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Rapid detection of KRAS (Kirsten Rat Sarcoma) gene mutation

A gene and reaction reagent technology, applied in the fields of biotechnology and medicine, can solve the problems of time-consuming, easy pollution, and cumbersome KRAS gene mutation detection procedures.

Active Publication Date: 2010-11-03
JIANGSU MICRODIAG BIOMEDICINE TECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0011] The purpose of the present invention is to provide a fast, accurate, easy-to-operate and pollution-free detection kit for detecting KRAS gene mutations, which solves the cumbersome, expensive, time-consuming, low-sensitivity and easy-to-pollution procedures for KRAS gene mutation detection in the prior art etc., especially providing highly sensitive detection of mutations in non-invasive serum or plasma in addition to pathological tissue

Method used

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  • Rapid detection of KRAS (Kirsten Rat Sarcoma) gene mutation
  • Rapid detection of KRAS (Kirsten Rat Sarcoma) gene mutation
  • Rapid detection of KRAS (Kirsten Rat Sarcoma) gene mutation

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0090] Verification and screening of embodiment 1 primers

[0091] Primers were designed according to the sequences of the second exon and the third exon of the KRAS gene. The different sequence lengths of the primers were amplified and compared, and the primers with good specificity were screened. The specificity results of the PCR amplification of the primers with the size of the primer fragments are as follows and figure 1 Shown:

[0092] The primers of table 1 primer fragment size carry out the specificity result of PCR amplification

[0093] Primer large

small (bp)

5-9

13-16

18-23

24-28

30-37

39-45

specificity

very specific

Difference

poor specificity

good specificity

specificity

it is good

poor specificity

specificity

very poor

[0094] figure 1 a is the electropherogram of the primer PCR amplification of 5-9bp, figure...

Embodiment 2

[0096] The extraction of embodiment 2 negative control DNA

[0097] Negative control DNA was extracted according to the following steps: Take 2ml of mixed anticoagulated whole blood from normal people, and then use TIANamp Genomic DNA Kit (TIANGEN BIOTECH CO., LTD) Genomic DNA Extraction Kit to extract DNA. DNA concentration was quantified using a Nano 1000 quantifier.

[0098] Negative control DNA can be considered qualified when it meets the following conditions: 1. OD value A260 / A230: 2.0-2.2; A260 / A280: 1.8-2.0; 2. The main band of the electrophoresis band is clear (loaded sample 6ul, 1.0 % agarose gel, 120V voltage, 25 minutes), the electrophoresis is as follows image 3 3. Sequencing identified no abnormal mutations in exons 2 and 3 of the KRAS gene. Sequencing results such as Figure 4 shown.

Embodiment 3

[0099] Example 3 Extraction of Tumor Tissue DNA

[0100] The sample collection is carried out according to the following steps: Take 50-100 mg of fresh tissue block and wash it with PBS or normal saline. The tissue used must be cut to a thickness of less than 0.5 cm, put it into a cryopreservation tube with a volume of 1.5 ml RNAlater, and mix well (Complete within 30 minutes). Store at room temperature for 1-2 hours, freeze overnight at 4°C, and transfer to -20°C for long-term storage the next day.

[0101] DNA extraction was carried out according to the following steps: use TIANamp Genomic DNA Kit (TIANGENBIOTECH CO., LTD) Genomic DNA Extraction Kit, and follow the instructions of the kit to extract DNA. Then carry out DNA purification and detection. If the OD value of the extracted DNA is not within the standard range of A260 / A230: 2.0-2.2; A260 / A280: 1.8-2.0, a DNA purification step is required.

[0102] DNA purification was carried out as follows: add an equal volume o...

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Abstract

The invention discloses a PCR (Polymerase Chain Reaction) reaction kit for detecting KRAS (Kirsten Rat Sarcoma) gene mutation, which is characterized by comprising a plurality of primer pairs used for specifically amplifying a targeting sequence of the KRAS gene, and each primer pair contains a continuous nucleotide sequence formed by at least 15 continuous nucleotides in a second exon KRAS2 or athird exon KRAS 3 of the KRAS gene, wherein the KRAS2 has the continuous nucleotide sequence of SEQ ID No:1, and the KRAS3 has the continuous nucleotide sequence of the SEQ ID No: 2. The kit completes the judgment on a genotype sample by adopting a saturated probe and a high resolution fusion curve analysis technology so as to provide guidance on medicine selection and diagnosis for various tumors including lung cancers and colorectal cancers.

Description

technical field [0001] The invention relates to the fields of biotechnology and medicine, in particular to a rapid detection of KRAS gene mutations related to selection and diagnosis of tumor medication. Background technique [0002] The ras gene family has three genes related to human tumors—HRAS, KRAS and NRAS, which are located on chromosome 11, 12 and 1, respectively. KRAS is also known as the p21 gene because it encodes a 21kD ras protein. Among the ras genes, KRAS has the greatest impact on human cancer. It is a molecular switch: when normal, it controls the pathways that regulate cell growth; when abnormal, it causes cells to continue to grow and prevents cells from self-destruction. It participates in intracellular signal transmission. When the KRAS gene is mutated, the gene will be permanently activated, unable to produce normal ras protein, causing intracellular signal transduction disorder, cell proliferation out of control and cancerous. The detection of KRAS g...

Claims

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Application Information

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IPC IPC(8): C12Q1/68G01N21/64
Inventor 王弢秦勇陈菲李晓燕
Owner JIANGSU MICRODIAG BIOMEDICINE TECH CO LTD
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