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Clinical Chemistry and Laboratory Medicine (CCLM)

Published in Association with the European Federation of Clinical Chemistry and Laboratory Medicine (EFLM)

Editor-in-Chief: Plebani, Mario

Editorial Board Member: Gillery, Philippe / Kazmierczak, Steven / Lackner, Karl J. / Lippi, Giuseppe / Melichar, Bohuslav / Schlattmann, Peter / Whitfield, John B.

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Application of an oligonucleotide microarray-based nano-amplification technique for the detection of fungal pathogens

Weiping Lu1, 2 / Dayong Gu3 / Xingyun Chen2 / Renping Xiong2 / Ping Liu2 / Nan Yang2 / Yuanguo Zhou2

1Department of Clinical Laboratory, Third Affiliated Hospital of Third Military Medical University, Chongqing, P.R. China

2The Molecular Biology Center, State Key Laboratory of Trauma, Burn and Combined Injury, Research Institute of Surgery and Daping Hospital, Third Military Medical University, Chongqing, P.R. China

3Research Institute of Disease Control and Prevention, International Travel Health Care Center, Shenzhen Entry-Exit Inspection and Quarantine Bureau, Shenzhen, P.R. China

Corresponding author: Yuanguo Zhou, PhD, The Molecular Biology Center, State Key Laboratory of Trauma, Burn and Combined Injury, Research Institute of Surgery and Daping Hospital, Third Military Medical University, 10 Changjiang Zhilu, Chongqing 400042, China Phone: +86-23-68757471, Fax: +86-23-68817159,

Citation Information: Clinical Chemistry and Laboratory Medicine. Volume 48, Issue 10, Pages 1507–1514, ISSN (Online) 1437-4331, ISSN (Print) 1434-6621, DOI: 10.1515/CCLM.2010.284, July 2010

Publication History

Received:
2009-11-03
Accepted:
2010-04-27
Published Online:
2010-07-12

Abstract

Background: The traditional techniques for diagnosis of invasive fungal infections in the clinical microbiology laboratory need improvement. These techniques are prone to delay results due to their time-consuming process, or result in misidentification of the fungus due to low sensitivity or low specificity. The aim of this study was to develop a method for the rapid detection and identification of fungal pathogens.

Methods: The internal transcribed spacer two fragments of fungal ribosomal DNA were amplified using a polymerase chain reaction for all samples. Next, the products were hybridized with the probes immobilized on the surface of a microarray. These species-specific probes were designed to detect nine different clinical pathogenic fungi including Candida albicans, Candida tropocalis, Candida glabrata, Candida parapsilosis, Candida krusei, Candida lusitaniae, Candida guilliermondii, Candida keyfr, and Cryptococcus neoformans. The hybridizing signals were enhanced with gold nanoparticles and silver deposition, and detected using a flatbed scanner or visually.

Results: Fifty-nine strains of fungal pathogens, including standard and clinically isolated strains, were correctly identified by this method. The sensitivity of the assay for Candida albicans was 10 cells/mL. Ten cultures from clinical specimens and 12 clinical samples spiked with fungi were also identified correctly.

Conclusions: This technique offers a reliable alternative to conventional methods for the detection and identification of fungal pathogens. It has higher efficiency, specificity and sensitivity compared with other methods commonly used in the clinical laboratory.

Clin Chem Lab Med 2010;48:1507–14.

Keywords: fungus; identification; internal transcribed spacer; microarray; nano-gold; rRNA

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