Primer and probe composition for detecting V600E of BRAF (V-Raf Murine Sarcoma Viral Oncogene Homolog B1) gene and detecting method of primer and probe composition

Abstract

The invention provides a primer and probe composition for detecting V600E of a BRAF (V-Raf Murine Sarcoma Viral Oncogene Homolog B1) gene and a detecting method of the primer and probe composition. The primer and probe composition comprises a mutant probe which aims at a V600E site of the BRAF gene and is as shown in SEQ ID NO.1, a wild type probe which aims at the V600E site of the BRAF gene andis as shown in SEQ ID NO.2 and specific primers which aim at the V600E site of the BRAF gene and are as shown in SEQ ID NO.3 to SEQ ID NO.4. Primer designing and probe modification are carried out ona specific zone aiming at the V600E site, a primer and a probe are matched with each other, ddPCR (Differential Display Polymerase Chain Reaction) is combined with a high-specificity primer and probecomposition, extraction of cfDNA (Cell-free DNA), recycle number of PCR and annealing temperature are optimized, all steps are in a synergistic effect, the accuracy, the stability and the sensitivityfor detection are finally increased, and wide application prospect and a market value are obtained.

Description

technical field [0001] The invention relates to the fields of medicine and biotechnology, in particular to a primer probe composition for detecting BRAF gene V600E and a detection method thereof. Background technique [0002] The BRAF gene is located on chromosome arm 7q34 and encodes the BRAF protein, while the serine-threonine kinase is part of the Ras-Raf-Mek-Erk-MAPK (mitogen-activated protein kinase) signaling cascade, the activation of which involves promoting cellular growth, proliferation and differentiation. There are three functional RAF proteins in humans, ARAF, BRAF and CRAF. Among them, BRAF has the highest basal kinase activity and is the most potent activator of the MAPK pathway. The BRAF gene consists of 18 exons, and the most common activating mutation is located in exon 15 at nucleotide position 1799, converting thymine to adenine, which results in the substitution of glutamic acid for valine at position 600, It was named V600E. The mutation occurs with...

AI Technical Summary

Problems solved by technology

[0005] The existing cancer gene detection methods have the following defects and deficiencies: First, the traditional detection optimization method mainly relies on the experience of technicians to interpret the results, which cannot directly, objectively and accurately reflect the detection results, nor can it perform absolute quantification of DNA; Second, the existing detection optimization method based on the BRAF gene V600E site usually has high requirements on the total amount and quality of cfDNA in the sample, and is easily affected by a large amount of blood-related DNA and reaction inhibitors; third, the existing BRAF gene V600E-based The detection optimization method of the site is cumbersome, and the detection sensitivity is low, and there will be a certain amount of false positive detection results, especially the cfDNA false positive detection rate in plasma samples is higher, because cfDNA is mainly derived from benign germline origin. cells, the proportion of ctDNA is relatively low; fourth, there are literatures showing that the addition of carrier RNA in the cfDNA extraction process does not increase the total amount of cfDNA extraction, and it is likely to cause large fragments of gDNA to aggregate.

Fourth, the steps of liquid biopsy are cumbersome, the cycle is long, and the cost is high, so it is not suitable for the detection of single or two sites

These detection methods have many disadvantages, for example: the detection of the whole genome or the whole exome, although the detection content is rich, but the cost is high, and most of the detected information has nothing to do with the corresponding cancer of the patient; the accuracy of FISH detection results The accuracy is highly dependent on the experience and skills of the testing personnel; the second-generation high-throughput sequencing detection steps are cumbersome, the workload is heavy, the detection cycle is long, and the cost of mutation information for only 1-2 gene loci is high

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