111979327A - Detection kit and detection method for human thyroid gland extraction-free oncogene mutation - Google Patents
Detection kit and detection method for human thyroid gland extraction-free oncogene mutation Download PDFInfo
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Abstract
The invention discloses a kit and a detection method for detecting human thyroid gland non-extraction oncogene mutation, belonging to the technical field of molecular biology, wherein the kit adopts a specific nucleic acid releaser and a PCR reaction solution matched with the nucleic acid releaser, combines a multiplex PCR capture technology and an NGS sequencing technology, is used for qualitatively detecting various variations of BRAF, TERT, RET, TP53, NRAS, HRAS, KRAS and PIK3CA genes in a fresh tissue sample with uncertain thyroid nodule puncture biopsy cytology, and provides help for auxiliary diagnosis and treatment of thyroid cancer through multi-gene multi-site detection and combination with clinical pathological analysis results.
Description
Technical Field
The invention relates to a detection kit and a detection method for human thyroid gland extraction-free oncogene mutation in the technical field of molecular biology.
Background
Thyroid cancer is a common head and neck malignant tumor, accounts for 1.0% -1.5% of the malignant tumor of the whole body, and can be comprehensively evaluated mainly by clinical palpation, ultrasound, nuclide imaging, fine needle cell biopsy and other methods at present, so that the accuracy of thyroid cancer diagnosis can be improved to a certain extent, but 10% -30% of thyroid cancer cannot be diagnosed clearly. Thyroid cancer guidelines both at home and abroad suggest that thyroid nodules cannot be diagnosed by thyroid nodule fine needle cytology biopsy (FNAB) detection, and molecular marker detection can be utilized to improve diagnosis accuracy, such as BRAF, RAS, RET/PTC, PAX8/PPAR gamma and other mutation detection are conventional molecular detection items, and the molecular marker detection is used as an important supplement of FNAB and provides important help for diagnosis and prognosis judgment of thyroid cancer. However, the tissue puncture needle head commonly used in clinic at present is 22-25G, wherein the outer diameter of 25G is only 0.5mm, and only hundreds of available cells can not meet the requirements of the existing nucleic acid detection kit on the market, so that a thyroid nodule puncture tissue sample detection kit with extremely low initial amount and high sensitivity is urgently needed. At present, the detection kit on the market basically extracts the puncture sample and then establishes a library, and the method can increase the loss of the nucleic acid extraction process, thereby further reducing the nucleic acid extraction amount. Research proves that direct library construction after a small amount of tissue sample cells are cracked can obviously improve the utilization rate of nucleic acid, and simultaneously can avoid interference of various salt ions in the kit on subsequent experiments.
The currently used target segment capture technology for the second generation sequencing technology mainly comprises multiplex PCR, hybridization capture and molecular inversion probes. Compared with a hybridization capture method, the multiple PCR technology has higher compatibility to the initial amount of nucleic acid, is more convenient to operate, and is suitable for the library construction of a small amount of samples.
The invention comprises 26 mutation sites of BRAF, TERT, NRAS, KRAS, HRAS, TP53, RET and PIK3CA 8 genes related to thyroid cancer. BRAF gene mutations are more common in thyroid benign tumors and papillary thyroid carcinoma, wherein BRAF V600E mutation is a promoter gene in the development process of papillary thyroid carcinoma, and a more effective diagnosis is made under the conditions of atypical cell morphology and poor sampling. RET gene mutation is most closely related to medullary thyroid carcinoma, and RET gene mutation exists in about 95% of hereditary medullary thyroid carcinoma and more than 80% of sporadic medullary thyroid carcinoma. RAS genes including HRAS, KRAS, NRAS, RAS mutations and aggressive association studies of papillary carcinoma found that RAS mutations are associated with distant metastasis and high mortality. TERT promoter mutation is closely related to the malignancy degree and prognosis of thyroid cancer, and the mutation is more common in thyroid follicular adenocarcinoma and papillary carcinoma with high invasiveness and clearly indicates poor prognosis. If the mutation appears at the same time with other oncogenic mutations such as BRAF mutation, RAS mutation and the like, the tumor is suggested to have high invasiveness and poorer prognosis. The TP53 gene mutation is related to the dedifferentiation of thyroid gland malignant tumor, and the prognosis is poor. The PIK3CA gene mutation is often related to the malignancy degree of thyroid cancer, and the detection of the sites can provide important clinical significance for clinical diagnosis and treatment.
Disclosure of Invention
The invention aims to provide a kit for detecting specific capture genes of thyroid nodule punctured tissues or cells, which has high sensitivity and simple and convenient operation, and greatly improves the detection capability of a small amount of clinical thyroid nodule punctured tissue samples.
