Method for improving linear amplification detection accuracy through multiple detection, kit and application

Abstract

The invention discloses a method for improving linear amplification detection accuracy through multiple detection, a kit and application. The method comprises the following steps: respectively carrying out linear amplification on a targeted target sequence and a reference sequence of a detection sample and a targeted target sequence and a reference sequence of a control sample by using multiple primers; linkers are added to the obtained linear amplification products respectively; performing sequencing; and finally, analyzing a sequencing result by using a standardized algorithm. According to the method, by reasonably designing primers, carrying out multiple linear amplification detection, standardizing an algorithm and firstly carrying out linear amplification and then connecting a single-chain connector, accumulation of introduced base errors can be effectively avoided, and the problem of primer preference existing in linear amplification is relieved; target DNA molecule missing detection is reduced, and the copy number of a target sequence is detected more accurately. The method is widely applicable to CNV detection of any sample possibly containing CNV; particularly, a heterogeneous sample is used for detecting diseases including infectious diseases, cancers and the like.

Description

technical field [0001] The invention relates to the technical field of gene detection, in particular to a method, kit and application for improving the accuracy of linear amplification detection through multiple detection. Background technique [0002] DNA testing is a molecular diagnostic technology that can be used in many fields such as infectious diseases, malignant tumors, and prenatal screening. Before DNA detection, it is generally necessary to amplify the target DNA to ensure that the amount and concentration of the target DNA can be detected by the prior art. PCR amplification is the current mainstream DNA amplification technology. However, the characteristic of exponential amplification of PCR (the amplification product of the previous round can be used as the template of the next round of amplification) easily accumulates the introduced base errors, which affects the detection accuracy. Some DNA linear amplification technologies developed in recent years can fun...

AI Technical Summary

Problems solved by technology

[0005] Traditionally, CNV detection methods are mainly based on immunohistochemistry and fluorescence in situ hybridization. These methods are based on the detection of tissue samples with intact cells, and cannot be detected for samples with severe cell heterogeneity, such as blood samples.

At present, the relatively widely used PCR method is easy to operate and has a fast turnaround. Unfortunately, even the most sensitive digital PCR method can only distinguish about 1.2 times the number of CNVs, and cannot effectively detect low-frequency CNVs in blood cell-free DNA samples.

For example, when the content of circulating tumor DNA is only 1%, even if the CNV of tumor cells is as high as 11 times, the copy number change in the blood is only 1.1 times, which is difficult to detect by existing technologies

The high-throughput sequencing technology developed in recent years can detect a large number of genome sequences at the same time, which is expected to improve the accuracy of CNV detection, but limited by the errors introduced by the library construction method, it is still difficult to detect low-frequency CNVs

Therefore, there are currently no high-resolution techniques that can effectively detect CNVs in heterogeneous samples

Images