Multilaboratory Validation Study of Standardized Multiple-Locus Variable-Number Tandem Repeat Analysis Protocol for Shiga Toxin–ProducingEscherichia coliO157: A Novel Approach to Normalize Fragment Size Data Between Capillary Electrophoresis Platforms

Abstract

The PulseNet USA subtyping network recently established a standardized protocol for multiple-locus variable-number tandem repeat analysis (MLVA) to characterize Shiga toxin-producing Escherichia coli O157. To enable data comparisons from different laboratories in the same database, reproducibility and high quality of the data must be ensured. The aim of this study was to test the robustness and reproducibility of the proposed standardized protocol by subjecting it to a multilaboratory validation process and to address any discrepancies that may have arisen from the study. A set of 50 strains was tested in 10 PulseNet participating laboratories that used capillary electrophoresis instruments from two manufacturers. Six out of the 10 laboratories were able to generate correct MLVA types for 46 (92%) or more strains. The discrepancies in MLVA type assignment were caused mainly by difficulties in optimizing polymerase chain reactions that were attributed to technical inexperience of the staff and suboptimal quality of reagents and instrumentation. It was concluded that proper training of staff must be an integral part of technology transfer. The interlaboratory reproducibility of fragment sizing was excellent when the same capillary electrophoresis platform was used. However, sizing discrepancies of up to six base pairs for the same fragment were detected between the two platforms. These discrepancies were attributed to different dye and polymer chemistries employed by the manufacturers. A novel software script was developed to assign alleles based on two platform-specific (Beckman Coulter CEQ8000 and Applied Biosystems Genetic Analyzer 3130xl) look-up tables containing fragment size ranges for all alleles. The new allele assignment method was validated at the PulseNet central laboratory using a diverse set of 502 Shiga toxin-producing Escherichia coli O157 isolates. The validation confirmed that the script reliably assigned the same allele for the same fragment regardless of the platform used to size the fragment.

Multilocus Genotype Analysis of Escherichia coli O157 Isolates from Australia and the United States Provides Evidence of Geographic Divergence

“…MLVA was performed as outlined by HyytiaTrees et al (39). Targets were analyzed in Australia using Peak Scanner software (Version 1.0; Applied Biosystems) and in the United States using GeneMarker software (Version 1.70; SoftGenetics, State College, PA).…”

“…Targets were analyzed in Australia using Peak Scanner software (Version 1.0; Applied Biosystems) and in the United States using GeneMarker software (Version 1.70; SoftGenetics, State College, PA). The number of allele repeats for each target was determined using a platformspecific reference table described by PulseNet (39). Partial fragments and fragment sizes differing from those listed in the reference table were confirmed by Sanger sequencing using an Applied Biosystems 3130 Genetic Analyzer (Applied Biosystems).…”

Precise Dissection of an Escherichia coli O157:H7 Outbreak by Single Nucleotide Polymorphism Analysis

“…All study isolates were subjected to XbaI and Bln1 PFGE at the Missouri State Public Health, Kansas Health and Environmental, or Washington State Public Health laboratories, and stx and stx-bacteriophage insertion site genotyping (17) was done at Washington University. Most study isolates underwent MLVA at the Minnesota Department of Health Public Health Laboratory, using a validated eight-locus interrogation on a Beckman Coulter CEQ 8000 electrophoresis platform (18).…”

Potentially Human-Pathogenic Escherichia coli O26 in Norwegian Sheep Flocks

“…However, MLST has been shown to differentiate poorly between E. coli bacteria that are clonally highly conserved but epidemiologically unlinked, such as isolates of serotype O26:H11 (20). For STEC belonging to serotype O157:H7, multilocus variable number tandem repeat analysis (MLVA) has been shown to be a rapid and relatively simple method with a high level of coclustering with the PFGE method (22,30,35). For other serotypes of E. coli, a generic MLVA protocol has been published by Lindstedt et al (29).…”