[Preliminary study on the use of DNA suspension array for the identification of corneal pathogenic fungi]

To develop a multiplex, microsphere-based DNA suspension array for the identification of important pathogenic fungi of cornea and to study the feasibility of its application in the clinical diagnosis of fungal keratitis.

Fusarium solani, Fusarium moniliforme, Fusarium oxysporum, Aspergillus fumigatus and Aspergillus flavus, which covered about 80% of pathogenic fungi of fungal keratitis, were chosen as target species of this study. Five species-specific capture probes were designed in the internal transcribed spacer (ITS) regions of ribosomal DNA and synthesized with 5' amino modifier C12 to covalently bind to different sets of fluorescent beads. Biotinylated amplicons of 5 reference strains and 42 clinical strains were generated with a pair of universal primers to yield fragments for detection. Comparison between single species detection and multiplex detection were designed, as well as detection between array and agarose gel electrophoresis (AGE). Spearman rank correlation analysis and t test were applied to evaluate the specificity, sensibility and reproducibility of suspension array.

Five reference strains and 40 of 42 (95.2%) clinical strains were correctly identified within 3 h post-PCR amplification while 2 other clinical strains were not identified because of their high background fluorescence intensity. Positive S/B ranged from 5.6 to 13.3. There was no significant difference between respective detection and mixed detection of 5 species (t = 0.2524, P = 0.8132). The sensitivity limit for this assay was determined to be 0.94 ng PCR products. The MFI presented positive correlation with amount of PCR products (r(s) = 1.0000, P < 0.01). Coefficient variation of four repeated detections was 1.8% - 13.7%.

The suspension array is a rapid, sensitive and specific method for the identification of the most important species of corneal pathogenic fungi and might be used in the clinical laboratories.