Optimizing Two Step And One-pot SHERLOCK Nucleic Acid Detection Assays And Their Applications In Detecting Influenza A And B Viruses

Background and objective:SHERLOCK(Specific High Sensitivity Enzymatic Reporter UnLOCKing),integrating reverse-transcription recombinase polymerase amplification(RT-RPA)with clustered regularly interspaced short palindromic repeats/associated protein 13a(CRISPR/Cas13a),is an ultra-sensitive and specific nucleic acid detection technology.Two-step SHERLOCK and one-pot SHERLOCK are classified according to whether RT-RPA nucleic acid amplification and CRISPR/Cas13a detection are combined into the same reaction system.Two-step SHERLOCK is more sensitive while one-step SHERLOCK is faster and less risk of contamination,easier to obtain quantitative results.However,SHERLOCK is still less sensitive to real-time quantitative PCR,especially detecting RNA,and it takes longer time than isothermal amplification technologies.In this study,we aimed to determine the optimal reaction conditions for two-step and one-pot SHERLOCK to improve the sensitivity and shorten the detection time.Then,we established assays for detecting influenza A and B viruses to evaluate the specificity and sensitivity of the optimized SHERLOCK nucleic acid detection platform.Methods:1.Targeting influenza A virus-specific M gene and influenza B virus NS1 gene,we designed three forward primers and three reverse primers and three crRNAs for each target.Primers and crRN A were screened based on two-step SHERLOCK.2.Determined the optimal RNase H concentration for the reverse transcriptase TransScript Ⅳ and M-MLV respectively in RT-RPA based on two-step SHERLOCK,and then compared the amplification efficiency of the two reverse transcripts.3,Determined the optimal reaction buffer for CRISPR/Cas13a fluorescence detection in two-step SHERLOCK,including pH value(6.8～8.5),salt type(sodium chloride vs.potassium chloride),salt concentration(20～100 mM),and magnesium ion concentration(0～9 mM).4.Identified the optimal reaction conditions for one-pot SHERLOCK,including,molecular weight of polyethylene glycol(PEG8000 vs.PEG20000),pH value(6.8-8.0),salt type(potassium acetate vs.potassium chloride),salt concentration(40～120 mM)and reaction temperature(37～43℃).5.Evaluation specificity and analytical sensitivity of SHERLOCK-based influenza virus detection assays.6.Compared to RT-qPCR,the optimized two-step and one-step SHERLOCK assays were evaluated for clinical application by testing clinical swab samples of influenza virus.Results:1.The best RPA primer combination for influenza A viruses was AM-Fc plus AMRa,while NS-Fc plus NS-Rb for influenza B virus.Using AM-crRNAl plus AMcrRNA3 can cover the detection of four influenza A virus subtypes,H1N1,H3N2,H5N1,and H7N9.2.2 U/μl TransScript Ⅳ reverse transcriptase with 0.2 U/μl RNase H can significantly improve RT-RPA amplification efficiency.3.In the CRISPR/Cas13a fluorescence detection system of two-step SHERLOCK,the fluorescence rising rate was increased by 43%using low concentration of potassium chloride(0～40 mM)than high concentration(60～100 mM).The optimal reaction buffer was 40 mM Tri-HCl(pH 7.4),0～40 mM potassium chloride,and 3～6 mM magnesium chloride.4.In one-pot SHERLOCK,the fluorescence intensity was more consistent using PEG20000 than PEG8000;The time for the fluorescence to rise was 20%shorter using potassium acetate than potassium chloride and the optimal concentration of potassium acetate was 80～100 mM.the optimal reaction temperature was 41℃.5.SHERLOCK-based influenza A and B viruses detection assays can specifically detect influenza viruses,and no cross reaction with other common respiratory pathogens.The analytical sensitivities of the two-step and one-pot SHERLOCK influenza A virus assays were 1 copy per reaction and 10 copy per reaction,respectively.The analytical sensitivity of two-step and one-pot SHERLOCK influenza B virus assays was 1 copy per reaction and 10 copy per reaction,respectively.6.Compared to quantitative polymerase chain reaction(qPCR)in detecting clinical samples,the sensitivity of the optimized two-step and one-pot SHERLOCK assays for influenza A virus were 98.75%and 93.75%,respectively,and the specificity of both were 100%.The sensitivity of the optimized two-step SHERLOCK and the one-pot SHERLOCK assays for influenza B virus were 98.72%and 92.95%,respectively,and the specificity of both were 100%within 50 minutes.Conclusions:The lower limit of detection of the optimized two-step SHERLOCK was 200 copy per milliliter,and the reaction time was 50 minutes.The lower limit of detection of the optimized one-pot SHERLOCK was 2000 copy per milliliter.