title: Specific detection of live Escherichia coli O157:H7 using tetracysteine-tagged PP01 bacteriophage.
abstract: Sensitive and rapid detection of Escherichia coli O157:H7, one of the most notorious bacterial pathogens, is urgently needed for public health protection. Yet, the existing methods are either lack of speed or limited in discriminating viable and dead cells. Using a recombinant bacteriophage, here we report the development of a rapid and sensitive method for live E. coli O157:H7 detection. First, the wild-type PP01 phage was engineered with a tetracysteine (TC)-tag fused with the small outer capsid (SOC) protein. Then, this PP01-TC phage was used to inoculate bacterial sample for 30min. Specific infection and rapid replication of PP01-TC phage in viable E. coli O157:H7 host cell yields a large number of progeny phages with capsids displaying TC tags that can be fluorescently labeled by a membrane permeable biarsenical dye (FlAsH). The bright green fluorescence of single E. coli O157:H7 cells can be readily detected by flow cytometry (FCM) and fluorescence microscopy. High specificity of the assay was verified with seven other bacterial strains. Practical application in E. coli O157:H7 detection in drinks was successfully demonstrated with artificially contaminated 100% apple juice. In less than three hours (including sample preconcentration) and with 40mL of sample volume, as low as 1cfu/mL E. coli O157:H7 can be detected in the presence of large excess of other nontarget bacteria via fluorescence microscopic measurement. The as-developed TC-PP01-FlAsH approach shows a great potential in the safeguard of liquid food products by providing rapid, sensitive, and specific detection of live E. coli O157:H7.
authors.affiliation: The MOE Key Laboratory of Spectrochemical Analysis & Instrumentation, The Key Laboratory for Chemical Biology of Fujian Province, Department of Chemical Biology, College of Chemistry and Chemical Engineering, Xiamen University, Xiamen, Fujian 361005, PR China.
authors.forename: Lina
authors.initials: L
authors.lastname: Wu
authors.affiliation: The MOE Key Laboratory of Spectrochemical Analysis & Instrumentation, The Key Laboratory for Chemical Biology of Fujian Province, Department of Chemical Biology, College of Chemistry and Chemical Engineering, Xiamen University, Xiamen, Fujian 361005, PR China.
authors.forename: Yiyi
authors.initials: Y
authors.lastname: Song
authors.affiliation: The MOE Key Laboratory of Spectrochemical Analysis & Instrumentation, The Key Laboratory for Chemical Biology of Fujian Province, Department of Chemical Biology, College of Chemistry and Chemical Engineering, Xiamen University, Xiamen, Fujian 361005, PR China.
authors.forename: Tian
authors.initials: T
authors.lastname: Luan
authors.affiliation: The MOE Key Laboratory of Spectrochemical Analysis & Instrumentation, The Key Laboratory for Chemical Biology of Fujian Province, Department of Chemical Biology, College of Chemistry and Chemical Engineering, Xiamen University, Xiamen, Fujian 361005, PR China.
authors.forename: Ling
authors.initials: L
authors.lastname: Ma
authors.affiliation: The MOE Key Laboratory of Spectrochemical Analysis & Instrumentation, The Key Laboratory for Chemical Biology of Fujian Province, Department of Chemical Biology, College of Chemistry and Chemical Engineering, Xiamen University, Xiamen, Fujian 361005, PR China.
authors.forename: Liuqin
authors.initials: L
authors.lastname: Su
authors.affiliation: The MOE Key Laboratory of Spectrochemical Analysis & Instrumentation, The Key Laboratory for Chemical Biology of Fujian Province, Department of Chemical Biology, College of Chemistry and Chemical Engineering, Xiamen University, Xiamen, Fujian 361005, PR China.
authors.forename: Shuo
authors.initials: S
authors.lastname: Wang
authors.affiliation: The MOE Key Laboratory of Spectrochemical Analysis & Instrumentation, The Key Laboratory for Chemical Biology of Fujian Province, Department of Chemical Biology, College of Chemistry and Chemical Engineering, Xiamen University, Xiamen, Fujian 361005, PR China. Electronic address: xmyan@xmu.edu.cn.
authors.forename: Xiaomei
authors.initials: X
authors.lastname: Yan
mesh_terms: D001152:Arsenicals / Q000737:chemistry*; D015373:Bacterial Typing Techniques / Q000295:instrumentation*; D001435:Bacteriophages / Q000302:isolation & purification*; D001681:Biological Assay / Q000295:instrumentation; D003545:Cysteine / Q000737:chemistry; D004867:Equipment Design; D019544:Equipment Failure Analysis; D019453:Escherichia coli O157 / Q000302:isolation & purification* / Q000821:virology*; D005504:Food Analysis / Q000295:instrumentation*; D005506:Food Contamination / Q000032:analysis; D005516:Food Microbiology / Q000295:instrumentation; D008856:Microscopy, Fluorescence / Q000295:instrumentation; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D013194:Staining and Labeling / Q000379:methods
chemical_list: D001152:Arsenicals; D003545:Cysteine
keywords: Bacteriophage; Biarsenical dye; E. coli O157:H7; Flow cytometry; Tetracysteine