A new closed-type visual nucleic acid detection method coupled with nucleic acid amplification reaction, nucleic acid invasion reaction and nanoparticle color reaction

Abstract

The invention discloses a novel closed type nucleic acid visual detecting method for coupling a nucleic acid amplification reaction, a nucleic acid intrusive reaction and a nano-particle chromogenic reaction. The detection method comprises the following steps: 1) target nucleic acid amplification and signal conversion phase: further amplifying the target nucleotide sequence, and converting the detection of the target nucleic acid to the detection of a hairpin probe fragment; 2) nanoprobe hybridization phase: after the amplification reaction, under the corresponding conditions, generating a gathering or dispersing phenomenon by the special nanoprobe, and utilizing the phenomenon to realize distinguishing detection to the target nucleic acid. With adoption of the detection method disclosed by the invention, the nucleic acid target sequence can be detected; furthermore, the nucleic acid intrusive reaction is high in specificity, and the nucleic acid sequence with single different basic groups near the intrusive loci can be distinguished and detected, so that the method not only can be used for detecting a template, but also can be used for single basic group distinguished SNP genotyping and gene mutation.

Description

Technical field [0001] The invention belongs to the field of molecular biology, and relates to a new closed visual nucleic acid detection method that couples nucleic acid amplification reaction, nucleic acid invasion reaction and nanoparticle color reaction. Background technique [0002] At present, nucleic acid-based detection technology has been widely used in clinical diagnosis, environmental monitoring, and the prevention and control of infectious diseases, such as polymerase chain reaction (Polymerase Chain Reaction, PCR) technology, which can realize template detection. Exponential amplification, magnification by a million times, but this type of technology is usually not satisfied with the detection of some samples that distinguish single-base target nucleic acids due to specific limitations. For example, when detecting samples with genetic mutations, PCR is developed Real-time PCR (RT-PCR) technology is not only limited in specificity, low in discrimination, requires spec...

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Problems solved by technology

[0002] At present, detection technology based on nucleic acid has been widely used in many aspects such as clinical diagnosis, environmental monitoring, and prevention and control of infectious diseases, such as polymerase chain reaction technology (Polymerase Chain Reaction, PCR) technology, which can realize template Exponential amplification, magnification of millions of times, but this type of technology is usually limited by specificity, so that it cannot be satisfied with the detection of some samples that distinguish single-base target nucleic acids. For example, when detecting gene mutation samples, it was developed by PCR. The real-time PCR (Real Time PCR, RT-PCR) technology not only has limited specificity, low discrimination, requires special equipment, but also has high detection cost.

The nucleic acid invasion reaction based on signal amplification relies on a signal amplification reaction of a special endonuclease. This reaction not only has good specificity, but also has the advantages of constant temperature and low reaction cost. At present, there is a nucleic acid invasion reaction based on Technology coupled with bio-nano-gold technology for molecular detection (see the patent "cascade invasion signal amplification reaction combined with nano-gold-oligonucleotide probe visual nucleic acid detection method", patent application number "201110161736.3"), but due to the The technical sensitivity is low, and only 6×10 template content can be detected 4 Samples with more than one copy / μL still cannot meet the requirements of most clinical samples for direct detection

Therefore, in the detection process of the actual sample, it is necessary to pre-amplify the template to be tested, take out the amplified product after opening the tube, and then perform the nucleic acid invasion reaction and the bionano gold color reaction; but take out the amplification product after opening the tube. The product is very easy to cause cross-contamination of the amplification product, resulting in false positive results

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