Dataset Information

Single-Cell-Based Digital PCR Detection and Association of Shiga Toxin-Producing Escherichia coli Serogroups and Major Virulence Genes.

ABSTRACT: 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.

SUBMITTER: Liu X

PROVIDER: S-EPMC7041568 | biostudies-literature | 2020 Feb

REPOSITORIES: biostudies-literature

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Single-Cell-Based Digital PCR Detection and Association of Shiga Toxin-Producing Escherichia coli Serogroups and Major Virulence Genes.

Liu Xuming X Noll Lance L Shi Xiaorong X Porter Elizabeth E Wang Yin Y Stoy Colin C Lu Nanyan N Nagaraja T G TG Anderson Gary G Bai Jianfa J

Journal of clinical microbiology 20200224 3

Escherichia coli serogroups O157, O26, O45, O103, O111, O121, and O145, when carrying major virulence genes, the Shiga toxin genes stx1 and stx2 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 ...[more]

PMID: 31896667

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Project description:BackgroundThe accuracy and metrology traceability of DNA quantification is becoming a critical theme in many fields, including diagnosis, forensic analysis, microorganism detection etc. Thus the research of DNA reference materials (RMs) and consistency of DNA quantification methods has attracted considerable research interest.ResultsIn this work, we developed 3 plasmid candidate RMs, containing 3 target genes of Escherichia coli O157:H7 (E. coli O157:H7) and other Shiga toxin-producing Escherichia coli (STEC): stx1, stx2, and fliC (h7) respectively. Comprehensive investigation of the plasmid RMs was performed for their sequence, purity, homogeneity and stability, and then the concentration was quantified by three different methods: ultraviolet spectrophotometer (UV), high resolution inductively coupled plasma mass spectrometry (HR-ICP-MS) and digital PCR. As a routinely applied method for DNA analysis, UV was utilized for the quantification (OD260) and purity analysis for the plasmids. HR-ICP-MS quantified the plasmid DNA through analysing the phosphorus in DNA molecules. Digital PCR distributed the DNA samples onto a microarray chip containing thousands of reaction chambers, and quantified the DNA copy numbers by analysing the number of positive signals without any calibration curves needed.ConclusionsBased on the high purification of the DNA reference materials and the optimization of dPCR analysis, we successfully achieved good consistency between UV, HR-ICP-MS and dPCR, with relative deviations lower than 10 %. We then performed the co-quantification of 3 DNA RMs with three different methods together, and the uncertainties of their concentration were evaluated. Finally, the certified values and expanded uncertainties for 3 DNA RMs (pFliC, pStx1 and pStx2) were (1.60 ± 0.10) × 10(10) copies/μL, (1.53 ± 0.10) × 10(10) copies/μL and (1.70 ± 0.11) × 10(10) copies/μL respectively.Graphical abstractWe developed 3 plasmid candidate RMs, containing 3 target genes of Escherichia coli O157:H7 (E. coli O157:H7) and other Shiga toxin-producing Escherichia coli (STEC): stx1, stx2, and fliC (h7) respectively, and the quantification of three different methods (UV, dPCR, ICP) was studied.

Project description:Shiga toxin-producing Escherichia coli (STEC) have been linked to food-borne disease outbreaks. As PCR is routinely used to screen foods for STEC, it is important that factors leading to inconsistent detection of STEC by PCR are understood. This study used whole genome sequencing (WGS) to investigate causes of inconsistent PCR detection of stx1, stx2, and serogroup-specific genes. Fifty strains isolated from Alberta feedlot cattle from three different studies were selected with inconsistent or consistent detection of stx and serogroup by PCR. All isolates were initially classified as STEC by PCR. Sequencing was performed using Illumina MiSeq® with sample library by Nextera XT. Virtual PCRs were performed using Geneious and bacteriophage content was determined using PHASTER. Sequencing coverage ranged from 47 to 102x, averaging 74x, with sequences deposited in the NCBI database. Eleven strains were confirmed by WGS as STEC having complete stxA and stxB subunits. However, truncated stx fragments occurred in twenty-two other isolates, some having multiple stx fragments in the genome. Isolates with complete stx by WGS had consistent stx1 and stx2 detection by PCR, although one also having a stx2 fragment had inconsistent stx2 PCR. For all STEC and 18/39 non-STEC, serogroups determined by PCR agreed with those determined by WGS. An additional three WGS serotypes were inconclusive and two isolates were Citrobacter spp. Results demonstrate that stx fragments associated with stx-carrying bacteriophages in the E. coli genome may contribute to inconsistent detection of stx1 and stx2 by PCR. Fourteen isolates had integrated stx bacteriophage but lacked complete or fragmentary stx possibly due to partial bacteriophage excision after sub-cultivation or other unclear mechanisms. The majority of STEC isolates (7/11) did not have identifiable bacteriophage DNA in the contig(s) where stx was located, likely increasing the stability of stx in the bacterial genome and its detection by PCR.

Dataset Information

Single-Cell-Based Digital PCR Detection and Association of Shiga Toxin-Producing Escherichia coli Serogroups and Major Virulence Genes.

Publications

Single-Cell-Based Digital PCR Detection and Association of Shiga Toxin-Producing Escherichia coli Serogroups and Major Virulence Genes.

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