POLYMERASE-CHAIN-REACTION AND RESTRICTION-FRAGMENT-LENGTH-POLYMORPHISM MEDIATED DETECTION AND SPECIATION OF CANDIDA SPP CAUSING INTRAOCULARINFECTION

PURPOSE. To determine the usefulness of polymerase chain reaction (PCR ) and restriction fragment length polymorphism (RFLP) analysis in the identification and speciation of Candida spp that causes ocular infect ion. METHODS. Oligonucleotide primers based on the cytochrome P450 L-1 A(1) demethylase gene were used to successfully amplify by PCR a sing le 1.0-kb and a single 500-bp DNA fragment from C. albicans, C. tropic alis, C. krusei, C. glabrata, C. parapsilosis, and C. pelliculosa geno mic DNA. RFLPs within the PCR product were identified after restrictio n enzyme digestion. RESULTS. The sensitivity of the amplification reac tion after two rounds of PCR was 10 fg genomic C. albicans DNA or one copy of the gene. No amplification product was obtained when DNA from C. guilliermondii, Aspergillus fumigatus, Fusarium solani, human leuko cytes, or 10 species of bacteria was used as a template. Experiments w ith spiked normal vitreous demonstrated equal sensitivity as long as t he volume of vitreous did not exceed 20% of the total PCR volume. RFLP analysis of the PCR product generated from each species obtained from the first-and second-round amplification products enabled species ide ntification after digestion with specific endonucleases. Application o f the technique to four clinical samples was successful. CONCLUSIONS. It is expected that the simplicity of the DNA extraction technique all ied with the broad specificity of the outer printers for all ophthalmi cally relevant Candida spp and the sensitivity of the second-round PCR will aid in the detection of fungal DNA in small intraocular samples. PCR-RFLP analysis has great potential in the rapid detection and iden tification of Candida spp and in the provision of a useful laboratory tool for the future.