Study On Detection Of SARS-CoV-2 And Mutation Sites By CRISPR Immunochromatography

ObjectiveSevere Acute Respiratory Syndrome Coronavirus-2（SARS-CoV-2）infection caused COVID-19 pandemic,the situation of epidemic prevention and control is still very grim.In this study,CRISPR immunochromatographic detection techniques for SARS-CoV-2 and its four mutations（H69/V70del,T478K,N501Y and P681H）were discussed and established to meet the needs of epidemic prevention and control for rapid and accurate detection of SARS-CoV-2 infection based on Clustered Regularly Interspaced Short Palindromic Repeats-CRISPR-associated system（CRISPR-Cas）,Multienzyme Isothermal Rapid Amplification（MIRA）and Human internal Reference（HR）immunochromatographic strips.Methods1.The whole genome sequences of SARS-CoV-2 and 6 human coronaviruses were analyzed by MEGA7 software to find the specific conserved sequence of novel coronavirus nucleocapsid（Nucleocapsid,N）protein gene.2.The recombinant plasmids carrying N gene,S gene of SARS-CoV-2 wild type and4 variation sites sequences were constructed,and the RNA reference materials of variation sites were obtained by T7 in vitro transcription using these plasmids.3.The detection target of Cas13a protein was designed in the specific conservative sequence of SARS-CoV-2 N gene,and the detection targets of Cas12a protein were designed at four variation sites of S gene of SARS-CoV-2.The corresponding CRISPR-RNA（crRNA）were chemically synthesized.4.The CRISPR/Cas fluorescence detection systems and RNA MIRA systems were established.Cr RNA and amplification primers of each detection target were screened.5.HR immunoassay strips were designed and CRISPR immunoassay systems were established.RNA reference materials transcribed in vitro and SARS-CoV-2 RNA reference materials were used to evaluate the limitation of detection（LOD）of mutation sites and N gene CRISPR nucleic acid detection systems,respectively.6.The sensitivity and specificity of CRISPR immunoassay detections for SARS-CoV-2 N gene and T478K mutation were evaluated using RT-PCR validated clinical samples.Results1.The detection systems of Cas13a fluorescence and immunochromatographic targeting SARS-COV-2 N gene were constructed.The optimal final concentrations of taurine,Mg Cl₂,T7 RNA polymerase and RNase inhibitor in detection systems were determined to be 10 mM,20 mM,1.5 U/μL and 0.8 U/μL,respectively.2.HR immunochromatographic strip was designed and Cas13a immunochro-matographic method was used to detect SARS-CoV-2 RNA reference material a nd positive clinical samples.The results showed that the positive clinical sampl es showed color in the control line 2 of the strip,and the standard sample did not show color in the control line 2 of the strip.3.The LOD of Cas13a immunochromatographic detection targeting SARS-CoV-2 N gene was 0.25 copies/μL.This method could specifically distinguish SARS-CoV-2from influenza A（BH3N2）,influenza B（Victoria）and 6 human coronaviruses.In 52positive and 101 negative clinical samples confirmed by RT-PCR,the sensitivity and specificity of Cas13a immunochromatography detection were 100%.4.The LOD of Cas12a immunochromatography detection targeting SARS-CoV-2T478K mutation site was 10 copies/μL.In the detection of 28 Delta positive and 24Delta negative clinical samples of SARS-CoV-2 patients confirmed by RT-PCR,the sensitivity and specificity of Cas12a immunochromatography detection were 92.8%and 100%,respectively.5.The LOD of Cas12a immunochromatography detections targeting H69/V70 del,N501Y and P681H variant sites of SARS-CoV-2 were 10 copies/μL,10 copies/μL and100 copies/μL,respectively.Conclusions1.The CRISPR immunochromatographic detection based on CRISPR,MIRA and HR immunochromatographic detection can distinguish nucleic acids from human clinical samples and samples from other sources,so as to control the quality of human samples,such as sampling,nucleic acid extraction and amplification.2.Based on Cas13a protein,CRISPR immunochromatographic detection of The CRISPR immunochromatographic detection targeting SARS-CoV-2 N gene was established,which can achieve highly sensitive and specific field detection.3.Based on Cas12a protein,CRISPR immunochromatographic detections of H69/V70 del,T478K,N501Y and P681H mutations of SARS-CoV-2 were established,which can identify these four mutations in the field.