Kit for detecting K-ras gene mutation and detection method with kit

A kit and mutation-type technology, applied in biochemical equipment and methods, microbiological determination/inspection, DNA/RNA fragments, etc., can solve problems that are not well applicable, difficult to detect, and low mutation rate of K-ras gene and other issues to achieve the effects of reducing the risk of contamination, high sensitivity, and fast detection speed

Active Publication Date: 2013-05-22
CHANGSHA 3G BIOTECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, detection methods with a sensitivity of >5% such as sequencing are still mostly used clinically, and the detection sensitivity of the mutant gene-specific amplification PCR method (ARMS) is generally between 1% and 5%, which is not well applicable to serum or plasma. K-ras gene mutation detection
In the June 2012 issue of Nature, two research teams f

Method used

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  • Kit for detecting K-ras gene mutation and detection method with kit
  • Kit for detecting K-ras gene mutation and detection method with kit
  • Kit for detecting K-ras gene mutation and detection method with kit

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0051] Example 1: Establishment of a high-sensitivity K-ras gene mutation detection system

[0052] 1. Design and synthesis of primers, PNA and probes

[0053] For the No. 1 exon region of K-ras gene, 4 fluorescent probes and corresponding PCR primer pairs were designed, and a probe and its corresponding primer pair were optimized through a large number of experiments. Both primers and fluorescent probes were synthesized by a professional company, and the fluorescent reporter group at the 5' end of the labeled probe was FAM; the fluorescent quencher group at the 3' end of the labeled probe was MGB. The PNA sequence was synthesized by a domestic professional company.

[0054] 2. Preparation of K-ras gene wild type and its seven common mutant plasmid template standards

[0055] 1) Colorectal cancer tumor tissue specimens of K-ras wild type (SEQ ID NO:5) and seven common mutant types (SEQ ID NO:6~SEQ ID NO:12) were collected from the hospital, and DNA was extracted, wild type D...

Embodiment 2

[0087] Example 2: Comparison between this method and the sequencing method for detecting K-ras gene mutation in colorectal cancer

[0088] 1. DNA extraction

[0089] Seventy-eight fresh colorectal cancer tissue specimens were collected from a cancer hospital, and DNA extraction was performed using tissue DNA extraction kits.

[0090] 2. The method of the present invention detects K-ras gene mutation

[0091] Refer to Table 1 to configure a 50 μl PCR reaction system, 10 3 wild-type K-ras plasmid as a negative control, 2 μl ddH 2 O was used as a blank control, and the mixed plasmid with a 1% mutation ratio was used as a positive control. ℃ 30s) for 40 cycles; the fluorescence detection channel was set to FAM, and the gain was set to 6.

[0092] 3. The method of the present invention detects the qualitative judgment of K-ras gene mutation

[0093] The CT value without adding PNA in the reaction system is defined as CT -PNA , the CT value of adding PNA to the reaction syst...

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Abstract

The invention discloses a kit for detecting K-ras gene mutation and a detection method with the kit. The kit comprises an amplification primer pair, a TaqMan-MGB (minor groove binder) probe and PNA (pentose nucleic acid), wherein the sequences of amplification primers are shown as SEQ ID NO:1 and SEQ ID NO:2; and the sequence of the TaqMan-MGB probe is shown as SEQ ID NO:3; and the sequence of PNA is shown as SEQ ID NO:4. The detection method comprises the following steps of: extracting DNA (deoxyribonucleic acid) of a sample to be detected; preparing K-ras wild and mutation plasmid standard products; and detecting the K-ras gene mutation, performing qualitative judgment on the K-ras gene mutation, drawing a standard curve, performing quantitative analysis on the mutation, and the like. The kit has the characteristics of quickness, simplicity, convenience, high sensitivity and high flux, and can be used for performing qualitative or quantitative detection on the K-ras gene mutation of clinical samples of tissue, urea, serum or plasma.

Description

technical field [0001] The invention belongs to the technical field of molecular detection, and in particular relates to a kit for quantitatively detecting whether K-ras mutant gene is contained in tissue, urine, serum or plasma and a detection method thereof. Background technique [0002] The mutational activation of K-ras is one of the main reasons for the malignant transformation of various human tumor cells. It is like a molecular switch, which can control and regulate the path of cell growth in normal conditions; when it is abnormal, it will lead to uncontrolled cell proliferation and prevent cell self-destruction. K-ras gene mutations mostly occur at the 12th and 13th codons, and this abnormality is found in pancreatic cancer (75%-95%), colorectal cancer (40%-50%) and lung cancer (20%-30%) and other tumors The incidence rate in tissues is relatively high, and K-ras gene mutations can often be detected in the serum or plasma of patients with these tumors. [0003] At p...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12N15/11G01N21/64
Inventor 张戈
Owner CHANGSHA 3G BIOTECH
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