A Study On Differentiation And Detection Four Types Of Respiratory Tract Virus Based On Toehold Switch Sensors In A Paper-based Cell-free System

Acute Respiratory Tract Infection（ARTI）is a common infectious disease mainly caused by respiratory tract viruses.As ARTI is severely threatening the human life and property security,in recent years,World Health Organization（WHO）has been continuingly appealing for more attention and efforts to prevent and control respiratory tract viruses.Post-infectious clinical syndromes of Respiratory syncytial virus（RSV）,Human coronavirus HKU1（HCo V-HKU1）,and Severe acute respiratory syndrome coronavirus 2（SARS-Co V-2）share high similarity,and therefore other methodologies are required for etiological diagnosis.Therefore,for respiratory tract viruses screening and diagnosis,establishing an effective,convenient,specific,and sensitive point-of-care testing（POCT）method is extremely critical.Based on the combination of toehold switch sensor,cell-free system and paper-based material,this study realizes the visual detection for HCo V-HKU1,SARS-Co V-2,RSVA,and RSVB.In addition,nucleic acid sequence-based amplification（NASBA）isothermal amplification is incorporated to increase the sensitivity of the detection method.Finally,the toehold switch sensor was applied onto a paper-based cell-free system that can further get rid of the dependence on complicated facilities and professional operators.NUPACK software was used to design candidate sequences for toehold switch sensor from highly conserved and specific sequences of HCo V-HKU1,SARS-Co V-2,RSVA,and RSVB.Recombinant plasmids bearing different switch sequence candidates were constructed with the design of p ET28a-switch-Lac Z and screened for background leakage and detection efficiency in cell-free system.According to the relative absorbance difference of A₅₇₀/A₄₂₀ and the colorimetric response of the liquid reaction,SC10,SR7,SA13,and SB2 toehold switch sensors were selected for HCo V-HKU1,SARS-Co V-2,RSVA,and RSVB,respectively.The NASBA isothermal amplification systems were established,and then the amplification primers and time were optimized.After that,the target sequence of toehold switch sensor was amplified under optimal conditions.The detection limits of NASBA combined with 15%urea denaturing polyacrylamide gel are 78 a M,63 a M,520 p M,and 910 a M for HCo V-HKU1,SARS-Co V-2,RSVA,and RSVB,respectively.Combined with NASBA,the specificity,cross reactivity,and sensitivity of the toehold switch sensors were comprehensively evaluated based on the response in a liquid or a paper-based cell-free system.The results showed that SC10,SR7,SA13,and SB2 toehold switch sensors have a high specificity without cross reaction.The sensitivity of the detection method for HCo V-HKU1,SARS-Co V-2,RSVA,and RSVB is 78 a M,63 a M,52a M,and 91 a M in a liquid cell-free system and the detection limits are 78 a M,63 a M,5.2 f M,and 91 a M in a paper-based cell-free system.In conclusion,this study established a colorimetric detection method for respiratory tract viruses based upon the combination of NASBA and toehold switch sensor in a paper-based cell-free system.A two-stage signal cascade amplification and twofold selectivity recognition significantly increase the sensitivity and specificity of the detection method.The cell-free system is freeze-dried on a portable paper-based material to facilitate stable storage and transportation.In consequence,this detection method is suitable for POCT in primary medical units lacking complicated equipment and operational experiences.Moreover,this study is meaningful in terms of promptly prevention and control of respiratory tract viruses,epidemiological analysis,and vaccine development.