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Volume 22, Number 12—December 2016
Research

Whole-Genome Characterization and Strain Comparison of VT2f-Producing Escherichia coli Causing Hemolytic Uremic Syndrome

Laura GrandeComments to Author , Valeria Michelacci, Roslen Bondì, Federica Gigliucci, Eelco Franz, Mahdi Askari Badouei, Sabine Schlager, Fabio Minelli, Rosangela Tozzoli, Alfredo Caprioli, and Stefano Morabito
Author affiliations: Istituto Superiore di Sanità, Rome, Italy (L. Grande, V. Michelacci, R. Bondi, F. Gigliucci, F. Minelli, R. Tozzoli, A. Caprioli, S. Morabito); National Institute for Public Health and the Environment, Bilthoven, the Netherlands (E. Franz); Faculty of Veterinary Medicine, Garmsar Branch, Islamic Azad University, Garmsar, Iran (M. Askari Badouei); Austrian Agency for Health and Food Safety, Graz, Austria (S. Schlager)

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Table 3

PCR and restriction fragment length polymorphism analysis conditions used to verify VT2f phage structure in a comparative analysis of the virulence profile of human and zoonotic VT2f-producing Escherichia coli strains*

Analysis Primer name Sequence, 5′→3′ Position Thermal profile Amplicon size, bp Restriction enzyme (obtained fragments, bp + bp)
PCR1 φ-vtx2f_1FW caccatatcccagcaactgc 1,985–2,005 95°C for 2 min, 30× (94°C for 30 s, 53°C for 30 s, 70°C for 9 min); 72°C for 10 min
6,331
PvuI (1,773 + 4,558)

φ -vtx2f_1RV
gttggcggttccgactacaa
8,315–8,296
PCR2 φ -vtx2f_2FW gcgcatcaccacttcatctt 8,337–8,357 95°C for 2 min, 30× (94°C for 30 s, 53°C for 30 s, 70°C for 9 min), 72°C for 10 min
8,166
HindIII (1,855 + 6,311)

128–1
agattgggcgtcattcactggttg
16,502–16,479
PCR3 φ -vtx2f_3FW ggagtggatattgccgacct 16,808–16,827 95°C for 2 min, 30× (94°C for 30 s, 53°C for 30s, 70°C for 9 min), 72°C for 10 min
3,927
BgIII (1,310 + 2,617)

φ -vtx2f_3RV
gtcttcctgctgaggcgatc
20,734–20,715
PCR4 φ -vtx2f_4FW taatcgcggccgtactcaag 22,172–22,191 95°C for 2 min, 30× (94°C for 30 s, 53°C for 30 s, 70°C for 9 min), 72°C for 10 min 8,808 NcoI (5,029 + 3,779)
φ -vtx2f_4RV tgttcagctccaccttacgg 30,979–30,960

*Analysis for PCR2, primer 128–1 from (5); all other data were compiled for this study. All the long PCR described were performed with the GoTaq Long PCR Master Mix (Promega, Madison, WI, USA) according to manufacturer’s instructions. Primer positions refer to the phage sequence deposited into the EMBL database (accession no. LN997803).

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