Composition for thyroid cancer detection, kit and application

Abstract

The invention belongs to the field of biological medicine, and relates to a composition for thyroid cancer detection, a kit and application, the primer composition provided by the invention can be used for simultaneously detecting mutation or fusion conditions of five gene loci of a BRAF gene and an RET-PTC gene, can be used for detecting thyroid cancer, and has relatively high sensitivity and specificity; meanwhile, by adopting the fluorescent PCR technology, the detection efficiency is improved, the detection time is shortened, and accurate diagnosis of thyroid cancer is realized.

Description

Technical Field

[0001] The present invention belongs to the technical field of gene detection, and in particular relates to a composition, a kit and an application thereof for thyroid cancer detection. Background Art

[0002] Thyroid cancer is a common malignant tumor of the endocrine system, and its main histological types include papillary carcinoma and follicular carcinoma. At present, the diagnosis of benign and malignant thyroid nodules mainly relies on ultrasound and ultrasound-guided fine needle aspiration (FNA). In the Bethesda system of thyroid cell pathology diagnosis criteria, categories III to V are uncertain diagnoses. The Chinese Thyroid Cancer Diagnosis and Treatment Guidelines (2018 Edition) believes that for thyroid nodules that cannot be determined to be benign or malignant by FNA, certain thyroid cancer markers can be tested on the puncture specimens.

[0003] In recent years, BRAF gene mutation and RET-PTC fusion gene have been found to be closely related to the occurrence of thyroid cancer. BRAF gene mutation has been studied most in thyroid FNA cell pathology, with a mutation rate of more than 80% in papillary carcinoma and a RET-PTC fusion rate of 15% to 20% in papillary carcinoma. The V600E / K mutation of the BRAF gene is significantly associated with the occurrence of papillary thyroid carcinoma, while the RET-PTC fusion gene is common in medullary thyroid carcinoma. The detection of these gene mutations can provide a deep understanding of the mechanism of cancer occurrence and help develop individualized treatment plans.

[0004] Although traditional gene mutation detection methods such as histopathological examination and immunohistochemical staining have certain accuracy, they have defects such as complex operation and low sensitivity. Therefore, the development of highly sensitive and highly specific gene detection technology is of great significance to improve the early diagnosis and treatment of thyroid cancer. Summary of the invention

[0005] The purpose of the present invention is to provide a composition, a kit and an application for thyroid cancer detection, which can accurately amplify and detect the mutation or fusion of the target gene through the efficient combination of specific primers and probes, and can be used to assist clinicians and pathologists in differential diagnosis of benign and malignant thyroid nodules, and achieve accurate diagnosis of thyroid cancer. Using the method of the present invention, 5 gene mutations or fusion sites of 2 genes can be detected simultaneously, and the mutations and fusion sites of the genes are shown in Table 1:

[0006] Table 1

[0007]

[0008] In order to achieve the above object, the present invention adopts the following technical solutions:

[0009] In the first aspect of the present invention, the present invention provides a composition for thyroid cancer detection, comprising a thyroid cancer detection reagent for detecting the mutation or fusion status of three BRAF gene mutation sites V600E, BRAF gene mutation sites V600K and RET-PTC gene fusion sites. The present invention jointly detects the mutation or fusion status of the BRAF mutation gene and the RET-PTC fusion gene, which can effectively improve the detection performance compared with single gene methylation detection, and the sensitivity and specificity are both above 90%.

[0010] In a second aspect of the present invention, the present invention provides a kit for diagnosing thyroid cancer, wherein the kit comprises the composition for detecting thyroid cancer according to the first aspect.

[0011] In a third aspect of the present invention, the present invention provides a method for determining the gene mutation or fusion status in a sample, wherein the gene includes BRAF gene mutation site V600E, BRAF gene mutation site V600K and RET-PTC fusion gene, and the method comprises:

[0012] (1) extracting using a co-extraction kit to obtain RNA and DNA, wherein the genomic DNA comes from the sample;

[0013] (2) performing reverse transcription on the genomic DNA to obtain a reverse transcription product;

[0014] (3) Using primer sets and probes to perform fluorescence quantitative PCR detection on the reverse transcription product to determine the gene mutation or fusion status in the sample.

