An interlaboratory comparison of ITS2-PCR for the identification of yeasts, using the ABI Prism 310 and CEQ8000 capillary electrophoresis systems

Thierry De Baere,Gerda Verschraegen,Anne Van Keerberghen,An Boel,Geert Claeys,Peter Van Hauwe,Hans De Beenhouwer,Mario Vaneechoutte

doi:10.1186/1471-2180-5-14

Thierry De Baere, Gerda Verschraegen + Show 6 more

Open Access

https://doi.org/10.1186/1471-2180-5-14

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Abstract

BackgroundCurrently, most laboratories identify yeasts routinely on the basis of morphology and biochemical reactivity. This approach has quite often limited discriminatory power and may require long incubation periods. Due to the increase of fungal infections and due to specific antifungal resistence patterns for different species, accurate and rapid identification has become more important. Several molecular techniques have been described for fast and reliable identification of yeast isolates, but interlaboratory exchangeability of identification schemes of molecular techniques has hardly been studied. Here, we compared amplified ITS2 fragment length determination by an ABI Prism 310 (Applied Biosystems, Foster City, Ca.) capillary electrophoresis system with that obtained by a CEQ8000 (Beckman Coulter, Fullerton, Ca.) capillary electrophoresis system.ResultsAlthough ITS2 size estimations on both systems differed and separate libraries had to be constructed for each system, both approaches had the same discriminatory power with regard to the 44 reference strains, identical identifications were obtained for 39/ 40 clinical isolates in both laboratories and strains from 51 samples were correctly identified using CEQ8000, when compared to phenotypic identification.ConclusionIdentification of yeasts with ITS2-PCR followed by fragment analysis can be carried out on different capillary electrophoresis systems with comparable discriminatory power.

Highlights

Most laboratories identify yeasts routinely on the basis of morphology and biochemical reactivity
Construction of an Internal Transcribed Spacer 2 (ITS2)-length library using CEQ8000 Since ITS2-fragment length determination on CEQ8000 was expected to be different from that obtained on ABI310, we determined the length of a set of 44 reference strains, representing 33 species and subspecies, using the CEQ8000 capillary electrophoresis apparatus
The obtained lengths on CEQ8000 are listed in Table 1 (Additional file 1) and are compared with the lengths obtained on ABI310, as reported earlier [16], and with the theoretically expected lengths, as derived from published sequences

Summary

Introduction

Most laboratories identify yeasts routinely on the basis of morphology and biochemical reactivity This approach has quite often limited discriminatory power and may require long incubation periods. As a solution for those problems, several PCR-based methods have been described Some of these approaches rely on species-specific probes [7,8] – being limited to those species for which probes are available, but most of them are based on amplification using universal fungal primers, followed by post-amplification analysis like probe hybridization [9], sequencing [10,11], restriction analysis [12,13], temperature gradient gel electrophoresis [14] or – most and least laborious – fragment length determination [15,16,17,18]. Techniques like real-time PCR [22,23], pyrosequencing [24] and Luminex flow cytometry [25] have been used for identification of yeasts

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