Double-system coupling reaction tube and double-system coupling method for nucleic acid amplification combined CRISPR

Abstract

The present invention discloses a double-system coupling reaction tube and a double-system coupling method for nucleic acid amplification combined CRISPR. The double-system coupling reaction tube comprises a tube body and a cover body matched and sealed with the tube body, an inner cavity of the tube body comprises a reaction area at a lower part and a storage area at an upper part; and the storage area is provided with a storage chamber for temporarily storing a CRISPR detection reagent during nucleic acid amplification and a release mechanism for releasing the CRISPR detection reagent from the storage chamber to the reaction area after the nucleic acid amplification is completed. During nucleic acid amplification, a heating part of the reaction tube by an instrument is only arranged in the reaction area at the lower part of the tube body, influence on the CRISPR detection reagent in the storage chamber arranged at the storage area at the upper part is relatively small, the nucleic acid amplification reagent and the CRISPR detection reagent can be added into a corresponding area in the reaction tube together at the beginning, so that secondary uncovering is avoided, problems of aerosol pollution and false positive results are reduced, and a whole detection operation process is simpler and more convenient.

Description

technical field [0001] The invention relates to the technical field of biotechnology detection, in particular to a reaction tube for dual-system coupling of nucleic acid amplification combined with CRISPR detection and a dual-system coupling method. Background technique [0002] Nucleic acid amplification detection technology is the basis of molecular biology research, which can be used for qualitative and quantitative analysis and detection of trace nucleic acids. It plays an important role in various fields related to clinical medicine, laboratory medicine, molecular biology, genomics and food safety. It is an important test method that is indispensable for the development of life sciences. Existing nucleic acid amplification techniques can be divided into two categories according to whether temperature cycling is required: the first category is variable temperature amplification systems, including polymerase chain reaction (Polymerase chain reaction, PCR), ligase chain re...

AI Technical Summary

Problems solved by technology

However, this technology requires specific instruments and has certain professional requirements for operators and experimental conditions.

The second type is isothermal amplification system, including loop-mediated isothermal amplification (LAMP), rolling circle amplification (Rolling Circle Amplification, RCA) and recombinase polymerase amplification (Recombinase Polymerase Amplification) , RPA), etc., this technology does not require thermal cycling process, does not require special instruments, but its amplification is prone to false positives

However, this technical solution is only used for fluorescence detection after PCR amplification to avoid the diffusion of PCR reaction products into the air in the form of aerosol, and is not suitable for the dual-system coupling method of nucleic acid amplification combined with CRISPR detection.

And the operation is more complicated and cannot be operated automatically. The PCR tube is put into the centrifuge tube after the reaction is completed. After a long time of operation, the outer surface of the PCR tube is easily contaminated with pollutants, resulting in false positives.

And, the process of puncturing the PCR tube in this technical scheme is not easy to control, it is likely that the PCR tube has been punctured before the cap of the centrifuge tube is closed, or the PCR tube is not punctured after the cap of the centrifuge tube is closed, resulting in impact on results

On the other hand, this technical solution can only be operated manually, but cannot realize automatic operation

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