[0015] In a fourth aspect of the present invention, the present invention provides an isolated nucleic acid sequence, wherein the isolated nucleic acid sequence comprises a primer set and a probe combination for BRAF gene mutation site V600E, BRAF gene mutation site V600K and RET-PTC fusion gene;

[0016] The PCR primer-probe combination includes at least one of the following sequences:

[0017]

[0018]

[0019] Preferably, the 5' end of the primer probe carries a fluorescent group, the 3' end carries a quenching group, and the fluorescent probe sequence has FAM and CY5 fluorescent groups.

[0020] Preferably, the kit further comprises an internal standard primer-probe combination for detecting an internal reference gene GAPDH, and the fluorescent group is selected from VIC.

[0021] The internal standard primer probe combination includes:

[0022]

[0023]

[0024] Preferably, the kit further comprises an amplification premix;

[0025] The amplification premix includes Taq polymerase;

[0026] The content of the Taq polymerase is 2% to 4% based on the total volume of the amplification premix, and the Taq polymerase uses deoxyribonucleotides (dNTPs) as substrates.

[0027] Preferably, the kit further comprises a positive control, which is a mutation / fusion positive nucleic acid.

[0028] Preferably, the kit further comprises a negative control substance, and the negative control substance is a wild-type nucleic acid.

[0029] The sample used is the solid tissue of thyroid cancer of the test subject.

[0030] Preferably, the sample used can be one or more of fresh surgical tissue and / or puncture tissue, frozen surgical tissue and / or puncture tissue, and formalin-fixed paraffin-embedded (FFPE) tissue samples. More preferably, the test sample is fresh surgical tissue and / or puncture tissue.

[0031] A fifth aspect of the present application provides a non-diagnostic detection method for thyroid cancer, using the mutation or fusion of the BRAF gene mutation site V600E, the BRAF gene mutation site V600K and the RET-PTC fusion gene as described in the first aspect, the detection method comprising:

[0032] Obtain RNA and genomic DNA from the sample to be tested and confirm the sample type;

[0033] Adding the nucleic acid sample to be tested into the reverse transcription reaction solution to obtain a reverse transcription product;

[0034] Adding the reverse transcription product to a PCR amplification reagent to obtain a PCR reaction mixture;

[0035] Set the PCR reaction conditions, and after the PCR reaction mixture is amplified by PCR, interpret and analyze the test results of the kit;

[0036] The PCR amplification reagents include a PCR primer-probe combination, an internal standard primer-probe combination and an amplification premix.

[0037] Compared with the existing technology, the BRAF gene mutation site V600E, BRAF gene mutation site V600K and RET-PTC fusion gene in the present invention utilize fluorescent PCR technology, through the efficient combination of specific primers and probes, to accurately amplify and detect the mutation or fusion of the target gene, with high sensitivity and specificity, reducing the occurrence of false negatives and false positives, and at the same time, the detection work is simple and fast, and the use of fluorescent PCR technology improves the detection efficiency and shortens the detection time, which is suitable for clinical rapid screening of thyroid cancer. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] Figure 1 It is a sample amplification diagram without gene mutation / gene fusion provided by the present invention;

[0039] Figure 2 It is a sample amplification diagram of BRAF-V600K mutation provided by the present invention;

[0040] Figure 3 It is a sample amplification diagram of BRAF-V600E mutation provided by the present invention;

[0041] Figure 4 It is a sample amplification diagram of CCDC6 exon1 / RET exon12 fusion provided by the present invention;

[0042] Figure 5 It is a sample amplification diagram of PRKAR1A exon7 / RET exon12 fusion provided by the present invention;

[0043] Figure 6 This is a sample amplification diagram of the NCOA4 exon8 / RET exon12 fusion provided by the present invention. DETAILED DESCRIPTION

[0044] The following examples are for illustrative purposes only and are not intended to limit the scope of the present invention.

[0045] It should be noted that this kit is only used to detect target gene sequences in patients suspected of thyroid cancer. The test results are for clinical reference only and should not be used as the sole basis for individualized treatment of patients. Clinicians should make a comprehensive judgment on the test results based on factors such as the patient's condition, drug indications, treatment response, and other laboratory test indicators.

