Establishment Of A CRISPR/Cas12a-based Rapid Detection Method For Group A Streptococcus Pyogenes

Objective(s):Group A streptococcus pyogenes,also known as Streptococcus pyogenes,is a purulent gram-positive coccus and the most pathogenic of the streptococci.It mainly invades the human upper respiratory tract and skin and causes acute tonsillitis,scarlet fever,sepsis and some non-suppurative diseases such as acute rheumatic fever,glomerulonephritis,and in rare cases,serious invasive diseases.Meanwhile,acute Streptococcus pyogenes pharyngitis caused by group A streptococcus pyogenes is a common pediatric disease with a high incidence in children.Early diagnosis and treatment are essential to prevent invasive infections.This study aims to establish a CRISPR/Cas-based rapid detection method for group A streptococcus pyogenes to quickly and accurately detect infections caused by group A streptococcus pyogenes,to improve the test’s adaptability and effectiveness,and to offer new approaches and ideas for the clinical diagnosis and treatment of group A streptococcus pyogenes.Methods:A vacuum-dried lyophilized powder of group A streptococcus pyogenes purchased from Henan Industrial Microbial Strain Engineering Technology Research Center was activated and cultured;1 ml of the activated culture was prepared,and nucleic acid was extracted using the centrifugal column method;the extracted DNA concentration was measured using a fluorometer Qubit 4.0.After reviewing the literature,DNase B of group A streptococcus pyogenes was chosen as the target sequence.Select the Cas12a recognition target and construct the matching cr RNA;design RAA primers using the Primer-BLAST software tool.Set up the RAA amplification reaction system,amplify the nucleic acid obtained from the standard strain as the template,screen the primers,and check the specificity of each primer.Create the reaction system for the GAS-CRISPR/Cas12a fluorescence assay and GAS-CRISPR/Cas12a test strip assay system,which includes the RAA amplification product,Cas12a protein,cr RNA,buffer,single-stranded DNA fluorescent probe,and enzyme-free water.Using tests to test the sensitivity and specificity,the two GAS-CRISPR/Cas12a assays were evaluated.To investigate the sensitivity of the two GAS-CRISPR/Cas12a assays,standard strain DNA extracts were diluted into five concentration gradients in accordance with the tenfold gradient dilution method;nucleic acids were extracted from Streptococcus agalactiae,Shigella sonnei,Staphylococcus aureus,and Escherichia coli isolated and preserved in the microbiology room of our hospital to obtain the specificity results.The bacterial samples that were isolated from the clinic were subjected to the GAS-CRISPR/Cas12a fluorescence assay and the GAS-CRISPR/Cas12a test strip assay.Results:Obtain the standard strain with satisfactory growth in culture for 24hours.Then,using the centrifugal column method,extract the DNA extract of the standard strain to a concentration of 7.48ng/μl.set up the RAA amplification reaction system,amplify under favorable conditions,and electrophorese the amplification products;screen primer 2 as the best primer pair,and obtain an amplification product of 255 bp,which corresponds to the size of the target gene,indicating successful amplification and primer pair.The best cr RNA was screened and detected in two ways,respectively,using the GAS-CRISPR/Cas12a fluorescence method and GAS-CRISPR/Cas12a test strip method detection system.The results demonstrated that the GAS-CRISPR/Cas12a fluorescence method maintained a high and well-characterized fluorescence curve at a 10~2-fold DNA dilution,indicating that the method’s sensitivity could reach 74.8 pg/μl;the GAS-CRISPR/Cas12a test strip method continued to produce positive results at a DNA concentration of 100 pg/μl,demonstrating that the method’s sensitivity reached 100 pg/μl.When the target strains from the clinic were examined,both approaches produced favorable results.Conclusion(s):In this study,the GAS-CRISPR/Cas12a fluorescence assay and GAS-CRISPR/Cas12a test strip assay were created using the RAA amplification approach in conjunction with CRISPR/Cas12a.With its features of high sensitivity,high specificity,and quick detection,the GAS-CRISPR/Cas12a method has a lot of potential for the advancement of POCT diagnostics.It has a lot of development potential.