Multiplex fluorescence melting curve based PCR detection method

Abstract

The invention relates to the technical field of polymerase chain reaction (PCR), and in particular relates to a multiplex fluorescence melting curve based PCR detection method. According to the detection method, a bar code probe capable of specifically hybridizing with to-be-detected target genes and a fluorescence detection probe capable of specifically binding with the bar code probe are adopted for simultaneously detecting the plurality of target genes in a to-be-detected sample, in the DNA amplification process, a specific bar code sequence corresponding to each to-be-detected target gene is generated, then the bar code sequence is subjected to DNA amplification, so that specific DNA products labeled with different fluorescent groups and quenching groups and having different lengths are formed, and through the final multiplex fluorescence melting curve analysis, the PCR multiple detection based on the melting curve analysis is realized.

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...

AI Technical Summary

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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