Liquid-stage chip constant-temperature detection method for tiny RNA

Abstract

The invention discloses a liquid-stage chip constant-temperature index amplification and detection method for tiny RNA target molecules. By fixing upstream and / or downstream primers to the surfaces of encoding microspheres and marking the two sides of notch agent recognition sequences of the primers fixed to the surfaces of the encoding microspheres with fluorescence report molecules which have the fluorescence resonance energy transfer effect and cancellation molecules, finally the high-throughput detection of various target molecules is achieved through target molecule specificity detectable fluorescence signals on the surfaces of the encoding microspheres. The design difficulty of miRNA inherent attributes on the amplification detection method for the target molecules is lowered, a liquid-state constant-temperature index amplification and detection system based on oligonucleotides primers is provided, and the method for achieving constant-temperature index amplification and absolute quantification under the constant-temperature condition can be achieved.

Description

technical field [0001] The invention belongs to the field of life science and biotechnology, and relates to a liquid chip constant temperature detection method for qualitatively and quantitatively detecting various microRNAs (microRNA, miRNA) through liquid chip constant temperature exponential amplification. Background technique [0002] Compared with nucleic acid molecules such as genomic DNA, mRNA, and long noncoding RNA (lncRNA), microRNA (microRNA, miRNA) molecules have their particularities, such as: nucleic acid molecular fragments are extremely small (≤22nt), and GC content is widely distributed ( 5-95%), resulting in large differences in melting temperature (melting temperature, Tm), it is difficult to design matching amplification primers and detection probes; the proportion of total RNA is extremely low (≤0.01%), requiring a highly sensitive detection method ; Family molecular differences are small (may only have a single base difference), and the requirements for...

AI Technical Summary

Problems solved by technology

When the liquid chip is applied to the detection of nucleic acid target molecules including miRNA, it often relies solely on the way of nucleic acid hybridization, the target molecule needs to be amplified in advance, and the amplification product or the hybridization of the target molecule and the microsphere requires multiple washings, etc. Steps to remove non-specific hybridization and background signal interference, there are many problems such as cumbersome detection steps and long time-consuming, which restrict the application of liquid chips in the field of nucleic acid detection to a certain extent

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