Gene methylation detection kit based on multiple fluorescent quantitative PCR detection means and application thereof

Abstract

The invention belongs to the field of gene detection, and particularly relates to a gene methylation detection kit based on multiple fluorescent quantitative PCR detection means and application of the gene methylation detection kit. The kit comprises a CRC-PCR reaction solution, a primer probe mixed solution 1, a positive quality control product, a negative quality control product and five pairs of primers of five genes. Based on the primer and probe design and the experimental verification of a reaction program, the kit can detect the DNA template amount as low as 5ng, and when the methylation rate in the template is as low as 1%, the DNA template amount can be detected.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of gene detection, and particularly relates to a gene methylation detection kit based on a multiplex fluorescence quantitative PCR detection method and application thereof. BACKGROUND

[0002] Colorectal cancer (CRC) is the fourth most common cancer and the third leading cause of cancer death worldwide, causing more than 500,000 deaths each year, accounting for 10% of new cancer diagnoses; more than 10% of patients are diagnosed with advanced disease, and about 30% of early CRC patients will develop metastatic disease; due to the fact that clinical symptoms are usually identified in the late stage, late diagnosis leads to a decrease in the survival rate of cancer patients; the 5-year relative survival rate for early CRC is about 90%; for advanced stage IV CRC, the relative survival rate decreases to about 14%; although most CRC patients achieve remission in initial treatment, 30% to 40% of patients will relapse. Therefore, in order to detect relapse early, patients usually enter a follow-up program, including regular blood tests, radiological examinations, and colonoscopy. The currently used blood biomarker carcinoembryonic antigen (CEA) has limited sensitivity and specificity for relapse. Detecting better blood markers should help identify patients at risk of relapse or in need of prolonged initial treatment, such as increased or prolonged chemotherapy, for insufficient initial treatment.

[0003] DNA methylation is an important way to regulate processes such as gene expression, cell development, and cell differentiation at the level of epigenetics. Abnormal methylation plays an important role in the process of cancer occurrence. In addition to identifying the potential role of tumor occurrence, methylation of specific genes can serve as a marker for different cancer types for diagnosis and classification of cancer. The development of colon cancer is accompanied by extensive epigenetic changes.

[0004] The main means for detecting colorectal cancer at present is colonoscopy, but this detection method causes people to have different degrees of pain during the process, and even causes complications in severe cases. At the same time, colonoscopy requires more endoscopes, and the universality of colonoscopy for colorectal cancer screening is a challenge. Fecal occult blood testing is another relatively desirable detection method. However, this method detects globin in hemoglobin, not hematin, making it more specific to human blood. Due to its convenience and quantitative analysis, it is more sensitive to advanced colorectal cancer tumors. Carcinoembryonic antigen detection (CEA) has limited clinical application due to its lack of sensitivity and specificity. SUMMARY

[0005] To solve the above problems, the application provides a kit, which comprises a CRC-PCR reaction solution, a primer probe mixture 1, positive quality control, negative quality control and primers; the sequence of the primers is shown in SEQ. No. 1, SEQ. No. 2, SEQ. No. 3, SEQ. No. 4, SEQ. No. 5, SEQ. No. 6, SEQ. No. 7, SEQ. No. 8, SEQ. No. 9, SEQ. No. 10, SEQ. No. 11 and SEQ. No. 12.

[0006] The application further provides application of the kit in detection of DNA methylation.

[0007] The application further provides application of the kit in detection of recurrent or metastatic colorectal cancer.

[0008] The application further provides application of the kit in detection of recurrence of colorectal cancer after surgery.

[0009] The application has the following beneficial effects:

[0010] The application adopts the method of multiplex PCR detection, and specific primers are designed and screened for the screened target sequences, thereby overcoming the problem of low sensitivity of single gene detection and improving the sensitivity and specificity of the kit detection.

[0011] In the primer design, the fluorescence quenching group is selected as MGB, which can improve the Tm value of the fluorescence probe, so that the PCR reaction amplification program is compatible with higher annealing / extension temperature, and high-temperature annealing can eliminate the non-specific amplification of the primers and improve the specificity of the kit detection.

