Quantification of Fungal DNA by Using Fluorescence Resonance Energy Transfer and the Light Cycler System

Abstract

The Light Cycler technique combines rapid in vitro amplification of DNA in glass capillaries with real-time species determination and quantification of DNA load. We have established a quantitative PCR protocol for two clinically important pathogens, Candida albicans and Aspergillus fumigatus. The sensitivity of the assay was comparable to those of previously described PCR protocols (5 CFU/ml). Specific detection of C. albicans andA. fumigatus could be achieved. The assay showed a high reproducibility of 96 to 99%. The assay was linear in a range between 101 and 104 Aspergillus conidia. As capillaries do not have to be reopened for post-PCR analysis, the risk of carryover contaminations could be minimized. The Light Cycler allowed quantification of the fungal loads in a limited number of clinical specimens from patients with hematological malignancies and histologically proven invasive fungal infections. Five of nine positive samples had fungal loads between 5 and 10 CFU/ml of blood, two of nine positive samples had fungal loads between 10 and 100 CFU/ml of blood, and two of nine samples had fungal loads of more than 100 CFU/ml of blood. All samples were also found to be PCR positive by PCR–enzyme-linked immunosorbent assay analysis.

Diagnosis of common dermatophyte infections by a novel multiplex real-time polymerase chain reaction detection/identification scheme

“…The new assay's analytical sensitivity of 100 fg DNA per 50 lL reaction volume is comparable to the sensitivities of published assays for the detection of Candida and Aspergillus in systemic infections. 17,18 It is superior to the sensitivity of 10-35 pg DNA per reaction found in PCR assays for detection of dermatophytes. 6,19 The combination of direct microscopy and culture, considered the gold standard in dermatophyte diagnostics, has an unsatisfactory sensitivity level, especially in onychomycosis, where studies show that 15-50% of microscopy positive nail specimens fail to yield positive cultures.…”

Identification of pathogenic yeast species by polymerase chain reaction amplification of theRPS0gene intron fragment

“…Several studies have described probes, restriction fragment length polymorphisms or other methods to identify unique ribosomal DNA (rDNA) sequences (Hopfer et al 1993;Prariyachatigul et al 1996;Kappe et al 1998;Turenne et al 1999;Velegraki et al 1999;Evertsson et al 2000;Kauffman et al 2000;Loeffler et al 2000;Martin et al 2000;Turin et al 2000). The most common approaches have targeted portions of the rDNA of species of yeast (Prariyachatigul et al 1996;Tanaka et al 1996;Elie et al 1998;Flahaut et al 1998;Walsh and Chanock 1998;Evertsson et al 2000;Baquero et al 2001).…”

A real-time PCR assay for the differentiation of Candida species

“…Primer sequences and hybridization probes for the detection of 18S rRNA gene for Candida spp. were used as previously described (Loeffler et al 2000b). Standard PCR was performed on a T gradient thermo block (Biometra, Göttingen, Germany) in a 50-ll reaction mix containing 1 ll of the isolated DNA, 0AE25 ll Taq DNA polymerase (5 U ll )1 , AmpliTaq Ò , 1AE25 ll PCR nucleotide mix (10 mmol l )1 of each dNTP) (all Roche, Mannheim, Germany), 0AE2 ll of each primer (0AE01 nmol ll )1 , Biometra), 5 ll 10 · PCR buffer (200 mmol l )1 Tris-HCl, 500 mmol l )1 KCl, pH 8AE4), 4 ll MgCl 2 (50 mmol l )1 , Invitrogen, Karlsruhe, Germany) and 38AE1 ll dH 2 0 (Sigma-Aldrich).…”

“…Early identification of Candida species improves the survival of individual patients and is necessary for initiation of an optimal antifungal therapy (Schabereiter-Gurtner et al 2007) or for the development of specific cell therapy strategies. However, current PCR protocols have been criticised because of poor sensitivity, lack of standardization and excessive time and labour efforts (Loeffler et al 2000b). Improvements of the methodology for clinical routine diagnostics are warranted.…”