The invention is realized by the following technical scheme:
a thyroid cancer gene mutation detection point, which is characterized in that: the thyroid cancer gene variation site is shown as a detection gene and a site in table 1,
TABLE 1 Gene and locus detection by this kit
| Gene | Detection site/region |
| BRAF | Val600 |
| TERT | C228T C250T |
| NRAS | Codon12/13/61 |
| HRAS | Codon12/13/61 |
| KRAS | Codon12/13/61 |
| PIK3CA | Codon545/1047 |
| RET | Met918 |
| TP53 | Arg273 |
A kit for extracting oncogene mutation without human thyroid is characterized in that: the kit comprises a nucleic acid releasing agent, PCR reaction liquid, a specific amplification primer, a purified magnetic bead, an NGS specific primer joint and a high-fidelity amplification reagent.
A kit detection process for detecting thyroid cancer mutant genes comprises the following steps:
1) obtaining a thyroid nodule puncture tissue sample, adding a nucleic acid releasing agent after centrifugation, placing for 10 minutes at room temperature, adding a prepared PCR reaction solution matched with the nucleic acid releasing agent and a specific amplification primer for direct amplification, and selecting an amplification product by using a magnetic bead chip to obtain a first round of PCR product; the components of the nucleic acid releasing agent are 300-500mmol/L NaOH and 0.1-2mol/L formamide; the PCR amplification buffer composition was 50mmol/L Tris-HCl, 30mmol/L KCl, 3.5mmol/L MgCl2(ii) a The concentration of dNTPs in the PCR reaction solution is 200-; the concentration of the primer for amplifying the target fragment is 30-50 pmol/L; the PCR amplification enzyme is a high-tolerance hot start Taq enzyme.
2) Taking the first round PCR product as a template, taking an NGS specific primer and a nucleotide sequence of Barcode as primers, selecting a high-fidelity amplification reagent to carry out second round PCR amplification, and purifying the PCR product to obtain a DNA sequencing library;
3) and (4) analyzing the data result by using a computer sequencing and bioinformatics analysis software.
Preferably, the concentration of dNTPs in the PCR reaction solution is preferably 600 mu mol/L;
preferably, the concentration of the primer for amplifying the target fragment is preferably 40 pmol/L.
The invention has the beneficial effects that:
the kit adopts nucleic acid-free extraction combined with a multiplex PCR capture technology and an NGS sequencing technology, is used for qualitatively detecting various variations of BRAF, TERT, RET, TP53, NRAS, HRAS, KRAS and PIK3CA genes in a fresh tissue sample with uncertain thyroid nodule puncture biopsy cytology, and provides help for auxiliary diagnosis and treatment of thyroid cancer through multi-gene multi-site detection and combined with clinical pathological analysis results.
The kit provides a rapid and simple library construction method, and the basic principle is to invent a nucleic acid releasing agent capable of rapidly releasing cell nucleic acid substances, and meanwhile, the nucleic acid releasing agent can denature impurity proteins, so that the interference of interfering substances on subsequent PCR amplification is reduced, and the rapid PCR amplification process is realized.
Drawings
FIG. 1 shows the principle of detection of thyroid cancer gene mutation.
FIG. 2 fragments of the library.
Detailed Description
The technical solutions adopted by the present invention and the effects thereof are further described below with reference to the drawings and the examples, and the technical solutions of the present invention are further described by the specific embodiments, but the present invention is not limited within the scope of the examples.
Material
1. Nucleic acid releasing agent produced by Rui Jing organism, having a product number of RJ 001S-A;
2. rui Jing biological PCR reaction solution, cat # RJ 001J-B;
3. purified magnetic beads Beackman XP beads, cat # A63881;
4, KAPA high fidelity amplification reagent, cat # KK 2602;
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 specific for amplification of a mutant site of a thyroid cancer-associated gene were synthesized by Shanghai.
