CRISPR/Cas9 typing PCR method for DNA homogeneous detection and application of CRISPR/Cas9 typing PCR method

Abstract

The invention discloses a CRISPR / Cas9 typing PCR method for DNA homogeneous detection and an application of the CRISPR / Cas9 typing PCR method. The method comprises the following steps: adding all reaction components into a PCR tube for mixing, putting a mixture on a fluorescent quantitative PCR instrument, and running a PCR program to obtain a detection result immediately, wherein during the method, a test tube does not need to be reopened for addition of other reagents, or other operations do not need to be additionally set, and after the detection is finished, the PCR reaction tube is directly discarded without opening the cover. According to the method disclosed by the invention, only several new components including Cas9 nuclease, two sgRNAs and insertion oligonucleotides are added inthe current PCR reaction, and by adding the new components, the tedious primer design is eliminated. A single universal primer is used for replacing a traditional primer and is used for amplifying alltarget DNAs. The method provided by the invention is simple and convenient, is rapid in detection, can homogeneously detect the target DNA with high specificity and high sensitivity, and overcomes the limitation of PCR.

Description

technical field [0001] The invention belongs to the field of biotechnology, in particular to a CRISPR / Cas9 typing PCR method for DNA homogeneous detection and an application thereof. Background technique [0002] Polymerase chain reaction (PCR) is known to have high sensitivity due to exponential amplification of target DNA. Therefore, PCR has become an indispensable DNA detection tool in various fields related to life sciences. For example, reverse transcription PCR (RT-PCR) is widely used to detect SARS-CoV-2 and plays a key role in the diagnosis of the current COVID-19 pandemic. However, PCR has been found to have certain drawbacks, which has prompted continued development of the technology. So far, three types of PCR techniques have been developed, including traditional PCR (tPCR), quantitative PCR (qPCR), and digital PCR. Although these different types of PCR techniques have been developed, the basic mechanism of PCR techniques remains the same, that is, a pair of sp...

AI Technical Summary

Problems solved by technology

Each of these ctPCRs has its own advantages and special application value, but single-tube homogeneous detection has not been realized

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