Multilocus Variable-Number Tandem-Repeat Analysis of Enterohemorrhagic<i>Escherichia coli</i>Serogroups O157, O26, and O111 Based on a<i>De Novo</i>Look-Up Table Constructed by Regression Analysis

Wakabayashi, Yuki; Harada, Tetsuya; Kawai, Takao; Takahashi, Yusuke; Umekawa, Nao; Izumiya, Hidemasa; Kawatsu, Kentaro

doi:10.1089/fpd.2020.2921

Cited by 2 publications

(2 citation statements)

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“…E. coli O157:H7 has been reported in several outbreaks worldwide, in both developing and developed countries, including in the USA [34], Canada [35], and Europe [36]. Currently, there several molecular techniques are commonly used for food-borne disease surveillance and the subtyping of E. coli, such as the PulseNet [37,38], Multilocus Sequence Typing [39], and Multilocus Variable-Number Tandem-Repeat Analysis [40] techniques. PulseNet is based on the characterization of whole bacterial genomes using enzymatic digestion patterns that are separated by pulsed-field gel electrophoresis [12,20,38].…”

Section: Introductionmentioning

confidence: 99%

“…Similarly, Multilocus Sequence Typing (MLST) is widely used as a genotyping method and has been applied successfully to E. coli [22,23]; however, this sequencing procedure is expensive and time-consuming for routine monitoring [42]. Multilocus Variable-Number Tandem-Repeat analysis is another subtyping technique used for E. coli, but it has limitations in the development of a universal panel for MLVA [40]. Nevertheless, SNP analysis enables the classification of very similar genomes and the detailed classification of genomes of E. coli that are difficult to distinguish using conventional typing techniques [43].…”

Section: Introductionmentioning

confidence: 99%

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Development of Single Nucleotide Polymorphism (SNP)-Based Triplex PCR Marker for Serotype-specific Escherichia coli Detection

2022

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Single-nucleotide polymorphisms (SNPs) are one of the most common forms of genetic variation and as such are powerful tools for the identification of bacterial strains, their genetic diversity, phylogenetic analysis, and outbreak surveillance. In this study, we used 15 sets of SNP-containing primers to amplify and sequence the target Escherichia coli. Based on the combination of the 15-sequence primer sets, each SNP site encompassing forward and reverse primer sequences (620–919 bp) were aligned and an SNP-based marker was designed. Each SNP marker exists in at least two SNP sites at the 3′ end of each primer; one natural and the other artificially created by transition or transversion mutation. Thus, 12 sets of SNP primers (225–488 bp) were developed for validation by amplifying the target E. coli. Finally, a temperature gradient triplex PCR kit was designed to detect target E. coli strains. The selected primers were amplified in three genes (ileS, thrB, and polB), with fragment sizes of 401, 337, and 232 bp for E. coli O157:H7, E. coli, and E. coli O145:H28, respectively. This allele-specific SNP-based triplex primer assay provides serotype-specific detection of E. coli strains in one reaction tube. The developed marker would be used to diagnose, investigate, and control food-borne E. coli outbreaks.

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