Multiple digital nucleic acid analysis device and analysis method based on melting curve

Abstract

The invention discloses a multiple digital nucleic acid analysis device and analysis method based on a melting curve, and relates to the field of nucleic acid analysis. The device comprises a micro-fluidic chip, a flat plate PCR instrument, a fluorescence detection system and a processor, wherein the fluorescence detection system comprises an excitation light source, an optical filter and a camera. The multiple digital nucleic acid analysis method comprises the following steps: dispersing a PCR reaction system into a large number of reaction micro-units on a micro-fluidic chip, then performing digital PCR amplification on the reaction micro-units on a plate PCR instrument, and performing temperature control and fluorescence detection on an amplification product to obtain the melting curve and performing analysis. According to the invention, the nucleic acid amplification products in the micro-reaction units for digital nucleic acid detection are subjected to melting curve analysis, and the amplification product is distinguished and classified, so that multiple digital nucleic acid quantitative detection is realized. Through design of the nucleic acid amplification product, melting curves of different amplification products are different, so that the amplification products are distinguished.

Description

Technical field [0001] The present invention relates to the field of nucleic acid analysis, and more particularly to a multi-digital nucleic acid analysis apparatus and analysis method based on a melted curve. Background technique [0002] Digital PCR is a method of precise nucleic acid quantification. By assigning a standard PCR reaction into a large number of tiny reaction units such that each reaction unit contains or does not include a target molecule of one or more copies, after the amplification is completed, the fluorescent signal in the reaction unit of the template will be significantly enhanced. By setting the threshold, it is classified as a positive point, and the number of copies of the target molecule can be calculated by the Poisson distribution by the ratio and number of positive and negative reaction units. [0003] Digital PCR needs to be detected by end-point fluorescence, usually embedded fluorescent dyes, such as SybrGreen et al; or hydrolyzed probe. In the d...

AI Technical Summary

Problems solved by technology

[0007] 1. Due to the low specificity of fluorescent dye lamps in existing digital nucleic acid detection (digital PCR, etc.), it is difficult to perform multiple digital nucleic acid quantitative analysis and endpoint fluorescence quantitative detection;

[0008] 2. The multiple digital PCR quantitative method using hydrolyzed fluorescent probes is expensive, difficult to design probes, and limited by the number of fluorescent channels;

[0009] 3. The existing multiple digital PCR quantitative detection instruments with multiple fluorescent channels are expensive and the system is relatively complicated

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