A composition, kit and use for thyroid cancer detection

Abstract

The application belongs to the field of biological medicine, and relates to a composition, a kit and application for thyroid cancer detection. The primer composition can be used for simultaneously detecting mutation or fusion of five gene sites of two genes, BRAF gene and RET-PTC gene, and can be used for detecting thyroid cancer and has high sensitivity and specificity. Meanwhile, the fluorescence PCR technology is adopted to improve detection efficiency, shorten detection time and realize precise diagnosis of thyroid cancer.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of gene detection, and particularly relates to a composition, a kit and application for thyroid cancer detection. BACKGROUND

[0002] Thyroid cancer is a common malignant tumor of the endocrine system, and main histological types include papillary carcinoma and follicular carcinoma. At present, the diagnosis of benign and malignant thyroid nodules mainly relies on ultrasound and fine needle aspiration (FNA) under ultrasound guidance. In the Bethesda system for pathological diagnosis of thyroid cells, types III to V are uncertain diagnoses. According to the Chinese thyroid cancer diagnosis and treatment specification (2018 edition), thyroid nodules that cannot be determined as benign or malignant by FNA can be subjected to detection of certain thyroid cancer markers.

[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 is most studied in thyroid FNA cytology, and the mutation rate of papillary carcinoma can reach more than 80%, and RET-PTC fusion occurs in 15% to 20% of papillary carcinomas. BRAF gene V600E / K mutation is significantly associated with the occurrence of thyroid papillary carcinoma, while RET-PTC fusion gene is commonly found in medullary thyroid carcinoma. Detection of these gene variations can provide a deep understanding of the mechanism of cancer occurrence and help develop individualized treatment plans.

[0004] Traditional gene variation detection methods such as histopathological examination and immunohistochemical staining have certain accuracy, but have defects such as complex operation and low sensitivity. Therefore, developing a high-sensitivity and high-specificity gene detection technology is of great significance for improving the early diagnosis and treatment of thyroid cancer. SUMMARY

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

[0006] Table 1

[0007]

[0008] To achieve the above purpose, the present application adopts the following technical solutions:

[0009] In the first aspect of the present application, the present application provides a composition for thyroid cancer detection, comprising thyroid cancer detection reagents for detecting the mutation or fusion state of three sites of BRAF gene mutation site V600E, BRAF gene mutation site V600K and RET-PTC gene fusion site. The present application can effectively improve the detection performance by jointly detecting the mutation or fusion state of BRAF mutant gene and RET-PTC fusion gene, and the sensitivity and specificity are both above 90%.

[0010] In the second aspect of the present application, the present application provides a kit for thyroid cancer diagnosis, which comprises the composition for thyroid cancer detection of the first aspect described above.

[0011] In the third aspect of the present application, the present application provides a method for determining the mutation or fusion state of a gene in a sample, wherein the gene comprises 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 is from the sample;

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

[0014] (3) performing fluorescence quantitative PCR detection on the reverse transcription product using a primer set and a probe set to determine the mutation or fusion state of the gene in the sample.

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

[0016] The PCR primer probe set comprises at least one of the sequences shown in the table:

[0017]

[0018]

[0019] Preferably, the 5' end of the primer probe is provided with a fluorescent group, and the 3' end is provided with 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 set for detecting the internal reference gene GAPDH, and the fluorescent group is selected from VI C.

[0021] The internal standard primer probe combination comprises:

[0022]

[0023]

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

[0025] The amplification premix comprises 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 is a substrate for deoxynucleotides (dNTPs).

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

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

[0029] The sample used is a thyroid cancer solid tissue of a detection object.

[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 detection sample is fresh surgical tissue and / or puncture tissue.

[0031] The fifth aspect of the embodiments of the present application provides a non-diagnostic detection method for thyroid cancer, which applies 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 comprises:

[0032] Obtaining the RNA and genomic DNA of the sample to be tested, and confirming the sample type.

[0033] Adding the nucleic acid sample to be tested to a 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] Setting the PCR reaction conditions, and after the PCR reaction mixture is amplified by PCR, the detection result of the kit is interpreted and analyzed.

