Establishment Of Real-time PCR For Detecting Common Pathogenic Species Of Fungal Keratitis

Objective: Fungal keratitis is a common ophthalmic infectious cornealdisease caused by pathogenic fungi, with high rates of blindness. In ourcountry, Fusarium is the most common fungal pathogen, especiallyFusarium solani, Fusarium oxysporum and Fusarium moniliforme. Thisstudy aims to establish a real-time PCR for detecting common pathogenicspecies of fungal keratitis, including Fusarium solani, Fusariumoxysporum and Fusarium moniliforme.Methods: We searched GenBank for the sequences of fungal COI geneand designed the species-specific primers of Fusarium solani, Fusariumoxysporum and Fusarium moniliforme for the first time. The DNAtemplates were serial diluted to determine the sensitivity of the reaction.Penicillium, Aspergillus fumigatus, Candida albicans and other fungiwere tested by the real-time PCR to obeserve the specificity of theprimers. Optimization of experimental conditions were conducted,thusestablishing a method of SYBR Green Ⅰ real-time PCR for rapiddetection of Fusarium.Results: The primers were designed with specificity and highsensitivity and could be a quantitative analysis of target DNA of Fusarium solani, Fusarium oxysporum and Fusarium moniliforme. thesensitivity concentration of this detection was1pg level. Primers specificdetection results showed that only Fusarium solani, Fusarium oxysporumand Fusarium moniliforme were amplified, the other strains were notamplified, and melting curves were with a single peak, indicating that theprimers were with high specificity.Conclusion: The study of real-time PCR method can detect the clinicalfungal keratitis caused by Fusarium solani, Fusarium oxysporum andFusarium moniliforme specifically and sensitive, which provides a fastand accurate method for the clinical diagnosis of Fusarium infection.