Research On Extraction-Free Visualization And Rapid Nucleic Acid Detection Technology Of SARS-CoV-2

The coronavirus disease 2019(COVID-19)pandemic,caused by SARS-CoV-2,has rapidly spread around the world,bringing serious consequences for the normal lives of people all over the world and the global economy.The liberalization of foreign epidemic prevention policies has directed enormous pressure to China.Because of the huge domestic population,rashly adopting liberalized epidemic prevention policies will inevitably have a huge impact on the medical system.At present,the general policy of"Clear COVID-19 infections in a timely manner"is most suitable for China’s national conditions.In addition to implementing the strategy of"Guarding against imported cases",the domestic policy of"Preventing a resurgence of the outbreak"requires high-precision positioning of the source of infection and accurate screening of infected people.The detection method with rapidity,high sensitivity and good specificity is of great significance.Nucleic acid testing,as the gold standard for detecting SARS-CoV-2infection,is an important part of epidemic prevention and control,and undertakes a huge social responsibility.However,the currently commonly used qPCR nucleic acid testing method can only be completed in a qualified laboratory using high-precision instruments by relevant technicians.In addition,the centrifugal column-based extraction protocol has many problems,such as large consumption of reagents,high labor cost and time-consuming.In this chapter,a simple nucleic acid extraction platform was firstly constructed,which can process 16 samples simultaneously.The processing time was shortened to 10 min,without an extra step of RNA elution.Secondly,a colorimetric RT-LAMP isothermal amplification technology based on immunomagnetic bead method was constructed,without the need for professional instruments.Add 57.5μg of immunomagnetic beads to 600μL of sample,it only takes 10 minutes to enrich the virus particles,and then the magnetic beads are dispersed in the amplification reaction system for direct amplification,this assay shows a limit of detection(Lo D)as low as 2.7×10~3 copies/mL for detecting GX/P2V.In addition,using SARS-CoV-2 nucleic acid reference materials,compared with SARS、MERS、HCoV-NL63、GX/P2V,shows no cross-reaction and high specificity.A rapid,visual pathogen detection assay freed from RNA extraction for detection of SARS-CoV-2 is established,which can provide a new platform for point-of-care SARS-CoV-2 detection in the future.Finally,we established a visual nucleic acid testing based on the toehold-mediated strand displacement reaction.This chapter constructed a sandwich-structured nanocomplex probe(SNC Probe),when the target single-stranded DNA is present,it will replace the gold nanoparticles in the SNC Probe at room temperature,after the gold nanoparticles are released,the solution displays pink color after magnetic separation to realize the visual nucleic acid testing.The proposed strategy realized a sensitive detection of 110 nt synthetic SARS-CoV-2 ssDNA with the detection limit of16 nM by using the naked eye.In addition,a detection limit of 5 nM can be achieved with the help of UV-Vis Spectrophotometer.The PCR amplification-based target-induced strand-displacement reaction can realize the sensitive detection of nucleic acids with the detection limit of 400 aM.We further verified no cross-reaction between SARS-CoV-2 and other human coronaviruses,like SARS、MERS、HCoV-HKU1、HCoV-NL63.The visual nucleic acid testing based on the toehold-mediated strand displacement reaction can be combined with isothermal amplification technology to build a rapid,visual nucleic acid testing assay in the future,which has the significant potential to facilitate SARS-CoV-2 testing in various scenarios such as homes or community hospitals.