Pathogens Diagnostics Using CRISPR-Cas12a

Rapid,early and accurate detection of pathogens is crucial to the efficient control of animal diseases.In this study,a novel paltform for pathogen detection was developed by integrating the unique collateral activity of Cas12a with Polymerase Chain Reaction(PCR).The contents are as follows:1.Cas12a-based fluorescent detection systemThe LbCas12a protein was purified by affinity chromatography,Gel filtration and Heparin methods.The optimized reaction conditions for cleavage activity of LbCas12a were determined by employing fluorophore quencher(FQ)-labeled reporter assay to optimize the factors of protein concentration,buffers,metal ions,the length of target,etc.Species-specific genes(iap gene for L.monocytogenes,khe gene for K.pneumonia,tuf gene for S.pyogenes,nuc gene for S.aureus,chuA gene for E.coli,ipaH gene for S.dysenteriae and invA for E.typhi)with sequence regions highly conserved between 7 different strains were chosen for primers design and selection.The optimized conditions for PCR amplification were determined by optimizing the primer concentration,annealing temperature,and cycle number.PCR amplification was performed using genomic DNA,from target and non-target pathogen individually,as the template.In addition,PCR reaction was operated using different 10-fold serial dilutions of the target pathogen genomic DNA as the template.The PCR amplicon sequence regions were used for crRNA design.crRNAs were transcribed in vitro using T7 transcription synthesis reaction.Gram-positive bacteria(Staphylococcus aureus)were selected as a model to analyse specificity and sensitivity by fluorescence quantitive polymerase chain reaction(qPCR).Primer and crRNA specificity of seven different pathogens was then confirmed by Cas12a-based fluorescence detection system.The Cas12a-based fluorescent method had high selectivity and ultralow cross-reactivity for each pathogens.The limit of detection(LOD)of the Cas12a-based fluorescence detection system is ranged from 10~1 to 10~5 aM for 7 different pathogens.The LOD for S.aureus is 10~4aM,which is 100-fold higher sensitivity than that by qPCR.2.Cas12a-based lateral flow detection systemThe lateral-flow readout depends on the degradation of a FAM-biotin reporter,allowing for diagnoses using Cas12a-based lateral flow detection system.The PCR amplicon of 7 different pathogens were obtained by PCR,and then the Cas12a cleavage reaction was performed.After 15～20 minutes,the results were directly interpreted via lateral-flow readout.PCR amplification was operated using different 10-fold serial dilutions of Shigella dysenteriae genomic DNA as the template.Then the Cas12a cleavage reaction was performed and the results were interpreted via lateral-flow readout.Moreover,we applied S.dysenteriae sample directly treated with NaOH to Cas12a-based lateral flow detection system.The Cas12a-based lateral-flow readout method can specifically identify each pathogens no cross hybridization was observed.For the target of S.dysenteriae,the limit of detection of the system is 10~2 aM.The Cas12a cleavage reaction and the result interpretation only need 20～25 min.The rapid detection technique permitted sensitive detection of S.dysenteriae cells at 10~4 CFU/mL.The rapid detection technique enabling pathogens detection directly from bacterial samples within 2 hours.In conclusion,we created a Cas12a-based detection platform that will provide useful information for the efficient control of the aquatic disease and the sustainable development of aquaculture.