Method of detecting circular tumor DNA in free DNA of peripheral blood, kit and analytic method of sequencing result thereof

Abstract

The embodiment of the invention discloses a method of detecting circular tumor DNA in free DNA of peripheral blood, a kit and an analytic method of a sequencing result thereof. The method of detecting circular tumor DNA comprises the following steps: S1, filling the tail end of cfDNA, and adding a basic group A into 3' of cfDNA; S2, connecting a joint to the tail end of the cfDNA treated in the S1 to obtain a connecting product, wherein the joint contains a molecular tag sequence and an Index sequence; S3, amplifying the connecting product with universal primers Primer 1.0 and Primer7; S4, capturing and enriching the amplified product obtained in the S3 by means of a probe; and S5, amplifying the enriched product obtained in the S4 by using the Primer 7 and Primer 5 and performing sequencing. The system error induced by PCR and sequencing can be removed effectively, so that the sequencing sensitivity of ctDNA is improved effectively.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a method for detecting circulating tumor DNA from free DNA in peripheral blood, a kit and an analysis method for sequencing results thereof. Background technique [0002] Plasma free DNA, also known as cfDNA (cell free DNA), refers to the DNA that is free from the outside of the cell in the periphery. The source of cfDNA includes both normal cells and abnormal cells (such as tumor cells) or exogenous microorganisms (such as viral DNA). Specifically, it includes apoptotic bodies from normal cell metabolism, tumor cell fragments, exosomes, etc. [0003] Circulating tumor DNA (circulating tumor DNA) ctDNA refers to the DNA that breaks down from the primary tumor or even the new tumor cells formed by metastasis into the circulating peripheral blood. ctDNA is of great significance in clinical medicine. The detection of ctDNA has important clinical value in the early detection of tumors, ...

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Problems solved by technology

[0009] However, the current mainstream detection process using NGS methods (including the technical solutions disclosed in Chinese patent applications CN201410771006.9 and CN201610018232.9) will involve PCR amplification and sequencing, and some system errors will occur in these two processes, such as illumina The sequencing error rate of the Hiseq platform is 0.1%, which is not a big problem in the detection of ordinary large sample templates

However, in the early screening of tumors, since the template itself is within the range of 1 / 10,000 to 1 / 1,000, these sequencing errors are on the same order of magnitude as our detection offline, which will cause very serious damage to the results. Interference, and secondly, due to errors introduced by PCR, it is also impossible to effectively identify

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