Polymerase chain reaction and restriction fragment length polymorphism mediated detection and speciation of Candida spp causing intraocular infection.
OBJECTIVE
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 infection.


METHODS
Oligonucleotide primers based on the cytochrome P450 L1 A1 demethylase gene were used to successfully amplify by PCR a single 1.0-kb and a single 500-bp DNA fragment from C. albicans, C. tropicalis, C. krusei, C. glabrata, C. parapsilosis, and C. pelliculosa genomic DNA. RFLPs within the PCR product were identified after restriction enzyme digestion.


RESULTS
The sensitivity of the amplification reaction 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 leukocytes, or 10 species of bacteria was used as a template. Experiments with spiked normal vitreous demonstrated equal sensitivity as long as the 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 identification after digestion with specific endonucleases. Application of the technique to four clinical samples was successful.


CONCLUSIONS
It is expected that the simplicity of the DNA extraction technique allied with the broad specificity of the outer primers for all ophthalmically 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 identification of Candida spp and in the provision of a useful laboratory tool for the future.
Okhravi
N
N
Department of Clinical Ophthalmology, The Institute of Ophthalmology, London, United Kingdom.
Adamson
P
P
Mant
R
R
Matheson
M M
MM
Midgley
G
G
Towler
H M
HM
Lightman
S
S
D001082:Aqueous Humor / Q000382:microbiology; D001483:Base Sequence; D002175:Candida / Q000145:classification* / Q000235:genetics* / Q000302:isolation & purification; D002177:Candidiasis / Q000382:microbiology; D003577:Cytochrome P-450 Enzyme System / Q000235:genetics*; D017931:DNA Primers / Q000737:chemistry; D004271:DNA, Fungal / Q000032:analysis* / Q000302:isolation & purification; D009877:Endophthalmitis / Q000175:diagnosis / Q000382:microbiology; D015821:Eye Infections, Fungal / Q000175:diagnosis / Q000382:microbiology; D005784:Gene Amplification; D005800:Genes, Fungal; D006801:Humans; D007634:Keratitis / Q000175:diagnosis / Q000382:microbiology; D008969:Molecular Sequence Data; D016533:Mycological Typing Techniques; D010088:Oxidoreductases / Q000235:genetics*; D016133:Polymerase Chain Reaction / Q000379:methods*; D012150:Polymorphism, Restriction Fragment Length; D012680:Sensitivity and Specificity; D058886:Sterol 14-Demethylase; D014822:Vitreous Body / Q000382:microbiology
C548418:CYP51A1 protein, human; D017931:DNA Primers; D004271:DNA, Fungal; D003577:Cytochrome P-450 Enzyme System; D010088:Oxidoreductases; D058886:Sterol 14-Demethylase