Detection The EaeA Gene Of Diarrheagenic Escherichia Coli By LAMP-CRISPR Mathods

Diarrheagenic Escherichia coli is a major pathogenic bacterium that infects a variety of animals and humans and causes diarrhea in humans.It has important public health significance because of its wide distribution,especially by polluting food and water sources.The eaeA gene is an important virulence gene for the enteropathogenic E.coli（EPEC）and shiga toxin-producing E.coli（STEC,including enterohaemorrhagic E.coli O157:H7）.In this study,a LAMP-CRISPR,which combined the rapid and sensitive amplification by LAMP and the specific targeted cleavage by CRISPR,was developed to rapidly and accurately detect the eaeA gene of E.coli.Establishment of LAMP method for detecting eaeA gene of diarrheagenic Escherichia coli1.Development of a LAMP method for detecting the eaeA gene of diarrheagenic E.coli According to the nucleotide sequence of eaeA,encoding for the intimin of O157:H7,two sets of primers were designed and synthesized,and then each set of primers was screened by LAMP,and eaeA-1 primers were finally confirmed to be suitable for LAMP detection.The specificity tests showed that when only the genomic DNA of the E.coli O157:H7 ATCC43895 strain was used as the template,the LAMP amplification products showed obvious ladder-like bands by agarose gel electrophoresis,while the genomic DNA and from other non-E.coli foodborne pathogenic bacteria as the template and H2O as a blank control gave negative results.The sensitivity tests showed that the minimum detection limit of the developed LAMP was 4.3×10²CFU/m L,and the detection sensitivity was 100 times higher than that of conventional PCR.2.Development of a LAMP-CRISPR method for detecting eaeA gene of diarrheagenic E.coliTo avoid the impact of non-specific amplification that may occur during the LAMP amplification,a LAMP-CRISPR method by combining the above-mentioned LAMP detection method with Cas12b-based CRISPR,was developed to detect the eaeA gene of E.coli.First,sg RNA was designed and prepared according to amplification region in eaeA gene by F3/B3 primers within eaeA-1 primers set,and then the sg RNAs were screened by LAMP-CRISPR test,and finally eaeA-1-sg RNA-2 was screened out by measuring the trans-cleavage activity of Aap Cas12b protein.The specificity tests showed that only the genomic DNA of E.coli O157:H7 ATCC43895 strain could activate the trans-cleavage activity of Aap Cas12b protein,while both the genomic DNA of other non-E.coli foodborne pathogens and H₂O as a blank control gave negative results.The sensitivity tests showed that the minimum detection limit of the LAMP-CRISPR was 4.3×10²CFU/m L,which was consistent with that of LAMP,and the sensitivity was 100 times higher than that of conventional PCR.This developed method could complete the detection within one hour.In summary,in this study,both LAMP and LAMP-CRISPR detection methods were developed to detect eaeA gene of diarrheagenic E.coli.The LAMP-CRISPR method has the advantages of good specificity,high sensitivity,simplicity,and rapidity,providing a new method for rapid clinical detection of the eaeA gene of diarrheagenic E.coli.