Amplification-free RNA quantitative detection method

Abstract

The invention discloses an amplification-free RNA quantitative detection method. The method comprises the following steps: designing a crRNA sequence which is complementarily paired with a target RNA base and has a 5 '-terminal repetitive sequence stem-loop structure; preparing a Cas13 reaction system; mixing an object to be detected with the Cas13 reaction system, dispersing the mixed system into a large number of micro-reaction units which are uniform in size and do not exceed nano-flow, and providing proper temperature conditions to incubate the micro-reaction units; and reading signals of the micro-reaction units after the incubation reaction is completed, and calculating the content of the target RNA in the sample to be detected. According to the amplification-free RNA quantitative detection method disclosed by the invention, RNA quantitative detection at the single molecule level can be realized, internal reference correction and standard curve establishment are not needed, steps of reverse transcription and nucleic acid replication are not needed, marking and functional modification are not needed, thermal circulation is not needed in thermostatic reaction, the operation steps are simple, and the detection precision is high.

Description

technical field [0001] The invention relates to the field of molecular biological detection, in particular to an amplification-free RNA detection and a single-molecule quantitative detection method thereof. Background technique [0002] Ribonucleic acid RNA (Ribonucleic Acid) is a genetic information carrier widely present in biological cells and some viruses and viroids. There are four main bases in RNA, namely A (adenine), G (guanine), C (cytosine), and U (uracil), where U replaces T (thymine) in DNA. There are many forms of RNA, including genomic RNA, messenger RNA (mRNA), transfer RNA (tRNA), ribose RNA (rRNA) and a series of non-coding RNAs (such as microRNA, lncRNA, circRNA, snRNA, cfRNA, etc.), each of which is involved in life play different roles in the process. For example, microRNAs are involved in the regulation of a series of cellular functions such as cell proliferation, differentiation, and death. At the same time, miRNAs can participate in the cellular path...

AI Technical Summary

Problems solved by technology

[0003] However, due to some characteristics of RNA itself, such as short fragment length, high sequence similarity, large expression difference span, and complex environmental background, its detection, especially the quantitative analysis at the single-molecule level, presents many challenges.

Traditional Northern Blotting and high-throughput sequencing require a series of complex operations, large sample consumption, long analysis time, and low detection sensitivity

qRT-PCR and ddPCR methods based on RT-PCR technology and a series of nucleic acid amplification detection technologies based on CRISPR (clustered regularly interspaced short palindromic repeats) effector proteins (including "SHERLOCK system" and its improved version) have high However, the steps involved in the process, such as reverse transcription and nucleic acid amplification, are easily interfered by various factors and affect the accuracy of quantitative detection.

At present, a series of newly developed RNA quantitative detection methods based on labeling and modification technologies also limit their wide applicability to a certain extent due to the complexity of the labeling / modification process.

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