K-Ras gene mutation typing fluorescence quantitative PCR detection kit and detection method thereof

Abstract

The invention provides a K-Ras gene mutation typing fluorescence quantitative PCR detection kit which comprises PCR mixed reaction solution, a peptide nucleic acid probe, a mutant type K-Ras-AllgloTM fluorescent probe and a positive control sample. The invention further provides a K-Ras gene mutation typing fluorescence quantitative PCR detection method which comprises the following steps: firstly designing a pair of PCR primers and the peptide nucleic acid probe according to K-Ras genes, respectively designing the AllgloTM fluorescent probe against K-Ras gene mutation sites, and adding the PCR reaction solution, the PCR primers, the peptide nucleic acid probe and the AllgloTM fluorescent probe in a reaction system for carrying out fluorescence quantitative PCR detection. The method adopts the PNA technology and is combined with the technology of the AllgloTM fluorescent probe, thereby being capable of quickly and accurately detecting the K-Ras gene mutation sites in various cancer tissues with high sensitivity, and having short time, simple operation and clear, intuitive and safe judgment and reading.

Description

technical field [0001] The invention relates to the field of molecular biology, in particular to a fluorescent quantitative PCR kit for detecting gene mutations and a detection method thereof, in particular to a fluorescent quantitative PCR detection kit for K-Ras gene mutation typing and a detection method thereof. Background technique [0002] Ras oncogene is involved in the occurrence and development of human tumors. It was originally a transforming gene cloned from two strains of rat sarcoma virus, Harvey and Kirsten, in the acute transforming retrovirus experiment. Since Weinberg et al. discovered human bladder cancer cells in 1982 After the activation of H-ras gene in human tumors, people have paid great attention to the role of ras oncogene in the development of human tumors. The ras gene family has three genes related to human tumors - H-ras, K-ras and N-ras, which are located on chromosome 11, 12 and 1 respectively. Among them, K-Ras has the greatest impact on huma...

AI Technical Summary

Problems solved by technology

[0007] 1. The detection period is long (the shortest time is about 24-48 hours from the specimen submission to the result)

[0008] 2. The operation process is complicated and involves the operation after PCR amplification, so it is easy to be contaminated, resulting in unsatisfactory results

[0009] 3. Interpretation of sequencing results is complex and time-consuming (especially when the sample size is large)

[0010] 4. The sensitivity of detection is not high enough: Katsuhiko et al once reported on "Lung Cancer" ("Lung Cancer") published in August 2005, if the content of the mutated gene accounts for less than 10% of the total amount of genomic DNA, then use Presence of mutant samples not detected by direct sequencing

[0011] 5. The sample size of an experimental test is limited, at most 8-24 cases

[0012] 6. Unsafe: A variety of toxic substances are used in the whole experimental process, such as EB, acrylamide, etc., which are harmful to the operator and the environment

[0013] 7. The cost is relatively high (about 200 yuan per site)

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