[0036] The PCR amplification reagent comprises a PCR primer probe combination, an internal standard primer probe combination and an amplification premix.

[0037] Compared with the prior art, the BRAF gene mutation site V600E, the BRAF gene mutation site V600K and the RET-PTC fusion gene in the application can accurately amplify and detect the mutation or fusion of the target gene by the high-efficiency combination of specific primers and probes through the fluorescence PCR technology, have high sensitivity and specificity, reduce the occurrence of false negative and false positive, and are simple and fast in detection work, so that the detection efficiency is improved and the detection time is shortened, and are suitable for clinical rapid screening of thyroid cancer. BRIEF DESCRIPTION OF DRAWINGS

[0038] Figure 1 is a sample amplification graph provided by the application without gene mutation / gene fusion;

[0039] Figure 2 is a sample amplification graph of BRAF-V600K mutation provided by the application;

[0040] Figure 3 is a sample amplification graph of BRAF-V600E mutation provided by the application;

[0041] Figure 4 is a sample amplification graph of CCDC6 exon1 / RET exon12 fusion provided by the application;

[0042] Figure 5 is a sample amplification graph of PRKAR1A exon7 / RET exon12 fusion provided by the application;

[0043] Figure 6 is a sample amplification graph of NCOA4 exon8 / RET exon12 fusion provided by the application. DETAILED DESCRIPTION

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

[0045] It should be noted that the kit is only used for detecting the target gene sequence of a suspected thyroid cancer patient, and the detection result is only for clinical reference and should not be used as the only basis for individualized treatment of the patient. The clinician should comprehensively judge the detection result in combination with the patient's condition, drug indications, treatment response and other laboratory detection indexes and the like.

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

[0047] REFERENCE Figure 1The embodiment provides a thyroid cancer detection kit based on a BRAF mutant gene and a RET-PTC fusion gene mutation or fusion, and the kit comprises a PCR primer probe combination.

[0048] The PCR primer probe combination comprises:

[0049]

[0050]

[0051] The kit is used for qualitatively detecting a BRAF mutant gene and a RET-PTC fusion gene mutation or fusion in a thyroid puncture sample, and is used for assisting a clinician and a pathologist in differentiating a benign nodule from a malignant nodule of a thyroid, and realizing accurate diagnosis of thyroid cancer.

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

[0053] As an implementation form, 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.

[0054] The internal standard primer probe combination comprises:

[0055]

[0056]

[0057] As an implementation form, the kit further comprises an amplification premix.

[0058] The amplification premix comprises Taq polymerase.

[0059] The content of the Taq polymerase is 2% to 4% of the total volume of the amplification premix, and the Taq polymerase takes deoxynucleotides (dNTPs) as a substrate.

[0060] The kit further comprises a positive control and a negative control.

[0061] The positive control is a mutant / fusion positive nucleic acid, and the negative control is a wild-type nucleic acid. The detection results of the positive control and the negative control in each detection can be used as an important index of the performance of the kit. If the detection results of the positive control or the negative control do not meet the expectation, it may indicate that there is a problem in the kit, such as invalidation of reagents, nucleic acid degradation caused by improper storage or reduction of primer probe activity. This is helpful for timely finding the quality problem of the kit and avoiding incorrect detection results caused by use of unqualified kits.

[0062] In addition, the kit further comprises reagents for sample processing and preparation, including reverse transcription reaction solution, PCR primer probe mixture solution, and PCR reaction solution.

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

[0064] Table 2

[0065]

[0066]

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

[0068] In the present 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 12000 rpm / min for 5 min with the punctured sample;

[0072] (2) After removing the supernatant, perform nucleic acid extraction.

[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) After mixing and centrifuging, perform reverse transcription reaction according to the following table procedure.

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

[0077] Table 3

[0078] Step Temperature (°C) Time Cycle number First step 37 15 minutes 1 Second step 85 5 seconds 1 Third step 4 3 minutes (instrument cooling) 1

[0079] 3. Fluorescent PCR detection 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 for each reaction).