[0012] Based on the above primer, probe design and experimental verification of the reaction program, the kit can detect as low as 5 ng of DNA template amount, and when the methylation rate in the template is as low as 1%, it can be detected. BRIEF DESCRIPTION OF DRAWINGS

[0013] In order to more clearly illustrate the technical solutions in the embodiments of the application or the prior art, the drawings needed in the embodiments will be briefly introduced below. Obviously, the drawings in the following description only constitute some embodiments of the application, and other drawings can be obtained by those skilled in the art without creative labor on the basis of these drawings.

[0014] Figure 1 ACTB of different methylation degree samples;

[0015] Figure 2 BCAT1 of different methylation degree samples;

[0016] Figure 3IKZF1 in different methylation level samples;

[0017] Figure 4 Septin9 in different methylation level samples;

[0018] Figure 5 SDC2 in different methylation level samples;

[0019] Figure 6 VAV3 in different methylation level samples. DETAILED DESCRIPTION

[0020] Various exemplary embodiments of the present application will now be described in detail, with the methods being carried out in accordance with conventional methods unless otherwise specified, and with the reagents being conventional commercially available reagents or reagents prepared according to conventional methods unless otherwise specified. This detailed description is not to be taken in a limiting sense and is merely describing some aspects, features, and embodiments of the present application.

[0021] It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the present application. Additionally, the use of the term "about" in relation to a value or a range of values is to be understood to include each individual value or range of values falling within the range of the stated value or range of values. In any statement of a value or a range of values, the intermediate value or values between the stated value or range of values are included in the present application. The upper and lower limits of these smaller ranges can independently be included or excluded in the range.

[0022] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. Although preferred methods and materials are described, any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present application. All documents mentioned herein are incorporated by reference to disclose and describe in full the methods and / or materials which are described therein. In the case of conflict between the present specification and any document incorporated herein by reference, the present specification will control.

[0023] Various modifications and changes can be made to the specific embodiments of the present application described herein without departing from the scope or spirit of the application. Other embodiments of the application will be apparent to those of ordinary skill in the art from the description and examples herein. The description and examples are illustrative of the application and are not intended to limit the scope of the application.

[0024] As used herein, the terms "comprises", "comprising", "includes", "including", "has", "having", "contains", "containing", and the like are open-ended terms that are intended to denote the inclusion of elements or steps, but not to exclude any other elements or steps.

[0025] The kit combines bisulfite modification and Taqman probe technologies, uses bisulfite to convert unmethylated C bases to U bases, and converts them to T bases during PCR amplification, while methylated C bases remain unchanged to distinguish between methylated and unmethylated C bases; the Taqman probe is used to detect the amplification product, and through the use of an optimized reaction system and high-specificity Taq enzyme, specific detection of methylated DNA in the sample is realized on a real-time fluorescent PCR platform. The kit uses a multi-color fluorescent detection system design, FAM fluorescence detects the methylation DNA state of BCAT1 and Septin9 genes, VIC fluorescence detects the methylation DNA state of IKZF1 and IRF4 genes, and CY5 fluorescence detects the internal standard gene level.

[0026] Multiplex PCR (multiplex PCR), also known as multiplex primer PCR or composite PCR, is a PCR reaction in which two or more primers are added to the same PCR reaction system to amplify multiple nucleic acid fragments at the same time. The reaction principle, reaction reagents and operation process are the same as general PCR.

[0027] The application provides a primer pair set for detecting gene methylation.

[0028] Among them, BCAT1 can be significantly highly expressed when CRC metastasis occurs. It can detect recurrent / metastatic colorectal cancer with higher sensitivity, especially in patients with liver metastasis or lung metastasis, thus having a significant monitoring effect in predicting CRC recurrence / metastasis. At the same time, BCAT1 is more sensitive than CEA in detecting colorectal cancer recurrence (8, 9, 10), and its sensitivity is twice that of CEA. Studies have shown that significant methylation of BCAT1 can be observed in more than 95% of CRC tissues. Compared with mutation detection in genomics, monitoring means in epigenetics is more convenient and fast.

