KRAS gene mutation detection quantification standard product as well as preparation method and valuing method thereof
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A quantitative standard product and genome technology, applied in the direction of biochemical equipment and methods, microbial determination/inspection, etc., to achieve the effect of improving efficiency and reducing the cost of testing quality control
Active Publication Date: 2018-02-06
NAT INST OF METROLOGY CHINA
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[0004] At present, there is no quantitativ...
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Embodiment 1
[0100] Preparation and determination of quantitative standards for 7 mutations of KRAS gene
[0101] 1. Materials and methods:
[0102] 1. Cell Lines and Media
[0103] RPMI-8226, SUN-C2B, NCI-H157, SW1573, A549, SW620, HCT-116, PG LCL8, the first 7 cell lines were purchased from ATCC, and the required medium and culture conditions were carried out according to the instructions of ATCC. The eighth cell line is provided by Fudan University, the medium is 1640+10% FBS, 5% CO 2 , cultivated at 37 degrees.
[0104] 2. DNA extraction and PCR amplification
[0105] Genomic DNA of 8 cell lines was extracted using the Genome Purification Kit of Kangwei Century Company, and PCR amplification was performed using the genomic DNA of 7 cell lines containing target mutations as templates to obtain PCR products containing 7 target mutations. 20 μL of PCR amplification system, including 2 μL of upstream and downstream primers with a concentration of 5 uM, 2 μL of DNA template, 10 μL of 2×...
Embodiment 2
[0144] Applicability verification of KRAS gene mutation quantitative standard
[0145] 1. Materials and methods:
[0146] 1. Materials and Reagents
[0147] 7 kinds of mutation detection kits of human KRAS gene from three different manufacturers in the domestic market were selected, and the random numbers were A, B, and C. Quantitative standard substance (code K) and 5% quantitative standard substance (code P) with 7 kinds of mutation levels of human KRAS gene, negative control substance (NC1~NC11), quantitative standard substance and reference substance are all provided by China Metrology Science Institute preparation.
[0151] The following indicators of the kit were verified by real-time fluorescent quantitative PCR method:
[0152]1) Accuracy: 10 parts of 5% quantitative standard were measured, an...
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Abstract
The invention discloses a KRAS gene mutation detection quantification standard product as well as a preparation method and a valuing method thereof. The preparation method comprises the following steps: performing PCR (Polymerase Chain Reaction) amplification by taking seven common mutant cell line DNAs (Deoxyribonucleic Acids) containing KRAS genes as a template to obtain a PCR product, and performing Sanger sequencing verification; mixing the PCR product with wild type human genome DNAs being subjected to ultrasonic fragmentation according to a certain proportion through balance weighing toobtain a quantification standard product for seven mutations of a KRAS gene. The invention provides a preparation method of a quantification standard product. Fragmented wild type human genome DNAs are taken as background DNAs, so that the background of an actual detection sample can be better simulated. A valuing method of the quantification standard product has high precision and high accuracy,and is independent of a DNA standard product; a measurement result can be traced to the international basic unit (mass), so that the reliability and traceability of the measurement result are ensured.The quantification standard product can be applied to method verification and quality control of a KRAS gene mutation detection method.
Description
technical field [0001] The invention relates to the field of gene detection, in particular to a preparation method and a value determination method of a KRAS gene mutation detection standard product. Background technique [0002] The KRAS (K-ras, p21) gene is about 35kb in length, located on chromosome 12, and encodes a 21kd ras protein. The normal KRAS gene is involved in intracellular signal transmission. When the KRAS gene is mutated, the normal ras protein cannot be produced, which will lead to the disorder of intracellular signal transmission, resulting in the continuous growth of cells and the inability to self-apoptosis and canceration. The most common mutations detected in cancer patients were those located at codons 12 and 13 of the KRAS gene. About 65% to 100% of patients with pancreatic cancer carry mutations at these sites in the KRAS gene, and about 30% to 50% of patients with colorectal cancer carry these mutations. Mutations in the KRAS gene often occur earl...
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