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- DOI: 10.1128/JCM.02136-06
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A cautionary tale: the lack of consistency in allele sizes between two laboratories for a published Multi-Locus Microsatellite Typing (MLMT) system.
Pasqualotto AC, Denning DW, Anderson MJ
J Clin Microbiol. 2007;45:522-528.
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Full-text held externally
- DOI: 10.1128/JCM.02136-06
Abstract
For species with low genetic diversity, typing using the difference in PCR fragment length resulting from the variation in the number of short tandem repeats, has been shown to provide a high level of discrimination.This technique has been called Multi-Locus Microsatellite Typing (MLMT) or Multiple Locus Variable-number tandem repeat Analysis (MLVA) and studies usually employ genetic or sequence analyzers to size PCR fragments to a high degree of precision.We set out to validate one such system that has been developed for Aspergillus fumigatus [de Valk, H.A., J.F.G.M.Meis, I.M.Curfs, K.Muehlethaler, J.W.Mouton, and C.H.W.Klaassen.2005.J.Clin.Microbiol.43:4112-4120.].The sizes of alleles were compared both by sequencing and from two genotyping laboratories where they used capillary electrophoresis (CE) for sizing.Size differences of up to 6 bases were found between the actual size by sequencing and the size reported by CE.In addition, because the two genotyping laboratories used different machines and running conditions, differences of up to 3 bases were identified between them.As the microsatellite markers used vary by repeat units of 3 or 4 bases, it was not possible to assign PCR fragments to the correct allele without confirming the size of a range of alleles by direct sequencing.Lines of best fit were plotted for each CE machine against actual size and will therefore enable unsequenced PCR fragments to be assigned to the correct allele.This study highlights the care required to ensure that a MLMT system undergoes a suitable correction procedure before data can be merged between different laboratories involved in the typing of individual species.