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. 2008 Feb;46(2):713-20.
doi: 10.1128/JCM.00514-07. Epub 2007 Dec 12.

Efficient identification of clinically relevant Candida yeast species by use of an assay combining panfungal loop-mediated isothermal DNA amplification with hybridization to species-specific oligonucleotide probes

Affiliations

Efficient identification of clinically relevant Candida yeast species by use of an assay combining panfungal loop-mediated isothermal DNA amplification with hybridization to species-specific oligonucleotide probes

João Inácio et al. J Clin Microbiol. 2008 Feb.

Abstract

The occurrence of invasive mycoses has progressively increased in recent years. Yeasts of the genus Candida remain the leading etiologic agents of those infections. Early identification of opportunistic yeasts may contribute significantly to improved disease management and the selection of appropriate antifungal therapy. We developed a rapid and reliable molecular identification system for clinically relevant yeasts that makes use of nonspecific primers to amplify a region of the 26S rRNA gene, followed by reverse hybridization of the digoxigenin-labeled products to a panel of species-specific oligonucleotide probes arranged on a nylon membrane macroarray format. DNA amplification was achieved by the recently developed loop-mediated isothermal DNA amplification technology, a promising option for the development of improved laboratory diagnostic kits. The newly developed method was successful in distinguishing among the major clinically relevant yeasts associated with bloodstream infections by using simple, rapid, and cost-effective procedures and equipment.

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Figures

FIG. 1.
FIG. 1.
General location of the LAMP primer set in relation to previously defined regions of the target DNA. Forward (F3) and backward (B3) outer primers and forward (FIP) and backward (BIP) inner primers are indicated. The specially designed inner primers, FIP and BIP, contain two distinct sequences (F1c plus F2 and B1c plus B2, respectively) corresponding to sense and antisense segments of the target DNA, one for priming in the first stage and the other for self-priming in a subsequent amplification reaction stage (33).
FIG. 2.
FIG. 2.
Agarose gel electrophoresis of LAMP products from clinically relevant yeasts obtained by using the primer set designed in this work. Lanes: 1 to 3, Candida lusitaniae PYCC 2705T, PYCC 4093, and PYCC 4175; 4, Saccharomyces cerevisiae PYCC 4455T; 5, S. bayanus PYCC 4456T; 6, S. paradoxus PYCC 4570T; 7, Saccharomyces exiguus PYCC 2543T; 8 to 10, C. glabrata PYCC 2418T, PYCC 3109, and PYCC 2716; 11 to 14, C. albicans PYCC 3436T, PYCC 2411, PYCC 2746, and PYCC 4079; 15 to 17, C. tropicalis PYCC 3097T, PYCC 4672, and PYCC 2508; 18 and 19, C. parapsilosis PYCC 2545T and PYCC 5124; 20 to 22, Pichia anomala PYCC 4121T, PYCC 3294, and PYCC 5618; 23 to 25, C. krusei PYCC 3341T, PYCC 2631, and PYCC 4740; 26, C. viswanathii PYCC 2811; 27, C. maltosa PYCC 3860T; 28, C. oleophila PYCC 4296; 29, Lodderomyces elongisporus PYCC 4136T; 30, Kluyveromyces polysporus PYCC 3887T; 31, Stephanoascus ciferrii PYCC 3818; NC, negative control; M, molecular weight marker (GeneRuler DNA ladder mix; Fermentas).
FIG. 3.
FIG. 3.
LAMP sensitivity. (A and B) Different amounts of genomic DNA from C. albicans PYCC 3436T (lanes 1 to 5) and C. krusei PYCC 3341T (lanes 6 to 10), subjected (A) or not (B) to a previous thermal denaturation step, were used in the reaction mixture: 500 pg (lanes 1 and 6), 5 pg (lanes 2 and 7), 1 pg (lanes 3 and 8), 0.5 pg (lanes 4 and 9). and 0.05 pg (lanes 5 and 10). (C) LAMP sensitivity determined with heat-treated whole cells of C. albicans PYCC 3436T placed directly in the reaction mixture. Estimated numbers of cells in 10 μl of the reaction mixture are as follows: lane 1, 7 ×ばつ 103; lane 2, 3.5 ×ばつ 103; lane 3, 103; lane 4, 700; lane 5, 70; lane 6, 7; lane 7, 1; lanes 8 to 10, <1. Lane NC, negative control; lane M, molecular weight marker (GeneRuler DNA ladder mix; Fermentas).
FIG. 4.
FIG. 4.
Hybridization of DIG-labeled LAMP amplification products to species-specific probes. (Upper left panel) Spatial distribution of the DNA probes immobilized onto each nylon membrane strip (U210, universal panfungal probe; Cl180, C. lusitaniae probe; Sc176, S. cerevisiae probe; Cg175, C. glabrata probe; Ca170 and Ca176, C. albicans probes; Cd176, C. dubliniensis probe; Ct171, C. tropicalis probe; Cp171, C. parapsilosis probe; Pa176, P. anomala probe; Ck175, C. krusei probe). The species in strips 1 to 31 correspond to those in Fig. 2, lanes 1 to 31; strips 32 to 35, DNA mixtures from C. albicans PYCC 3436T plus S. cerevisiae PYCC 4455T, C. albicans PYCC 3436T plus C. tropicalis PYCC 3097T, C. albicans PYCC 3436T plus C. glabrata PYCC 2418T, and C. lusitaniae PYCC 2705 plus C. tropicalis PYCC 3097T, respectively; strip 36, negative control (DNA replaced with water).

References

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