Abstract
Multiple-locus variable-number of tandem repeats analysis (MLVA) is widely used for typing of pathogens. Methods such as MLVA based on determining DNA fragment size by the use of capillary electrophoresis have an inherent problem as a considerable offset between measured and real (sequenced) lengths is commonly observed. This discrepancy arises from variation within the laboratory set-up used for fragment analysis. To obtain comparable results between laboratories using different set-ups, some form of calibration is a necessity. A simple approach is to use a set of calibration strains with known allele sizes and determine what compensation factors need to be applied under the chosen set-up conditions in order to obtain the correct allele sizes. We present here a proof-of-concept study showing that using such a set of calibration strains makes inter-laboratory comparison possible. In this study, 20 international laboratories analysed 15 test strains using a five-locus Salmonella enterica serovar Typhimurium MLVA scheme. When using compensation factors derived from a calibration set of 33 isolates, 99.4% (1,461/1,470) of the MLVA alleles of the test strains were assigned correctly, compared with 64.8% (952/1,470) without any compensation. After final analysis, 97.3% (286/294) of the test strains were assigned correct MLVA profiles. We therefore recommend this concept for obtaining comparable MLVA results.
Highlights
Multiple-locus variable-number of tandem repeats analysis (MLVA) has become an increasingly popular method for fast, reproducible and inexpensive subtyping of many bacterial species including Salmonella enterica serovar Typhimurium [1,2]
Polymerase chain reaction (PCR) is used to amplify DNA containing the VNTR sites and electrophoresis is used to distinguish the alleles according to their sizes
We show that MLVA results from 20 laboratories using different laboratory MLVA primers and/or capillary electrophoresis (CE) equipment can be compared in a relevant way by the use of calibration strains
Summary
Multiple-locus variable-number of tandem repeats analysis (MLVA) has become an increasingly popular method for fast, reproducible and inexpensive subtyping of many bacterial species including Salmonella enterica serovar Typhimurium [1,2]. The principle of MLVA is a concurrent analysis of loci with tandem repeated DNA sequences (variable number of tandem repeats, VNTRs). Polymerase chain reaction (PCR) is used to amplify DNA containing the VNTR sites and electrophoresis is used to distinguish the alleles according to their sizes. It is known that CE, www.eurosurveillance.org as employed by common sequencing equipment, is notorious for having a set-up-dependent discrepancy between measured and real (sequenced) fragment lengths [3,4,5,6]. Production of data that are comparable between laboratories is crucial for the usefulness of typing methods for food-borne pathogens, e.g. to enable detection of common outbreaks in different regions or countries and to track the pathogens in the food production chain
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