A multi-color fluorescent melting curve PCR detection method

Abstract

The present invention relates to the technical field of polymerase chain reaction (PCR), in particular to a multi-color fluorescent melting curve PCR detection method, which uses a barcode probe capable of specifically hybridizing with a target gene to be detected and a In the DNA amplification process, a specific barcode sequence corresponding to each target gene to be detected is generated, and then the barcode sequence is amplified by DNA. DNA products of different lengths labeled with different fluorophores and quenching groups are amplified and analyzed by the final multicolor fluorescence melting curve, thereby realizing PCR multiplex detection based on melting curve analysis.

Description

technical field [0001] The invention relates to polymerase chain reaction (PCR), in particular to a multicolor fluorescent melting curve PCR detection method. Background technique [0002] Polymerase chain reaction (PCR) is a technology that uses heat-resistant DNA polymerase to amplify genes in vitro, and is widely used in many fields such as gene cloning / disease diagnosis / forensic identification. Ordinary PCR technology requires electrophoresis analysis of the product after amplification, and the analysis process is cumbersome and time-consuming. With TaqMan TM With the invention of hydrolysis probes and the use of double-stranded DNA fluorescent dyes, PCR products can be analyzed and detected by real-time fluorescence methods. This new PCR technology is called real-time fluorescent quantitative PCR. Compared with ordinary PCR technology, real-time fluorescent quantitative PCR has obvious advantages. The DNA amplification and result detection of real-time fluorescent qua...

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Problems solved by technology

However, based on TaqMan TM The disadvantage of the real-time fluorescent quantitative PCR detection method of the hydrolysis probe is that one color of the fluorescent group can only label one TaqMan TM Hydrolysis probe, detects one gene of interest, therefore, has limited multiplexing capability

However, because the fluorophore itself does not emit fluorescence of a single wavelength, and the PCR instrument has a limited degree of discrimination of the fluorescence emitted by different fluorophores, therefore, the existing PCR instrument can only detect the fluorescence emitted by 4 to 6 fluorophores. signal, which also enables the application of TaqMan TM The multiplex capability of real-time PCR with hydrolysis probes is limited

For real-time fluorescent quantitative PCR using double-stranded DNA fluorescent dyes, only one double-stranded DNA fluorescent dye can be used in the same reaction system because the dye does not have the ability to recognize specific sequences.

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