Application of an oligonucleotide microarray-based nano-amplification technique for the detection of fungal pathogens.
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.
Lu
Weiping
W
Department of Clinical Laboratory, Third Affiliated Hospital of Third Military Medical University, Chongqing, PR China.
Gu
Dayong
D
Chen
Xingyun
X
Xiong
Renping
R
Liu
Ping
P
Yang
Nan
N
Zhou
Yuanguo
Y
D004271:DNA, Fungal / Q000235:genetics* / Q000302:isolation & purification; D004275:DNA, Ribosomal / Q000235:genetics* / Q000302:isolation & purification; D005658:Fungi / Q000235:genetics / Q000472:pathogenicity*; D006046:Gold / Q000737:chemistry; D006801:Humans; D053768:Metal Nanoparticles / Q000737:chemistry; D036103:Nanotechnology / Q000379:methods*; D020411:Oligonucleotide Array Sequence Analysis / Q000379:methods*; D015345:Oligonucleotide Probes / Q000737:chemistry; D016133:Polymerase Chain Reaction; D012680:Sensitivity and Specificity; D012834:Silver / Q000737:chemistry
D004271:DNA, Fungal; D004275:DNA, Ribosomal; D015345:Oligonucleotide Probes; D012834:Silver; D006046:Gold