A New Technique For Detection Of Nucleic Acid Based On Surface Enhanced Raman Spectroscopy And CRISPR

At this stage,the nucleic acid detection technology of pathogens mainly relies on qPCR,the recognition mode of nucleic acid molecules still employs fluorescence,sensing sensitivity is limited,and several rounds of amplification reactions are required,pyrophosphate and other multiple byproducts can affect the reaction efficiency.Therefore,an increasing number of investigators are beginning to try new ydetection modalities that amplify or nonamplify a small number of target nucleic acid sequences and physical methods for novel molecular detection sensing of nucleic acids.With the increased attention given to Clustered Regularly Interspaced Short Ralindromic Repeats(CRISPR)and their related protein families(Cas),this system has been widely used for nucleic acid shearing,modification,and capture.CRISPR/Cas systems can insert a target nucleic acid sequence into the CRISPR,and the related proteins are transcribed and translated by the CRISPR specific sequence,and finally combined with it.Meanwhile,guide crRNA(CRISPR RNA)that specifically binds the target nucleic acid is synthesized,and finally assembled into CRISPR/Cas system to completethe cleavage and capture of nucleic acid.SERS technology can detect certain macromolecules.Thus,it has been developed as a new modality in nucleic acid detection.This is a kind of precious metal as the substrate,using laser irradiation to detect the sensitivity of the substance to be detected on the substrate.The advantages of this technology are small sample size,accurate detection,and short time.Herein,we present novel techniques based on SERS and CRISPR/dcas9 mediated nucleic acid detection that candirectly bind to target double stranded nucleic acid sequences and greatly enhance the corresponding signals through a silver substrate.In terms of probe design,the first probe is a SERS probe with no label on the surface.This probe does not require modification of the reporter molecule,and only needs to remove impurities such as residual sodium citrate on the surface,and use KI solution on the silver nanoparticles.The surface is modified with a layer of iodide ions,the DNA is combined with the SYBR Green I,then mixed with the probe and PCR is performed,and finally MgSO₄ is added for direct detection.the other probe is combined with gRNA on the surface of the material to form a capture probe,Then mix the probe with the target DNA and react at 37°C,and finally add SYBR Green I for detection.In this experiment,we completed the trace detection of ASFV and Orf1ab,N genes based on SERS technology and CRISPR/dCas9 system.The advantages of this sensing method:(1)It can directly detect double-stranded DNA;(2)Different from the use of fluorescence enhancement to display DNA detection results,the resonance effect occurs through the fluorescence enhancement of SYBR Green I and the electromagnetic enhancement between the probes.Thereby increasing the intensity of surface-enhanced Raman spectroscopy by 3 to 4 orders of magnitude,so that a lower sample size can be detected.The minimum detection limit for direct detection of Orf1ab gene and N gene by non-amplified method is 4×10⁵copies/?L and 3000copies/?L.This thesis uses CRISPR/dCas9 to directly capture the DNA double-strand of the target sequence,and enhances the detection signal through SERS resonance,which provides a certain technical basis and experimental reference for non-amplified nucleic acid detection methods,and it provides a new direction for the development of rapid non-amplification detection technology.