Primer sequence pair for BRAF:
BRAF-F
5’-GAACGACATGGCTACGATCCGACTTCAGTGGAAAAATAGCCTCAATTCTTACCA-3’
BRAF-R
5’-TCCTAAGACCGCTTGGCCTCCGACTTCTTCATGAAGACCTCACAGTAAAAATAGGT-3’
primer sequence pair for TERT:
TERT_F 5’-GAACGACATGGCTACGATCCGACTTAGCGCTGCCTGAAACTCG-3’
TERT_R 5’-TCCTAAGACCGCTTGGCCTCCGACTTCCTTCACCTTCCAGCTCCG-3’
primer sequence pairs for NRAS:
NRAS-F
5’-GAACGACATGGCTACGATCCGACTTCAAATGACTTGCTATTATTGATGGCAA-3’
NRAS-R
5’-TCCTAAGACCGCTTGGCCTCCGACTTGTTATAGATGGTGAAACCTGTTTGTTGG-3’
NRAS-F
5’-GAACGACATGGCTACGATCCGACTTTGGGATCATATTCATCTACAAAGTGGTT-3’
NRAS-R
5’-TCCTAAGACCGCTTGGCCTCCGACTTGATTACTGGTTTCCAACAGGTTCTTG-3’
primer sequence pairs for HRAS:
HRAS-F 5’-GAACGACATGGCTACGATCCGACTTCTGTACTGGTGGATGTCCTCAA-3’
HRAS-R 5’-TCCTAAGACCGCTTGGCCTCCGACTTGCCTGTTGGACATCCTGGATAC-3’
HRAS-F 5’-GAACGACATGGCTACGATCCGACTTGCCAGGCTCACCTCTATAGTG-3’
HRAS-R 5’-TCCTAAGACCGCTTGGCCTCCGACTTAGGAGCGATGACGGAATATAAGC-3’
primer sequence pairs for KRAS:
KRAS-F 5’-GAACGACATGGCTACGATCCGACTTATGTACTGGTCCCTCATTGCAC-3’
KRAS-R
5’-TCCTAAGACCGCTTGGCCTCCGACTTGTAATAATCCAGACTGTGTTTCTCCCTT-3’
KRAS-F
5’-GAACGACATGGCTACGATCCGACTTACCTCTATTGTTGGATCATATTCGTCCAC-3’
KRAS-R
5’-TCCTAAGACCGCTTGGCCTCCGACTTTATTATAAGGCCTGCTGAAAATGACTGAAT-3’
primer sequence pair for PIK3 CA:
PIK3CA-F
5’-GAACGACATGGCTACGATCCGACTTACAGAGTAACAGACTAGCTAGAGACAATGA-3’
PIK3CA-R
5’-TCCTAAGACCGCTTGGCCTCCGACTTTAGCACTTACCTGTGACTCCATAGAAA-3’
PIK3CA-F
5’-GAACGACATGGCTACGATCCGACTTATCTTTTGATGACATTGCATACATTCGAAA-3’
PIK3CA-R
5’-TCCTAAGACCGCTTGGCCTCCGACTTGTGGAAGATCCAATCCATTTTTGTTGTC-3’
primer sequence pairs for RET:
RET-F
5’-GAACGACATGGCTACGATCCGACTTCCCTCCTTCCTAGAGAGTTAGAGTAACTT-3’
RET-R 5’-TCCTAAGACCGCTTGGCCTCCGACTTCACCCACACTTACACATCACTTTG-3’
primer sequence pair for TP 53:
TP53-F 5’-GAACGACATGGCTACGATCCGACTTCTTTCTTGCGGAGATTCTCTTCCT-3’
TP53-R 5’-TCCTAAGACCGCTTGGCCTCCGACTTGATTTCCTTACTGCCTCTTGCTTCT-3’
NGS specific primers:
circularized primer sequences: 5' - [ P-GAACGACATGGCTACGATCCGACTT-3 ];
barcode primer sequence:
5’-TGTGAGCCAAGGAGTTGXXXXXXXXXXTTGTCTTCCTAAGACCGCTTGGCCTC-3’
p stands for phosphorylation
XXXXXXXXXXXX is 10 base sequences with different numbers, and is used for distinguishing different sample loading samples
Example 2: pretreatment of samples
The tissue sampled by a puncture needle of qualified professionals is obtained, and after the biopsy is finished, the tissue is completely soaked in PBS liquid as soon as possible. Before the samples were transported, the samples were stored in a refrigerator at-20 ℃ or-80 ℃.
Example 3: construction of the library
The library establishing process of the kit is as follows:
(1) treatment of punctured tissue samples with nucleic acid releasing agents
Taking out the puncture tissue sample, centrifuging at 5000rpm for 3min, and removing the supernatant; mu.l of a nucleic acid releasing agent (400mmol/L NaOH, 1mol/L formamide) was added to the precipitate, mixed well and left at room temperature for 10 minutes for the next experiment.
(2) Amplification of fragments of interest
The PCR reaction solution had a composition of 50mmol/L Tris-HCl, 30mmol/L KCl, 3.5mmol/L MgCl2600. mu. mol/L dNTPs, 20pmol/L primer and 5U PCR hot start Taq enzyme.
