Rapid detection of Staphylococcus aureus by aptamer fluorescence sensor mediated by CRISPR-Cas12a
Objective To take Staphylococcus aureus as the representative of typical foodborne pathogens,so as to establish a new detection technology of typical foodborne pathogens by using the method of clustered regularly interspaced short palindromic repeats(CRISPR)-associated protein(Cas).Methods In the presence of Staphylococcus aureus,it could bind to the aptamer of Staphylococcus aureus adapter adsorbed on magnetic beads.The originally bound complementary chains were released into the solution,and the supernatant containing free complementary chains was separated through magnetic separation.Specially designed CRISPR RNA(crRNA)was bound to activate the trans-cleaving DNA enzyme activity of Cas12a protein.the nucleic acid fluorescent probe was cleaved,and then the fluorescence intensity of the system was detected.Results After the pretreatment was completed,the detection reaction could be controlled within 1 hour,which was much shorter than the time required by the national standard method.Moreover,the required instruments and equipment were simple and convenient,and it was easy to develop detection kits.Compared with the commercially available rapid detection kit for Staphylococcus aureus,this experimental method did not need to carry out pre-enrichment and cracking steps,and it took less time,and the qualitative results could meet the needs of daily supervision and inspection work.Conclusion This method is easy to operate,easy to use,and takes a short time.A reagent kit has been developed,and it can be used together with a portable detection box to facilitate rapid qualitative screening of Staphylococcus aureus in canteens,training stations and other fields.This method also has a wide range of applicability.For other food-borne pathogens,different food-borne pathogens can be detected by modifying them into specific aptamers and designing corresponding crRNA primers.
Staphylococcus aureusAptamerSignal amplificationMagnetic microspheresCRISPR-Cas system
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维普