[0029] IKZF1 gene encodes a transcription factor protein with zinc finger structure, which regulates hematopoietic differentiation and immune system development, and has anticancer effect (11, 12). BCAT1 and IKZF1 hypermethylation occurs in 95% of colorectal cancer tumors and can be detected in the blood of colorectal cancer patients (13, 14). Methylation of BCAT1 / IKZF1 is a better prognostic marker than CEA after colorectal cancer surgery or during adjuvant chemotherapy. BCAT1 and IKZF1 have low methylation levels or even lack methylation in normal tissues. Studies have shown that qualitative blood tests for BCAT1 and IKZF1 methylation in cfDNA are more sensitive than CEA and can predict the recurrence of patients who are monitored after primary colorectal cancer resection (8, 10).

[0030] Methylation Septin9 is a reliable biomarker for detecting colorectal cancer, Septin9 is located on chromosome 17q25.3, is a conservative skeleton protein gene, involved in cell division and cytoskeleton organization. In the development of colorectal cancer, methylation Septin9 DNA is released from necrotic and apoptotic cancer cells into the peripheral blood, so the risk of colorectal cancer can be determined by detecting the degree of DNA methylation of Septin9 specific promoter region in peripheral blood. When the cancerous tissue is radically resected by surgery, if the higher methylation level of Septin9 is detected again, it may show a poor prognosis (11, 12) which can be used as an auxiliary monitoring means for postoperative recurrence diagnosis of colon cancer patients.

[0031] Main components of the kit:

[0032] CRC-PCR reaction solution, primer probe mixture 1, positive quality control, negative quality control

[0033] ACTB-F: 5'-GGAGTTTTTGTTTTTTGG TTAGTTG-3'

[0034] ACTB-R: 5'-CAAAATAAAATACAAAACAAACCTAATCC-3'

[0035] ACTB-P: 5'-CY5-ATGGAGGTTTAGTGGTAATATAGGTTTTGTT TGG-IABkFQ

[0036] BCAT1-F: 5'-ATGTAATTCGTTAGGTCGCG-3'

[0037] BCAT1-R: 5'-CAATACCCGAAACGACGACG-3'

[0038] BCAT1-P-: 5'FAM-TCGTCGCGAGAGGGTCGGTT-MGB-3'

[0039] IKZF1-F: 5'-GACGACGTATTTTTTTCGTGTTTC-3'

[0040] IKZF1-R: 5'-GCGCACCTCTCGACCG-3'

[0041] IKZF1-P: 5'-VIC-TTTGTAT C G-ZEN-GAGTAG C GATT C GGGAG G-MGB-3'

[0042] Septin9-F: 5'-GCGCGATTCGTTGTTTATTAGTT-3'

[0043] Septin9-R: 5'-AATAATCCCATCCAACTACGCG-3'

[0044] Septin9-P: 5'-Texas Red-TTAACCGCGAAATCCGAC-MGB-3' By designing long fragment forward and reverse primer pairs and fluorescence quenching groups, the Tm value of the fluorescence probe is improved, so that the qPCR amplification reaction program can be compatible with higher annealing / extension temperature, reducing the temperature difference between annealing / extension temperature and denaturation temperature, shortening the time required for heating and cooling, thereby effectively shortening the qPCR amplification reaction time. At the same time, high-temperature annealing further eliminates non-specific amplification and improves detection specificity.

[0045] A specific operation:

[0046] 1. Take the heavy methionine converted DNA of the sample to be tested and the positive and negative quality control products and shake them well. Centrifuge for standby.

[0047] 2. Thaw the reagent kit at room temperature, shake well, and centrifuge for standby.

[0048] 3. According to the number of detection samples n, plus the negative and positive controls and the loss, calculate the volume of PCR reaction liquid and primer probe mixed liquid needed. The following table is an example. Configure PCR Mix in two new EP tubes, mix well, and centrifuge.

[0049] PCR Mix preparation

[0050]

[0051] 4. Divide the two reaction Mixes into 15 μL / tube in PCR reaction tubes, respectively add 5 μL of DNA sample and blank control, positive quality control after conversion in step 1, press the tube cover, shake well, centrifuge, and remove the bubbles.

