title: Rapid identification and differentiation of non-O157 Shiga toxin-producing Escherichia coli using polymerase chain reaction coupled to electrospray ionization mass spectrometry.
abstract: A polymerase chain reaction (PCR)-mass spectroscopy assay was developed to identify non-O157 Shiga toxin-producing Escherichia coli (STEC) with Plex-ID biosensor system, a platform identifying short PCR amplicons by specific base compositions. This assay simultaneously amplifies five fragments of two housekeeping genes, two subunits of stx2 gene, and four other virulence genes of STEC. A total of 164 well-characterized STEC isolates were examined with the assay to build a DNA base composition database. Another panel of 108 diverse STEC isolates was tested with the established database to evaluate the assay's identification capability. Among the 108 isolates, the assay specificity was 100% for three (stx1, eae, and aggA) out of five tested virulence genes, but 99% for stx2 and 96% for hlyA, respectively. Main stx1/stx2 subtypes and multiple alleles of stx1/stx2 could be differentiated. The assay successfully identified several clinically significant serotypes, including O91:H14, O103:H25, O145:H28/NM, O113:H21, and O104:H4. Meanwhile, it was able to group isolates with different levels of pathogenic potential. The results suggest that this high-throughput method may be useful in clinical and regulatory laboratories for STEC identification, particularly strains with increased pathogenic potential.
authors.affiliation: College of Food Science and Engineering, Northwest A&F University, Yangling, Shaanxi Province, China.
authors.forename: Jinling
authors.initials: J
authors.lastname: Shen
authors.forename: Fei
authors.initials: F
authors.lastname: Wang
authors.forename: Feng
authors.initials: F
authors.lastname: Li
authors.forename: Roberta
authors.initials: R
authors.lastname: Housley
authors.forename: Heather
authors.initials: H
authors.lastname: Carolan
authors.forename: Irene
authors.initials: I
authors.lastname: Yasuda
authors.forename: Erik
authors.initials: E
authors.lastname: Burrows
authors.forename: Rachel
authors.initials: R
authors.lastname: Binet
authors.forename: Rangarajan
authors.initials: R
authors.lastname: Sampath
authors.forename: Jianmin
authors.initials: J
authors.lastname: Zhang
authors.forename: Marc W
authors.initials: MW
authors.lastname: Allard
authors.forename: Jianghong
authors.initials: J
authors.lastname: Meng
mesh_terms: D018829:Adhesins, Bacterial / Q000235:genetics; D000818:Animals; D004269:DNA, Bacterial / Q000235:genetics; D019453:Escherichia coli O157; D029968:Escherichia coli Proteins / Q000235:genetics; D036781:Fimbriae Proteins / Q000235:genetics; D005506:Food Contamination / Q000032:analysis; D005516:Food Microbiology; D006801:Humans; D016133:Polymerase Chain Reaction / Q000379:methods*; D012703:Serotyping; D022622:Shiga Toxin 1 / Q000235:genetics; D022641:Shiga Toxin 2 / Q000235:genetics; D054323:Shiga-Toxigenic Escherichia coli / Q000145:classification* / Q000235:genetics / Q000302:isolation & purification*; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods*; D037521:Virulence Factors / Q000235:genetics
chemical_list: D018829:Adhesins, Bacterial; C079725:AggA protein, E coli; D004269:DNA, Bacterial; D029968:Escherichia coli Proteins; D022622:Shiga Toxin 1; D022641:Shiga Toxin 2; D037521:Virulence Factors; C073819:eaeA protein, E coli; D036781:Fimbriae Proteins