[0082] Table 4

[0083] Reagent component Amount / person (μL) PCR reaction solution 10～20 PCR primer probe mixture 8～12 Total amount / test 18～32

[0084] 3.2 Add sample:

[0085] Take 2-10 μL of the positive control, negative control, and cDNA from the sample to be tested and add them to the aliquoted eight-tube strips. Tightly cap the eight-tube strips and centrifuge briefly to remove any liquid from the tube walls to the bottom.

[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 test is completed, the test result is determined 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 in 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 following methods can be used to effectively interpret the test results: if the internal standard gene test (VIC channel) shows typical amplification, the analysis can continue; if the internal standard gene test (VIC channel) does not show amplification, the test needs to be repeated.

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

[0097] 1. If the target gene of the sample (FAM channel and CY5 channel) does not have a 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 determined 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 sample test result 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 sample test result is determined to be positive for the BRAF gene mutation.

[0099] The results of this embodiment are shown below:

[0100] The 186 puncture samples are detected, 79 of which are thyroid benign lesion samples, and 107 of which are thyroid cancer patient samples, the sensitivity and specificity of the single gene mutation / fusion detection result and the two gene mutation / fusion combined detection result are analyzed, the comparison between the detection result of the application and the clinical result is shown in Table 6, and the comparison between the detection result of the application and the first generation sequencing detection result is shown in Table 7:

[0101] Table 6

[0102]

[0103]

[0104] Table 7

[0105]

[0106] The application can effectively improve the detection performance, the sensitivity and the specificity are both above 90% by jointly detecting the mutation or fusion state of the BRAF mutant gene and the RET-PTC fusion gene compared with single gene methylation detection, and the detection performance of the application is good.

[0107] The sensitivity and the specificity are both above 95% by comparing the detection of the BRAF mutant gene with the first generation sequencing result, and the sensitivity and the specificity are both above 95% by comparing the detection of the RET-PTC fusion gene with the first generation sequencing result.

[0108] The thyroid cancer detection method according to the second aspect of the application will be described below.

[0109] The application also provides a non-diagnostic detection method for thyroid cancer, which uses the mutation or fusion of the five gene sites of the two genes of the BRAF gene and the RET-PTC gene to diagnose the effect of thyroid cancer, and the detection method comprises the following steps.

[0110] Obtaining a nucleic acid sample (DNA / RNA) to be detected, and confirming the sample type;

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

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

[0113] Setting the PCR reaction conditions, and interpreting and analyzing the detection result of the kit after the PCR reaction mixture is amplified by PCR;

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

[0115] The method comprises the following steps:

[0116] The PCR primer probe combination, the internal standard primer probe combination and the amplification premix are mixed uniformly to obtain the PCR amplification reagent;

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

[0118] The above is only the preferred embodiment of the present application, and is not used to limit the present application, any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present application shall be included in the protection scope of the present application.

Claims

1. A composition for thyroid cancer detection, characterized in that: The composition is a reagent for detecting mutations or fusions at five gene sites of two genes, the BRAF gene and the RET-PTC gene; Among them, the mutation primers used to detect the BRAF gene mutation site V600E include the sequences shown in SEQ ID NO: 1 to SEQ ID NO: 4; The mutation primers for detecting the BRAF gene mutation site V600K include the 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 the RET-PTC fusion gene include the sequences shown in SEQ ID NO: 29 to SEQ ID NO: 32 and SEQ ID NO: 33 to SEQ ID NO:

36.

2. The composition according to claim 1, characterized in that The composition further includes a reagent for detecting an internal reference GAPDH gene, wherein the reagent for detecting an internal reference GAPDH gene includes a sequence shown in SEQ ID NO: 37 to SEQ ID NO: 48; wherein the probe sequence has a fluorescent group VIC.

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

4. The composition according to claim 1, characterized in that The composition further comprises at least one selected from the following: a reverse transcription reaction solution, a PCR primer-probe mixture; Wherein, the reverse transcription reaction solution includes reverse transcriptase and buffer.

5. The composition according to claim 1, characterized in that The composition further 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 The kit further comprises at least one of the following reagents: a DNA / RNA co-extraction reagent and a PCR amplification reagent.

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