[0052] 5. Carefully place the detection reaction tube in the PCR amplification detector, set the PCR amplification parameters according to the following table, and set the target gene fluorescence reporter group as FAM, VIC, Texas Red fluorescence, the internal reference gene fluorescence reporter group as CY5 fluorescence, and the quenched fluorescence group as NFQ-MGB.

[0053] FAM channel VIC channel Texas Red channel CY5 channel Reaction Mix BCAT1 IKZF1 Septin9 ACTB

[0054] 6. Set the PCR reaction program according to the following steps:

[0055]

[0056] 7. After the sample is put into the machine, save the file and run the program.

[0057] 8. ABI 7500 Software analysis software Threshold value setting: FAM channel Threshold value is set to 25000; CY5 channel Threshold value is set to 10000.

[0058] Two positive judgment values and test result interpretation

[0059] 1. The blank control quality control product FAM / VIC / Texas Red and CY5 channel has no signal, indicating that there is no pollution in the test, and the result analysis can continue.

[0060] 2. The positive quality control FAM / VIC / Texas Red channel Ct value is ≤33, and the CY5 channel Ct value is ≤35, indicating that the experimental system is normal, and the result analysis can continue.

[0061] 3. All detection sample reaction wells should have CY5 signal, and the CY5 signal Ct value is ≤35. If there is no CY5 signal, it indicates that the added DNA contains PCR inhibitors or DNA processing fails, and the DNA needs to be re-extracted and bisulfite treated.

[0062] 4. Detect the FAM channel of the sample well. If an S-shaped amplification curve appears and the Ct value is ≤40, the sample is BCA T1 gene methylation positive; if no amplification curve appears or the Ct value is >40, the sample is BCA T1 gene methylation negative.

[0063] 5. Detect the VIC channel of the sample well. If an S-shaped amplification curve appears and the Ct value is ≤40, the sample is IKZF1 gene methylation positive; if no amplification curve appears or the Ct value is >40, the sample is IKZF1 gene methylation negative.

[0064] 6. Detect the Texas Red channel of the sample well. If an S-shaped amplification curve appears and the Ct value is ≤40, the sample is Septin9 gene methylation positive; if no amplification curve appears or the Ct value is >40, the sample is Septin9 gene methylation negative.

[0065] 7. When detecting the sample, at least one of BCA T1, IKZF1, and Septin9 genes is methylation positive, then the sample is determined to be methylation detection positive.

[0066] 8. When detecting the sample, BCA T1, IKZF1, and Septin9 genes are all methylation negative, then the sample is determined to be methylation detection negative.

[0067] The three technical solutions of the application bring the beneficial effects

[0068] The application can realize high sensitivity and specificity of detection of BCAT1, IKZF1 and Septin9 gene methylation in human plasma free DNA, can realize rapid detection, and can reduce the probability of non-specific amplification and false positive results, and improve the detection sensitivity. The detection capability for low methylation degree samples is considerable: under the premise of 5ng cfDNA input in each reaction, the minimum detection limit of the methylation frequency of 6 target genes in the sample is 1%. As shown in the figure, 5ng 1%; 5ng 5%; 5ng 10%.

[0069] The above-described embodiments are only descriptions of the preferred modes of the application and do not limit the scope of the application. Without departing from the design spirit of the application, various modifications and improvements to the technical solutions of the application made by those skilled in the art shall fall within the protection scope determined by the claims of the application.

Claims

1. A kit, characterized in that: It includes CRC-PCR reaction solution, primer-probe mixture 1, positive quality control product, negative quality control product and primers; the sequences of the primers are shown in SEQ.NO.1, SEQ.NO.2, SEQ.NO.3, SEQ.NO.4, SEQ.NO.5, SEQ.NO.6, SEQ.NO.7, SEQ.NO.8, SEQ.NO.9, SEQ.NO.10, SEQ.NO.11 and SEQ.NO.

12.

2. Use of the kit as claimed in claim 1 in detecting DNA methylation.

3. Use of the kit according to claim 1 in detecting recurrent or metastatic colorectal cancer.

4. Use of the kit according to claim 1 in detecting postoperative recurrence of rectal cancer.

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