Construction Of Engineering Bacteriophage JL1and Its Detection On Escherichia Coli O157:H7

This work was supported by grants from the General Administration of QualitySupervision, Inspection and Quarantine of the People’s Republic of China (Project Code:No.2012IK163).Since E. coli O157:H7has been reported at the eighties of the last century, it has beenclassified as the one of the most serious foodborne pathogens as its pathogenicity by WHO.Several case of death have happened due E. coli O157:H7in food in Europe and otherdeveloped countries, also occurred in our country. How to detect E. coli O157:H7in foodor food material quickly and efficiently and avoid accidents has become the focus of healthdepartments around the world.Phage as the virus of bacteria can infect its host specificity. This specific relationshipbetween phage and its host bacteria is the base of detection methods. Detection offoodborne pathogens by phage methods have drow more and more attention as itsspecificity, short detection time, low cost and other advantages. It has become thesupplement or replacement of the current detection methods.In this study, we have isolated a phage from sewage using E. coli O157:H7as host,the host spectrum of JL1was performed with24differnet strains, suggested JL1has anarrow spectrum and it was specificity to E.coli O157: H7. We have studied the biologicalcharacteristics of JL1. We test the influence of pH, temperature and chloroform on JL1viability, by measuring phage titers after treatments; the one-step growth curve of phageJL1shows that the incubation time is about20min, the lytic time is about30min.Morphological analysis under transmission electron microscope (TEM) identified phageJL1as a member of the family Siphoviridae of the order Caudovirales, JL1had anicosahedral head approximately30±2nm in diameter and a long flexible non-contractiletail about110±10nm in length. Complete genome sequence of phage JL1indicted JL1iscomposed of a linear double-stranded DNA of43,457base pairs in length,240ORFs thatis long than100bp was found in its genome, contain60putative genes,19putative genescould be noted with functions,39putative genes could find high homologous sequenceswith unknown functions, only2didn’t find homologous sequences. The complete genome sequence of JL1is available in GenBank under accession number JX865427.2.Comparative analysis of the genomes using WebACT revealed JL1is different to otherfour phages that have high similarity with it significantly. Eight different E.coli O157:H7phages were found in Genbank. Comparison of the size of their genomes, GC content,gene density suggeste the genetic information of JL1is difference to other phagessignificantly.After analysis the gene structure of JL1, we selected three genes which related to capsidprotein as insertion sites for reporter gene, egfp was inserted into3’ of them byhomologous recombinant method to construct reporter phage. A reporter phage JL1-EGFPwas obtained. JL1-EGFP was applied to detect E.coli O157:H7in pure culture, greenfluorescence could be observed under fluorescence microscope, the minimum bacteriaconcentration can be detected positice is104CFU/mL, the host spectrum have not beenchanged after homologous recombinant, JL1-EGFP is specificity to E.coli O157:H7.