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001-es BibID:	BIBFORM074742
Első szerző:	Fidler Gábor (molekuláris biológus, genetikus)
Cím:	Validation of a simplex PCR assay enabling reliable identification of clinically relevant Candida species / Fidler Gabor, Leiter Eva, Kocsube Sandor, Biro Sandor, Paholcsek Melinda
Dátum:	2018
ISSN:	1471-2334 1471-2334
Megjegyzések:	Background: Fungal bloodstream infections (BSI) may be serious and are associated with drastic rise in mortality and health care costs. Candida spp. are the predominant etiological agent of fungal sepsis. The prompt and species-level identification of Candida may influence patient outcome and survival. The aim of this study was to develop and evaluate the CanTub-simplex PCR assay coupled with Tm calling and subsequent high resolution melting (HRM) analysis to barcode seven clinically relevant Candida species. Methods: Efficiency, coefficient of correlation and the limit of reliable detection were estimated on purified Candida EDTA-whole blood (WB) reference panels seeded with Candida albicans, Candida glabrata, Candida parapsilosis, Candida tropicalis, Candida krusei, Candida guilliermondii, Candida dubliniensis cells in a 6-log range. Discriminatory power was measured on EDTA-WB clinical panels on three different PCR platforms; LightCycler?96, LightCycler? Nano, LightCycler? 2.0. Inter- and intra assay consistencies were also calculated. Results: The limit of reliable detection proved to be 0.2?2 genomic equivalent and the method was reliable on broad concentration ranges (106 ?10 CFU) providing distinctive melting peaks and characteristic HRM curves. The diagnostic accuracy of the discrimination proved to be the best on Roche LightCycler?2.0 platform. Repeatability was tested and proved to be % C.V.: 0.14 ? 0.06 on reference- and % C.V.: 0.14 ? 0.02 on clinical-plates accounting for a very high accuracy. Reproducibility was % C.V.: 0.11 between reference- and % C.V.: 0.12between clinicalpanels which is highly acceptable. Conclusion: Our assay demonstrates recent advances on Tm calling and HRM analysis for the molecular identification of relevant Candida species. This unique, simplex PCR assay may be capable to outperform conventional phenotypic methods by reducing time and providing accurate and reliable results directly from blood (2 h) or from whole blood culture bottles (12?24 h).
Tárgyszavak:	Orvostudományok Elméleti orvostudományok idegen nyelvű folyóiratközlemény külföldi lapban Candida High resolution melting Tm calling Species level identification Simplex PCR
Megjelenés:	BMC Infectious Diseases. - 18 : 393 (2018), p. 1-13. -
További szerzők:	Leiter Éva (1976-) (biológus) Kocsubé Sándor Biró Sándor (1949-) (molekuláris genetikus) Paholcsek Melinda (1984-) (molekuláris biológus, genetikus)
Pályázati támogatás:	GINOP-2.3.2-15-2016-00042 GINOP
Internet cím:	Szerző által megadott URL DOI Intézményi repozitóriumban (DEA) tárolt változat
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001-es BibID:	BIBFORM072749
Első szerző:	Fidler Gábor (molekuláris biológus, genetikus)
Cím:	DNA Barcoding Coupled with High Resolution Melting Analysis Enables Rapid and Accurate Distinction of Aspergillus species / Fidler Gabor, Kocsube Sandor, Leiter Eva, Biro Sandor, Paholcsek Melinda
Dátum:	2017
ISSN:	1369-3786
Megjegyzések:	We describe a high-resolution melting (HRM) analysis method that is rapid, reproducible, and able to identify reference strains and further 40 clinical isolates of Aspergillus fumigatus (14), A. lentulus (3), A. terreus (7), A. flavus (8), A. niger (2), A. welwitschiae (4), and A. tubingensis (2). Asp1 and Asp2 primer sets were designed to amplify partial sequences of the Aspergillus benA (beta-tubulin) genes in a closed-, single-tube system. Human placenta DNA, further Aspergillus (3), Candida (9), Fusarium (6), and Scedosporium (2) nucleic acids from type strains and clinical isolates were also included in this study to evaluate cross reactivity with other relevant pathogens causing invasive fungal infections. The barcoding capacity of this method proved to be 100% providing distinctive binomial scores; 14, 34, 36, 35, 25, 15, 26 when tested among species, while the within-species distinction capacity of the assay proved to be 0% based on the aligned thermodynamic profiles of the Asp1, Asp2 melting clusters allowing accurate species delimitation of all tested clinical isolates. The identification limit of this HRM assay was also estimated on Aspergillus reference gDNA panels where it proved to be 10?102 genomic equivalents (GE) except the A. fumigatus panel where it was 103 only. Furthermore, misidentification was not detected with human genomic DNA or with Candida, Fusarium, and Scedosporium strains. Our DNA barcoding assay introduced here provides results within a few hours, and it may possess further diagnostic utility when analyzing standard cultures supporting adequate therapeutic decisions.
Tárgyszavak:	Orvostudományok Klinikai orvostudományok idegen nyelvű folyóiratközlemény külföldi lapban molecular barcoding high resolution melting Aspergillus species level identification standard cultures
Megjelenés:	Medical Mycology. - 55 (2017), p. 642-659. -
További szerzők:	Kocsubé Sándor Leiter Éva (1976-) (biológus) Biró Sándor (1949-) (molekuláris genetikus) Paholcsek Melinda (1984-) (molekuláris biológus, genetikus)
Internet cím:	DOI Intézményi repozitóriumban (DEA) tárolt változat
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