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KRAS 8 Mutations Detection Kit(Fluorescence PCR)

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KRAS 8 Mutations Detection Kit(Fluorescence PCR)

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Introduction

[Product Name] KRAS 8 Mutations Detection Kit (Fluorescence PCR)
[Packaging Size] 24 tests/kit, 48 tests/kit.
[Intended Use]
This kit is intended for in vitro qualitative detection of 8 mutations in codons 12 and 13 of K-ras gene in extracted DNA from human paraffin-embedded pathological sections.

Point mutations in the KRAS gene have been found in a number of human tumor types, about 17%~ 25% mutation rate in tumor, 15%~ 30% mutation rate in lung cancer patients, 20%~ 50% mutation rate in colorectal cancer patients. Because the P21 protein encoded by the K-ras gene is located downstream of the EGFR signaling pathway, after the K-ras gene mutation, the downstream signaling pathway is always activated and is not affected by the upstream targeted drugs on EGFR, resulting in continuous malignant proliferation of cells. Mutations in the K-ras gene generally confer resistance to EGFR tyrosine kinase inhibitors in lung cancer patients and resistance to anti-EGFR antibody drugs in colorectal cancer patients [1, 2, 3]. In 2008, the National Comprehensive Cancer Network (NCCN) issued a clinical practice guideline for colorectal cancer, which pointed out that the mutation sites that cause K-ras to be activated are mainly located in codons 12 and 13 of exon 2, and recommended that all patients with advanced metastatic colorectal cancer can be tested for K-ras mutation before treatment[4]. Therefore, rapid and accurate detection of K-ras gene mutation is of great significance in clinical medication guidance. This kit uses DNA as the detection sample to provide qualitative assessment of mutation status, which can assist clinicians in screening colorectal cancer, lung cancer and other tumor patients who benefit from targeted drugs. The test results of the kit are 

for clinical reference only and should not be used as the sole basis for individualized treatment of patients. Clinicians should make comprehensive judgments on the test results based on factors such as the patient's condition, drug indications, treatment response and other laboratory test indicators.

[Test Principle]

This detection kit uses a specific blocking technique for the wild-type sequence combined with Taqman probe to detect 8 mutations of 2 exon of K-ras gene. The Taqman probe is labeled with FAM that acts as a reporter and also has a second dye BHQ2 which acts as a quencher. When not bound to the target sequence, the fluorescent signals of the intact probes are suppressed by the quencher dye. During the PCR amplification step, hybridization of the probes to the specific single -stranded DNA template results in cleavage of the probe by the 5' to 3' exonuclease activity of the DNA polymerase resulting in separation of the reporter and quencher dyes and the generation of a fluorescent signal. The specific block in the K-ras reaction buffer can block the wild-type amplification through a specific PCR reaction program, while the mutant-type amplification is not affected. The detection of mutations in sample DNA is realized on a real-time PCR platform.

The detection system contains a certain proportion of dUTP, dNTP and uracil DNA glycosylase (UDG enzyme), this enzyme (also known as uracil-N-glycosylase or UNG enzyme) can interrupt PCR containing dUTP product, thus effectively preventing PCR contamination.

The internal control selects a relatively conserved segment on K-ras, about 150 bp, as a quality control for the reagents, DNA quality and the operation itself. Even if the DNA is degraded, the amount of DNA in the reaction system can be detected by the reaction system.

[ Storage Condition and Shelf-life]
The kit can be stored below -18°C and protected from light. The shelf life is 9 months (please use it before the expiration date). Repeated freezing and thawing cannot exceed 4 cycles. It can be transported stably for 5 days below -18°C and protected from light.
See the packaging label for the production date, production batch number and expiration date.

[Applicable Instruments]

Applied Biosystems 7500 Real-Time PCR Systems, Applied Biosystems 7300 Real-Time PCR Systems, QuantStudio®5 Real-Time PCR Systems, LightCycler®480 Real-Time PCR system, BioRad CFX96 Real-Time PCR System.
[Requirements for Samples]

First check that the paraffin-embedded pathological tissue or section contains tumorous cells, the part taken should be in the middle of the paraffin block as much as possible, and the number of paraffin sections should be no less than 3, and the thickness should be 5-10 μm. The surgically removed tissue should be fixed in 4-10% formalin as soon as possible, and the fixation time should be 8-24 hours; thorough dehydration should be performed before embedding. For paraffin-embedded pathological tissue or section samples, please select samples that have not been stored for more than 3 years.

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