Double-dye fluorescent PCR (Polymerase Chain Reaction) specific detection system for simultaneously detecting Acipenser stellatus and Acipenser baerii species and application thereof

Abstract

The invention discloses a fluorescent PCR (Polymerase Chain Reaction) specific detection system for simultaneously identifying Acipenser stellatus and Acipenser baerii species by using double fluorescent dyes and application thereof. According to the invention, two fluorescent dyes are used for performing species fluorescent PCR detection for the first time, and specific detection primers of the Acipenser stellatus species and the Acipenser baerii species are used for simultaneously detecting the Acipenser stellatus species and the Acipenser baerii species; mitochondrial genes of the Acipenser stellatus and the Acipenser baerii are amplified by virtue of EVA-Green and SybrGreen double-dye fluorescent PCR; After amplification, two specific amplification curves are respectively generated, so that the problems that the fish species detection specificity is not high, the detection limit cycle number is relatively low, near-source species are easy to confuse, the operation is repeated and the like are solved. The full combination of the DNA of the species and the dye can be ensured, and the conditions of off-target and amplification failure of the dye are prevented. The specific primer and the probe are reasonable in design, good in specificity and high in detection sensitivity.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a dual-dye fluorescent PCR specific detection system and application for simultaneously detecting EVA-Green and SybrGreen species of Acipenser sinensis and Acipenser siberia. Background technique [0002] SybrGreen green fluorescent dye is widely used in fluorescent PCR amplification, but the dye has a detailed inhibitory effect on PCR amplification. Considering the particularity of fish DNA detection, that is, there are many homologous species and high similarity, and the PCR reaction of related species is within 35 1 cycle to 40 cycles are prone to non-specific amplification curves, and re-amplification is often required in this case. In order to reduce the amplification time, prevent secondary amplification, and reduce off-target dyes and single dye amplification failures, it is urgent to develop a method for using two dyes for single detection. At present, there is n...

AI Technical Summary

Problems solved by technology

The present invention can provide two amplification curves for a single species, and at the same time solve the problems of low detection specificity of fish species, low detection limit cycle number, easy confusion of close species, repeated operations, etc.

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