Digital nucleic acid amplification detection method and integrated detection system based on CRISPR and Cas

Abstract

The invention discloses a digital nucleic acid amplification detection method and an integrated detection system based on CRISPR/Cas. The integrated detection system comprises an integrated reaction chip, a temperature control module, a light source and an optical signal detector. The method comprises the following steps of uniformly dividing a nucleic acid amplification system into amplificationmicro-droplets, mixing the amplification micro-droplets subjected to digital nucleic acid amplification with detection micro-droplets containing a CRISPR/Cas system to perform a CRISPR reaction, and detecting an optical signal to realize high-specificity detection of a target object after the reaction is finished, so that the concentration or copy number of nucleic acid molecules in a to-be-detected sample is obtained, and high-sensitivity absolute quantitative detection of the target object is realized. The method has the characteristic of absolute quantification of a digital amplification technology and the advantages of high sensitivity and high specificity of CRISPR/Cas detection, the detection process is integrated on one chip, the operation steps are simplified, and besides, the problems of cross contamination and the like are avoided.

Description

technical field [0001] The invention relates to the technical field of nucleic acid detection microfluidics, in particular to a CRISPR / Cas-based digital nucleic acid amplification detection method and an integrated detection system. Background technique [0002] Since Vogelstein et al. proposed the digital Polymerase Chain Reaction (dPCR) in 1999, it has shown great technical advantages and application prospects in food safety, forensic identification, precision medicine and other research fields. dPCR is an absolute quantification technique for nucleic acid molecules. By dispersing a sample into tens to tens of thousands of different reaction units, the number of nucleic acid templates in each unit is less than or equal to 1; each unit is separately amplified by PCR After the amplification, the reaction units with nucleic acid templates will emit fluorescent signals, and the reaction units without templates will have no fluorescent signals. Therefore, the number of nucleic ...

AI Technical Summary

Problems solved by technology

[0008] The purpose of the present invention is to provide a digital nucleic acid amplification detection method based on CRISPR / Cas and an integrated detection system in order to solve the problems of poor integration of the digital detection system in the background technology and the use of the CRISPR / Cas system to easily cause aerosol pollution. Using this method, CRISPR / Cas is combined with digital nucleic acid amplification technology to achieve absolute quantitative and highly specific detection of target nucleic acid molecules. Integrated detection system for CRISPR / Cas

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