Method for detecting multi-locus low-frequency mutation of free target DNA of lung cancer plasma and kit

Abstract

The invention relates to the technical field of biology and the field of nucleic acid detection and relates to method for detecting multi-locus low-frequency mutation of free target DNA of lung cancerplasma and a kit. The invention provides high-sensitivity and high-flux method for detecting multi-locus mutation of free DNA of lung cancer plasma once and a kit. According to the detection method,a ddPCR technique and a next generation sequencing technique are combined for detecting the mutation of lung cancer crDNA. The detection method comprises the following steps: (1) preparing a digital PCR mixed liquid including a to-be-tested sample DNA template, a primer and a PCR premixing liquid; (2) preparing a digital PCR micro-reaction drop, and carrying out PCR amplified reaction; (3) recycling and purifying a PCR product; (4) preparing and amplifying PCR premixing liquid for the second time; (5) carrying out purification and library quality control on the PCR product; and (6) carrying out computer sequencing and data analysis. According to the detection method, hotspot mutation information of 10 genes including EGFR / BRAF / KRAS / PIK3A / MET2 / ERBB2 / AKT1 / NRAS / TP53 / PTEN of lung cancer can bedetected for one time, the cost can be remarkably lowered, and the high-sensitivity and high-flue detection of mutation information of lung cancer ctDNA can be realized.

Description

technical field [0001] The invention relates to the fields of biotechnology and nucleic acid detection, in particular to a method for detecting low-frequency mutations at multiple sites in plasma free target DNA of lung cancer and a kit thereof. Background technique [0002] According to reports, lung cancer is the fastest growing malignant tumor in the world, and non-small cell lung cancer (NSCLC) accounts for about 80% of lung cancer. The latest research estimates that in 2015, there were about 733,000 new cases of lung cancer in my country (accounting for 17.1% of the overall new cancer cases), and 610,000 deaths (accounting for 21.1% of the overall cancer deaths), which seriously threatened the health of the Chinese population. life and health. About 75% of the patients were found in the middle and advanced stages, and surgical resection and chemotherapy are still the main treatment methods, but there are many complications, and the 5-year survival rate of patients is ve...

AI Technical Summary

Problems solved by technology

However, ctDNA only accounts for less than 1% of the total free DNA in plasma, especially in patients with early cancer, which is even lower at about 0.01%. Therefore, it is not easy to accurately detect tumor-associated low-frequency mutation information from such a high background free DNA. thing

Existing ctDNA detection technologies such as droplet digital PCR technology (ddPCR), digital PCR and flow-based emulsion magnetic amplification technology (BEAMing) have greatly improved the accuracy, but the number of detection sites at a time is small, which cannot meet Screening needs one by one; based on cancer targeted capture and deep sequencing technology (CAPP-seq), hybrid capture is required, the steps are relatively cumbersome, time-consuming, and costly, which limits its wide application in clinical practice

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