[0046] First, a detection kit for evaluating the effect of thyroid cancer based on the mutation or fusion of BRAF gene mutation site V600E, BRAF gene mutation site V600K and RET-PTC fusion gene according to the embodiments of the present application is described.

[0047] refer to Figure 1, This embodiment proposes a thyroid cancer detection kit based on BRAF mutation gene and RET-PTC fusion gene mutation or fusion, the kit comprising a PCR primer probe combination;

[0048] The PCR primer probe combination includes:

[0049]

[0050]

[0051] This kit is used to qualitatively detect the mutation or fusion of BRAF mutation gene and RET-PTC fusion gene in thyroid puncture samples. It is used to assist clinicians and pathologists in differential diagnosis of benign and malignant thyroid nodules and achieve accurate diagnosis of thyroid cancer.

[0052] As an embodiment, the 5' end of the primer probe carries a fluorescent group, and the 3' end carries a quenching group, and the fluorescent group is selected from FAM and CY5.

[0053] As an implementation manner, the kit of this example further includes an internal standard primer probe combination for detecting an internal reference gene GAPDH, and the fluorescent group is selected from VIC.

[0054] The internal standard primer probe combination includes:

[0055]

[0056]

[0057] As an implementation mode, the kit of this embodiment further includes an amplification premix solution;

[0058] The amplification master mix includes Taq polymerase;

[0059] The content of Taq polymerase is 2% to 4% of the total volume of the amplification premix solution, and Taq polymerase uses deoxyribonucleotides (dNTPs) as substrates.

[0060] The kit of this embodiment also includes a positive control substance and a negative control substance.

[0061] Among them, the positive control is a mutation / fusion positive nucleic acid, and the negative control is a wild-type nucleic acid. The test results of the positive control and the negative control in each test can be used as an important indicator of the performance of the kit. If the test results of the positive control or the negative control do not meet expectations, it may indicate that there is a problem with the kit, such as reagent failure, improper storage leading to nucleic acid degradation, or reduced activity of primers and probes. This helps to detect quality problems of the kit in a timely manner and avoid erroneous test results caused by the use of unqualified kits.

[0062] In addition, the kit also includes reagents for processing and preparing samples, and the reagents include reverse transcription reaction solution, PCR primer probe mixture and PCR reaction solution.

[0063] Specifically, the main components in this embodiment are shown in Table 2 below:

[0064] Table 2

[0065]

[0066]

[0067] The primers or probes mentioned above can be artificially synthesized, for example, using phosphoramidite solid support for synthesis or other chemical synthesis methods commonly used in the art.

[0068] In this embodiment, fresh samples are preferentially punctured, and the specific detection method is as follows:

[0069] 1. Sample preparation:

[0070] Fresh tissue sample processing steps:

[0071] (1) Centrifuge the storage tube containing the puncture sample at 12000 rpm / min for 5 min;

[0072] (2) Remove the supernatant and extract the nucleic acid.

[0073] 2. Reverse transcription:

[0074] (1) Take 2-10 μL of TC reverse transcription reaction solution and 2-10 μL of extracted nucleic acid sample into a PCR tube;

[0075] (2) Mix well and centrifuge, then perform reverse transcription according to the procedure in the table below.

[0076] Specifically, the steps of reverse transcription in this embodiment are shown in Table 3 below:

[0077] Table 3

[0078] step Temperature(℃) time Number of cycles first step 37 15 minutes 1 Step 2 85 5 seconds 1 Step 3 4 3 minutes (instrument cooling) 1

[0079] 3. Fluorescence PCR test method:

[0080] 3.1 Preparation of PCR amplification reagents:

[0081] Prepare the PCR reaction solution according to Table 4 below (positive and negative controls need to be set up for each reaction).

[0082] Table 4

[0083] Reagent ingredients Dosage / person (μL) PCR reaction solution 10～20 PCR primer probe mixture 8～12 Total / Test 18～32

[0084] 3.2 Add sample:

[0085] Take 2-10 μL of positive control, negative control and cDNA of the sample to be tested and add them to the PCR eight-tube. Tightly cover the eight-tube and centrifuge briefly to centrifuge the liquid on the tube wall to the bottom of the tube.

