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Single-Cell-Based Digital PCR Detection and Association of Shiga Toxin-Producing Escherichia coli Serogroups and Major Virulence Genes
Author(s) -
Xuming Liu,
Lance Noll,
Xiaorong Shi,
Elizabeth Porter,
Yin Wang,
Colin Stoy,
Nanyan Lu,
T. G. Nagaraja,
Gary A. Anderson,
Jianfa Bai
Publication year - 2020
Publication title -
journal of clinical microbiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 2.349
H-Index - 255
eISSN - 1070-633X
pISSN - 0095-1137
DOI - 10.1128/jcm.01684-19
Subject(s) - intimin , virulence , shiga toxin , escherichia coli , microbiology and biotechnology , biology , toxin , gene , virology , serotype , shiga like toxin , enterobacteriaceae , genetics
Escherichia coli serogroups O157, O26, O45, O103, O111, O121, and O145, when carrying major virulence genes, the Shiga toxin genes stx 1 and stx 2 and the intimin gene eae , are important foodborne pathogens. They are referred to as the "top 7" Shiga toxin-producing E. coli (STEC) serogroups and were declared by the USDA as adulterants to human health. Since top 7 serogroup-positive cattle feces and ground beef can also contain nonadulterant E. coli strains, regular PCR cannot confirm whether the virulence genes are carried by adulterant or nonadulterant E. coli serogroups. Thus, traditional gold-standard STEC detection requires bacterial isolation and characterization, which are not compatible with high-throughput settings and often take a week to obtain a definitive result. In this study, we demonstrated that the partition-based multichannel digital PCR (dPCR) system can be used to detect and associate the E. coli serogroup-specific gene with major virulence genes and developed a single-cell-based dPCR approach for rapid (within 1 day) and accurate detection and confirmation of major STEC serogroups in high-throughput settings. Major virulence genes carried by each of the top 7 STEC serogroups were detected by dPCR with appropriately diluted intact bacterial cells from pure cultures, culture-spiked cattle feces, and culture-spiked ground beef. Furthermore, from 100 randomly collected, naturally shed cattle fecal samples, 3 O103 strains carrying eae and 2 O45 strains carrying stx 1 were identified by this dPCR assay and verified by the traditional isolation method. This novel and rapid dPCR assay is a culture-independent, high-throughput, accurate, and sensitive method for STEC detection and confirmation.

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