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Specific primer pair and kit for detecting methylation of septin9 gene according to high-resolution melting curve

A technology of specific primer pairs and kits, applied in biochemical equipment and methods, microbial determination/inspection, DNA/RNA fragments, etc., can solve problems such as difficulty in achieving concentration, and achieve simple operation, good specificity and low cost. Effect

Pending Publication Date: 2020-12-11
BEIJING KANGMEI TIANHONG BIOTECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The dyes used in traditional melting curve technology will inhibit PCR at high concentrations, so it is difficult to achieve a sufficient concentration to saturate and combine with PCR products, while the saturated dyes used in HRM do not affect PCR amplification in a considerable concentration range, so they can be used with The PCR product saturates and binds to more accurately reflect the DNA melting process

Method used

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  • Specific primer pair and kit for detecting methylation of septin9 gene according to high-resolution melting curve
  • Specific primer pair and kit for detecting methylation of septin9 gene according to high-resolution melting curve
  • Specific primer pair and kit for detecting methylation of septin9 gene according to high-resolution melting curve

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0046] Embodiment 1, design and preparation of primers and preparation of standard plasmids

[0047] 1. Design and preparation of primers

[0048] Multiple primer pairs were designed based on the CpG island of septin9 gene, and the effects of multiple primer pairs were verified through preliminary experiments, and 3 primer pairs were obtained through preliminary screening.

[0049] Primer pair A consists of primer F1 and primer R1.

[0050] F1 (SEQ ID NO: 1): 5'-GTTAGTGCGAGATAGGGAGGTC-3';

[0051] R1 (SEQ ID NO: 2): 5'-CAATACAAAACTAACCGCCG-3'.

[0052] Primer pair B consists of primer F2 and primer R2.

[0053] F2: 5'-AGAAATTTTAGGATTTGGGCG-3';

[0054] R2: 5'-GACGTTTCCCACCTACTTAAACG-3'.

[0055] Primer pair C consists of primer F3 and primer R3.

[0056] F3: 5'-GTGTTGGGTTATAAGACGTTAGAATC-3';

[0057] R3: 5'-TAACAAAACAATAAACACCTCGAA-3'.

[0058] The above primers were synthesized separately.

[0059] 2. Preparation of standard quality particles

[0060] Preparation of...

Embodiment 2

[0062] Example 2. Establishment of a method for detecting methylation by methylation-specific PCR combined with HRM curve analysis

[0063] 1. Bisulfite treatment

[0064] Take 20 μl of the sample solution and perform bisulfite treatment to obtain 10 μl of the product solution, which is the template solution. The kit used for bisulfite treatment is: EZ-96 DNA Methylation-Gold MagPrep (ZYMO, product number D5042); operate according to the kit instructions.

[0065] 2. Perform PCR amplification and high-resolution melting curve analysis

[0066] The reaction system is shown in Table 1.

[0067] The instrument used for the reaction is 480II (Roche).

[0068] The amplification reaction program is shown in Table 2.

[0069] After amplification, high-resolution melting (high-resolution melting, HRM) curve analysis was performed. The parameters of the HRM curve analysis were set as follows: 65°C for 1s, then increased from 65°C to 95°C at a rate of 0.02°C / s (25 fluorescence co...

Embodiment 3

[0082] Embodiment 3, sensitivity detection

[0083] Prepare sample solution, also known as 100% methylated sample, with methylated plasmid and TE buffer. A sample solution was prepared by using methylated plasmids, unmethylated plasmids and TE buffer (the mass percentage of methylated plasmids to the total plasmids was 75%), also known as 75% methylated samples. A sample solution was prepared with methylated plasmids, unmethylated plasmids and TE buffer (the mass percentage of methylated plasmids in the total plasmids was 50%), also known as 50% methylated samples. A sample solution was prepared by using methylated plasmids, unmethylated plasmids and TE buffer (the mass percentage of methylated plasmids to the total plasmids was 25%), also known as 25% methylated samples. A sample solution was prepared by using methylated plasmids, unmethylated plasmids and TE buffer (the mass percentage of methylated plasmids in the total plasmids was 10%), also known as 10% methylated sampl...

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Abstract

The invention discloses a specific primer pair and kit for detecting methylation of a septin9 gene according to a high-resolution melting (HRM) curve. The invention firstly protects the specific primer pair, and the specific primer pair consists of a single-stranded DNA molecule as shown in SEQ ID NO:1 and a single-stranded DNA molecule as shown in SEQ ID NO:2. The invention further protects application of the specific primer pair to preparation of the kit. The kit has the functions of diagnosing or assisting in diagnosing colorectal carcinoma, detecting methylation of a promoter region of theseptin9 gene, and detecting CpG island methylation of the promoter region of the septin9 gene. By employing the primer pair provided by the invention, by combining methylation specific PCR with HRM curve, methylation of the septin9 gene is analyzed and detected, and the primer pair has the advantages of being simple and convenient to operate, good in specificity, high in sensitivity and low in cost. The specific primer pair and the kit can be used for early screening or auxiliary diagnosis of patients with the colorectal carcinoma, and have good prospects in health screening and clinical auxiliary diagnosis.

Description

technical field [0001] The invention belongs to the field of biotechnology, and relates to a pair of specific primers and a kit for detecting methylation of septin9 gene based on a high-resolution melting curve. Background technique [0002] Colorectal cancer is a common malignant tumor of the digestive tract, and its incidence rate is second only to gastric cancer and esophageal cancer. In the case fatality rate of malignant tumors in my country, colorectal cancer patients occupy the fifth place among men and the sixth place among women. In the past 20 years, the incidence of colorectal cancer has gradually increased. In my country, more than 80% of patients have developed to the middle and late stage when they are diagnosed, and the early diagnosis rate is only 10%-15%. Domestic surveys show that the early postoperative survival rate reaches 90%. -95%, compared to only 5% in the late stage. Surgical treatment can cure early colorectal cancer, but the average survival time...

Claims

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Application Information

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IPC IPC(8): C12Q1/6886C12Q1/6858C12N15/11
CPCC12Q1/6886C12Q1/6858C12Q2600/154C12Q2531/113C12Q2523/125C12Q2527/107C12Q2563/107
Inventor 柳辉
Owner BEIJING KANGMEI TIANHONG BIOTECH
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