Field-deployable Detection Methods For Novel Viral Nucleic Acids Based On Magnetic/Fluorescent Microspheres

The constant emergence of new outbreaks of infectious diseases has caused incalculable damage to human society.Early detection,isolation and treatment can effectively reduce the spread of infectious diseases and control the spread of epidemics.Timely,efficient and precise preventive control of infectious diseases is a fundamental guarantee for high-quality socioeconomic development.Primary health care institutions are the first gate for infectious disease surveillance.However,the low configuration of laboratory testing equipment and insufficient staff capacity in these institutions lead to their inability to identify infectious patients in a timely and sensitive manner,thus delaying epidemic prevention and control and even causing the spread of epidemics.Various field assays have been developed and play an important role in improving the diagnostic sensitivity of primary care institutions,but some shortcomings still exist.For example,the sensitivity of lateral flow immunochromatographic techniques is low and the specificity of isothermal amplification assays is insufficient.In response to the demand for field detection technologies,research and applications based on magnetic nanospheres to capture ultra-low concentrations of salmonella,fluorescent nanosphere test strips for simultaneous detection of zika and dengue viruses,and CRISPR/Cas12a-based rapid detection of SARS-Co V-2 were conducted in the following three areas.(1)A simple,efficient and reliable method was constructed for the capture and identification of bacteria at ultra-low concentrations.A highly controllable layer-by-layer assembly method was used to prepare quick-response magnetic nanospheres(MNs),which combined the advantages of magnetic microparticles and nanoparticles.The MNs do not require strong magnetic field separation tools or complex separation operation techniques,and capture almost all salmonella by attracting them with ordinary magnets for 1 min.The MNs have highly efficient recognition and superb binding power,with an incubation time of 20 minutes and salmonella capture efficiency of more than 95%,even if there is only one bacterium in the sample.In contrast,under the same conditions,the capture rate of magnetic microparticles for salmonella was less than 50%.The MNs have good anti-interference ability and stability,and can be directly applied to complex samples such as serum.Meanwhile,the captured salmonella can be directly used for downstream assays such as proliferation culture,polymerase chain reaction and fluorescence identification without the need of release process,which does not affect the downstream identification effect.This technique is a promising research tool for capturing ultra-low concentrations of pathogenic microorganisms.(2)A sensitive assay for the simultaneous identification of zika and dengue virus was constructed.Multiplex amplification of viral nucleic acids in the samples was performed by PCR,and the amplification products were detected using fluorescent nanosphere-lateral flow laminar analysis strips.The technique effectively integrates the advantages of PCR and the lateral flow laminar analysis strips,with good sensitivity and specificity.The visual method can detect 50 copies/m L of zika virus and 430 copies/m L of dengue virus,without non-specific reaction to related similar symptomatic viruses.In addition,this method can detect two viruses at the same time,effectively improving the sensitivity of clinical diagnosis and allowing clearer differentiation of virus species by different fluorescence.Furthermore,the PCR amplification product can be easily detected by the action of the lateral flow laminar analysis strips for 10 minutes.The method is expected to be used to carry out simple detection of viral nucleic acids under field conditions.(3)A new SARS-CoV-2 identification method based on CRISPR/Cas12 a and magnetic separation enhanced colorimetry was constructed.Visual detection of SARS-Co V-2 RNA was performed using a gold nanoparticle probe with a detection limit of 50 RNA copies per reaction.The CRISPR/Cas12a-assisted assay allows for specific differentiation of SARS-Co V-2 from other closely related viruses.Further analysis of clinical samples showed that this method was in 95% agreement with approved clinical RT-q PCR diagnostic kits.The method is not dependent on precision instruments and has potential value for application to screen for SARS-Co V-2 under field conditions.