| Listeria monocytogenes（LM）is one of the common pathogens among foodborne pathogens and the only one among the seven international Listeria spp.that can cause zoonotic diseases.At present,the laboratory detection method of Listeria monocytogenes is mostly traditional bacterial isolation and PCR identification,relying on professional personnel and instruments,which is time-consuming and laborious.In recent years,PfAgo（Pyrococcus furiosus Argonaute）protein has been used as a novel restriction endonuclease for nucleic acid detection,which has the advantages of high specificity,high sensitivity and rapid reaction.The virulence of Listeria monocytogenes is mainly due to the presence of various bacterial virulence genes,and the hly gene of Listeria monocytogenes is one of the most important virulence genes,and the listeriolysin O（LLO）protein encoded by the hly gene is closely related to the pathogenicity of Listeria monocytogenes.Therefore,this study was conducted to establish a rapid detection method for Listeria monocytogenes in food based on the virulence gene hly and PfAgo protein combined with LAMP isothermal amplification.In this study,pET-32a-PfAgo recombinant expression plasmid was constructed,expressed and purified by chemical transfer to E.coli BL21（DE3）,p MD-19T-hly recombinant plasmid was constructed based on hly gene,and the optimal amplification system was established by optimization of LAMP isothermal amplification system.The PfAgo protein detection system was determined by specific primer and probe screening,reaction temperature and time optimization.Finally,the results are visualized in combination with the test strips.The specific experimental methods and results are as follows:1 Construction and protein expression of pET-32a-PfAgo recombinant plasmidThe pET-32a-PfAgo recombinant expression plasmid was constructed and transferred toEscherichia coli BL21（DE3）for induction of expression and western-Blot identification,and the protein induction conditions were finally determined to be 15℃and 16h.The protein was purified using magnetic beads.2 pMD-19T-hly recombinant plasmid construction and LAMP amplification method optimizationThe pMD-19T-hly recombinant plasmid was constructed by designing full-length primers based on hly gene and PCR amplification.The hly sequences of 50 Listeria monocytogenes strains published in Gen Bank were compared and analyzed by Megalign software,and five different sets of LAMP-specific internal and external primers were designed by selecting the highly conserved region of the target gene sequence.The optimal primer concentration was8:1,and the optimal Mg2+concentration was 6 m M.3 Establishment of pET-32a-PfAgo protein assay systemFirstly,we verified that the protein has cleavage activity and can cut the target site arbitrarily,and the results of 20%TBE-PAGE gel electrophoresis showed that the 5’phosphorylated primer can cut the single-stranded DNA into two segments under the guidance of the protein;four primers were designed according to the highly conserved region of hly gene,and the results showed that all four primers can cut the target site under the action of the protein,regardless of the sequence site.The four primers were designed based on the highly conserved region of hly gene.The reaction system was then optimized,and the best combination of g DNA1 and the corresponding two primers was selected,with high fluorescence value;the temperature optimization showed the optimal temperature of 95℃;the time optimization showed the optimal reaction time of 40 min;the optimal final concentration of g DNA primer was 0.5μM,the buffer Mn2+concentration was 1m M,and the protein amount was 400U.The fluorescence assay of PfAgo protein was combined with test strips,and the results showed that the optimal concentration of the test strip probe was20n M.The specificity test showed no cross-reactivity with S.aureus,avian pathogenic E.coli,Salmonella typhimurium,Listeria innocua,Staphylococcus,and Enterococcus faecalis;The sensitivity of fluorometric assay showed that the minimum detection limit for p MD-19T-hly recombinant plasmid was 1.8×101copies/μL,and the detection limit of test strip method was 1.8×102copies/μL.This study successfully established the detection method of Listeria monocytogenes based on PfAgo protein combined with LAMP technology,which has the advantages of easy operation,high efficiency and accuracy,short reaction time,and can avoid the use of expensive instruments when combined with test strips,which can provide a new option for the rapid detection of Listeria monocytogenes and provide effective ideas for food safety detection in remote areas and grassroots laboratories. |