[0086] 3.3PCR amplification:

[0087] Set the PCR reaction conditions. The steps for setting the reaction conditions are shown in Table 5 below:

[0088] Table 5

[0089]

[0090] After the detection is completed, the method for determining the detection result in this embodiment is as follows:

[0091] 1. Baseline setting:

[0092] The baseline can be automatically output by the instrument, or manually adjusted according to the instrument's instructions.

[0093] 2.Threshold setting:

[0094] It should be set at the exponential amplification stage of the fluorescence signal, and the fluorescence curve above the threshold should have a typical "S" curve.

[0095] It is worth mentioning that the test results can be effectively interpreted in the following way: if the internal standard gene detection (VIC channel) has typical amplification, the analysis can continue; if the internal standard gene detection (VIC channel) has no amplification, the test needs to be repeated.

[0096] The results of the test kit detection in this example are as follows:

[0097] 1. If the target gene (FAM channel and CY5 channel) of the sample has no typical "S"-shaped amplification curve or has an amplification curve but the Ct value is greater than 23, the test result of the sample is judged to be negative;

[0098] 2. If the FAM channel of the target gene of the sample shows a typical "S"-shaped amplification curve and the Ct value is ≤23, the test result of the sample is determined to be positive for the RET-PTC fusion gene. If the CY5 channel of the target gene of the sample shows a typical "S"-shaped amplification curve and the Ct value is ≤23, the test result of the sample is determined to be positive for the BRAF gene mutation.

[0099] The results of this embodiment are shown as follows:

[0100] 186 biopsy samples were tested, including 79 samples of benign thyroid lesions and 107 samples of thyroid cancer patients. The sensitivity and specificity of the single gene mutation / fusion detection results and the combined detection results of two gene mutations / fusions were analyzed. The comparison between the detection results of the present invention and the clinical results is shown in Table 6, and the comparison between the detection results of the present invention and the first-generation sequencing detection results is shown in Table 7:

[0101] Table 6

[0102]

[0103]

[0104] Table 7

[0105]

[0106] The present invention jointly detects the mutation or fusion status of the BRAF mutant gene and the RET-PTC fusion gene. Compared with single gene methylation detection, the detection performance can be effectively improved, and the sensitivity and specificity both reach more than 90%. The detection performance of the present invention is good.

[0107] The present invention detects BRAF mutation gene and compares it with the first generation sequencing result, and the sensitivity and specificity both reach more than 95%. The present invention detects RET-PTC fusion gene and compares it with the first generation sequencing result, and the sensitivity and specificity both reach more than 95%.

[0108] The following describes a method for detecting thyroid cancer according to the second aspect of an embodiment of the present application.

[0109] This embodiment also provides a non-diagnostic detection method for thyroid cancer, which uses the mutation or fusion of the five gene sites of the above-mentioned BRAF gene and RET-PTC gene to diagnose thyroid cancer. The detection method includes:

[0110] Obtain the nucleic acid sample (DNA / RNA) to be tested and confirm the sample type;

[0111] Add the nucleic acid sample (DNA / RNA) to be tested into the reverse transcription reaction solution to obtain the reverse transcription product cDNA;

[0112] Adding the cDNA of the sample to be tested into the PCR amplification reagent to obtain a PCR reaction mixture;

[0113] Set the PCR reaction conditions, and after the PCR reaction mixture is amplified by PCR, interpret and analyze the test results of the kit;

[0114] The PCR amplification reagents include a PCR primer probe combination, an internal standard primer probe combination and an amplification premix.

[0115] The step of adding the sample DNA to be tested into a PCR amplification reagent to obtain a PCR reaction mixture comprises:

[0116] Evenly mixing the PCR primer probe combination, the internal standard primer probe combination and the amplification premix solution to obtain a PCR amplification reagent;

[0117] The sample DNA to be tested is added to the PCR amplification reagent to obtain a PCR reaction mixture.

