Application Of Phage 80α Receptor Binding Protein RBP And Spy System In The Detection Of Staphylococcus Aureus

Staphylococcus aureus(S.aureus)is widely distributed in nature and is one of the main pathogens causing foodborne diseases,which can lead to death in severe cases.The existing detection technology of Staphylococcus aureus has high detection cost and is difficult to popularize in areas with scarce resources.A fast and accurate detection technology of Staphylococcus aureus is needed to make up for the shortcomings of existing detection methods.Phages bind to their host bacterial cells with high specificity through receptor-binding proteins(RBPs),followed by a phage propagation cycle.With the continuous development of technology and the strong specificity of RBP,RBP has been used to detect bacteria.Magnetic bead enrichment technology is simple and fast,and is widely used in cell sorting,pathogenic microorganism detection,and drug-targeted therapy.Recombinase polymerase amplification(RPA)has been widely used in the detection of bacteria,fungi,parasites,viruses,drug resistance genes,etc.,and is becoming popular in molecular diagnosis.As a cutting-edge technology in the field of in vitro diagnostics,the CRISPR/Cas12 a system has a cr RNA-guided Cas protein that recognizes target DNA and stimulates non-specific single-strand DNA cleavage activity;the advantages of the above detection technologies are for the development of rapid and efficient detection methods for Staphylococcus aureus provided important support.In this study,the phage 80α receptor-binding protein(RBP)of Staphylococcus aureus was used as the research object,and the recombinant protein Spytag-RBP was constructed in combination with the Spycatcher-Spytag system,and its ability to bind Staphylococcus aureus was verified.At the same time,the optimal concentration of Spycatcher protein bound to NHS magnetic beads was explored.After capture of Staphylococcus aureus by self-made immunomagnetic beads,with the help of RPA isothermal amplification and emerging CRISPR/Cas12 a technology,it was aimed at constructing a new rapid strain of Staphylococcus aureus.Detection method.Taking advantage of the fact that the NHS group on the surface of the magnetic bead can form a stable peptide bond with the protein with a primary amine group,the Spycatcher protein prepared in the previous stage was incubated with the NHS magnetic bead,and then incubated with the Spytag-RBP protein after the incubation,prepare immunomagnetic beads,and finally capture and concentrate Staphylococcus aureus in the matrix.Since it was found that the concentration of Spycatcher protein had a significant effect on the binding ability of magnetic beads during the experiment,which in turn affected the efficiency of capturing Staphylococcus aureus,we optimized the incubation concentration of Spycatcher protein respectively.The results showed that when the protein concentration was 0.7μg/μL,the magnetic beads could achieve better capture efficiency.SDS-PAGE protein gel electrophoresis and PCR verification proved that the Spycatcher-Spytag RBP immunomagnetic beads in this study have a good ability to capture Staphylococcus aureus.Based on the above results,this study combined magnetic bead enrichment,RPA and CRISPR/Cas12 a technologies to construct a rapid detection technology for Staphylococcus aureus,and this technology can also detect golden yellow in milk,juice and other foods.Staphylococcus,the entire detection method can be completed within 2 hours at 37°C,and the lowest detection limit of Staphylococcus aureus in artificially contaminated milk samples is 10 CFU/m L.This technology does not require professional technicians or auxiliary equipment,has good development prospects,and is of great significance for the prevention and diagnosis of foodborne diseases.