The following system is prepared:
the PCR instrument is set with the following conditions for reaction:
(3) first round PCR product purification
1) After the end of the programmed reaction, the sample was briefly centrifuged, transferred to 21 μ l (0.7 ×) XP beads, vortexed, and incubated at room temperature for 2 min;
2) placing the sample on a magnetic frame, after the solution is clarified, transferring the supernatant into an XP magnetic bead containing 15 microliter (0.5X), 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;
(4) 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 were reacted using different Barcode number primer PCR instruments set up as follows:
(5) 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) the sample tube was placed on a magnetic rack and after clarification the supernatant was transferred to a new clearly labeled 1.5ml EP tube and the purified product was the constructed library.
(7) Library cyclization: accurately quantifying the constructed library, and performing quality control on the library fragments by using a fragment analyzer (figure 2); the library refers to the operation instruction of MGIEasy cyclization kit and the instruction of Huada MGI-200 computer kit, and the DNA amplification library of each sample is respectively allocated with 50M data quantity to carry out computer sequencing.
(8) And (4) performing data result analysis by using bioinformatics analysis software.
Example 4 Performance verification
1. Performance analysis
Thyroid cancer papillary TPC-1 cells (mutant wild type) are selected respectively, 50, 100, 200, 400 and 800 cells are selected as initial cell numbers after cell counting, each reaction is repeated for 10 times, and as a result, successful library establishment can be realized by as low as 100 cells.
Selecting several gene mutation site homozygous cell lines, such as BRAF V600E homozygous cell line BCPAP, TERT C228T homozygous cell line BHT-101, TERT C250T homozygous cell line ACT-1 and KRAS G12R homozygous cell line CAL-62, selecting 1, 3, 5, 10 and 20 homozygous cells from each cell line respectively, mixing the cells into TPC-1 cells, wherein the total number of each combined cell is 100, repeating each reaction for 10 times, and detecting mutation in 5 homozygous cells, namely detecting mutation sites in 5% in 100 cells by using the kit.
2. Clinical sample validation
50 clinical thyroid gland puncture tissue samples of Shanghai Ruiki hospital were collected. The database is built according to the detection method, the detection result is analyzed, the benign and malignant degree of the thyroid nodule of each patient is judged, the judgment result is compared with the pathological detection result of the patient, and the difference between the results is compared, wherein the difference is shown in the table 2:
TABLE 2 comparison of the test results of the clinical sample kit with the pathological results of the patients
As can be seen from the above, in the samples of 50 thyroid nodules, the sensitivity of the kit of the present invention is 92.8%, the specificity is 88.9%, and the accuracy is 90% by comparing the test kit of the present invention with the clinical pathology results, and thus the results of the clinical sample test examples can be seen: the detection result of the kit provides an important reference basis for further diagnosing the type of the thyroid disease lesion.
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.
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Claims (5)
1. A thyroid cancer gene mutation detection point, which is characterized in that: the thyroid cancer gene variation sites are shown in the detection genes and sites in table 1.
2. A detection kit for detecting human thyroid gland extraction-free oncogene mutation is characterized in that: the kit comprises a nucleic acid releasing agent, PCR reaction liquid, a specific amplification primer, a purified magnetic bead, an NGS specific primer joint and a high-fidelity amplification reagent.
3. A detection kit and a detection method for human thyroid gland extraction-free oncogene mutation are characterized in that: the detection process comprises the following steps:
1) obtaining a thyroid nodule puncture tissue sample, adding a nucleic acid releasing agent after centrifugation, placing for 10 minutes at room temperature, adding a prepared PCR reaction solution matched with the nucleic acid releasing agent and a specific amplification primer for amplification, and selecting an amplification product by using a magnetic bead chipObtaining a first round PCR product; the components of the nucleic acid releasing agent are 300-500mmol/L NaOH and 0.1-2mol/L formamide; the PCR amplification buffer composition was 50mmol/L Tris-HCl, 30mmol/L KCl, 3.5mmol/L MgCl2(ii) a The concentration of dNTPs in the PCR reaction solution is 200-; the concentration of the primer for amplifying the target fragment is 30-50 pmol/L; the PCR amplification enzyme is a hot start Taq enzyme with high tolerance;
2) taking the first round of PCR products as a template, taking the NGS specific primer and the nucleotide sequence of the Barcode as primers, selecting KAPA Hifi high fidelity enzyme for carrying out second round PCR amplification, and purifying PCR products to obtain a DNA sequencing library;
3) and (4) analyzing the data result by using a computer sequencing and bioinformatics analysis software.
4. The kit and the method for detecting the human thyroid gland non-extraction oncogene mutation according to claim 3, wherein the kit comprises: the concentration of dNTPs in the PCR reaction solution is preferably 600. mu. mol/L.
5. The kit and the method for detecting the human thyroid gland non-extraction oncogene mutation according to claim 3, wherein the kit comprises: the primer concentration for amplifying the target fragment is preferably 40 pmol/L.
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