[0118] The above are only preferred embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. A composition for thyroid cancer detection, characterized in that: Contains reagents for detecting mutations or fusions at five gene sites in two genes: the BRAF gene and the RET-PTC gene.

2. The composition according to claim 1, characterized in that The mutation primers used to detect the BRAF gene mutation site V600E include sequences shown in SEQ ID NO: 1 to SEQ ID NO: 4; The mutation primers for detecting the BRAF gene mutation site V600K include sequences shown in SEQ ID NO: 5 to SEQ ID NO: 8; The common downstream primer and probe sequences for detecting the BRAF gene include sequences shown in SEQ ID NO:9 to SEQ ID NO:12, and SEQ ID NO:13 to SEQ ID NO:16; The fusion primers used to detect the RET-PTC fusion gene include sequences shown in SEQ ID NO: 17 to SEQ ID NO: 28; The common downstream primer and probe sequences for detecting RET-PTC fusion gene include sequences shown in SEQ ID NO:29 to SEQ ID NO:32, and SEQ ID NO:33 to SEQ ID NO:36; Optionally, the composition further comprises a reagent for detecting an internal reference GAPDH gene, wherein the reagent for detecting the GAPDH gene comprises a sequence shown in SEQ ID NO:37 to SEQ ID NO:48, wherein the fluorescent probe sequence has a fluorescent group VIC.

3. The composition according to claim 1, characterized in that The 5' end of the primer probe carries a fluorescent group, and the 3' end carries a quenching group. The fluorescent probe sequences respectively include FAM, VIC and CY5.

4. The composition according to claim 1, characterized in that The composition comprises at least one selected from the following: The kit includes a reverse transcription reaction solution; The reverse transcription reaction solution includes reverse transcriptase and buffer; The kit also includes a PCR primer-probe mixture; The kit also includes a PCR reaction solution; The PCR reaction solution includes Taq polymerase and buffer; The content of the Taq polymerase is 2% to 4% based on the total volume of the amplification premix, and the Taq polymerase uses deoxyribonucleotides (dNTPs) as substrates.

5. The composition according to claim 1, characterized in that The composition comprises at least one selected from the following: PCR buffer, salt ions, dNTPs, DNA polymerase or target nucleic acid.

6. A kit for diagnosing thyroid cancer, characterized in that: The kit comprises the composition according to any one of claims 1 to 5.

7. The kit according to claim 6, characterized in that It further comprises at least one of the following reagents: a DNA / RNA co-extraction reagent, a reverse transcription reaction solution, and a PCR amplification reagent.

8. An application of a composition and a kit for detecting thyroid cancer, characterized in that: The genes include BRAF gene mutation site V600E, BRAF gene mutation site V600K, and RET-PTC fusion gene, and the application includes: (1) using a co-extraction kit to simultaneously obtain RNA and genomic DNA, wherein the RNA and genomic DNA are from the sample; (2) Reverse transcription of the extracted nucleic acid to obtain cDNA; (3) Using primer sets and probes to perform fluorescence quantitative PCR detection on the transformation products to determine the gene mutation or fusion status in the sample.

9. The use according to claim 8, characterized in that: The procedure of the fluorescent PCR reaction is: The reaction was performed at 98°C for 3 min; 15 cycles of reaction at 95°C for 30 s and then 66°C for 20 s; 30 cycles of reaction at 95°C for 30 s and then 62°C for 35 s, and the fluorescence signals were collected.

10. A method for evaluating the thyroid cancer status of a subject, characterized in that: The method comprises: a) providing a sample from a subject, wherein the sample contains a target nucleic acid of the subject, wherein the target nucleic acid includes nucleic acids from BRAF gene mutation site V600E, BRAF gene mutation site V600K and RET-PTC fusion gene; b) evaluating the mutation or fusion status of the target nucleic acid; c) evaluating the thyroid cancer status of the subject based on the mutation or fusion status of the target nucleic acid; isolating the target nucleic acid from the sample; amplifying the target nucleic acid; The internal reference gene is used to determine whether the DNA content meets the detection requirements, and the Ct value of the target gene is used to determine the gene mutation or fusion status.

Images