Abstract: Objective To develop and evaluate a PCR-reverse line blot hybridization (RLB) assay for rapid detection and identification of common species of Candida.Methods The common primers,labeled with biotin,were designed targeting the second internal transcribed spacer (ITS2) region between 28S rRNA and 5.8S rRNA of Candida,and used to amplify the DNA of 6 species of Candida (C.alblcans,C. troptcalis,C.krusei,C.glabrata,C.parapsilosis,and C.dubliniensis).The PCR products were identified by hybridization with 6 specific oligonucleotide probes labeled with amidogen and fixed on a nylon membrane. The method was also applied to detect 200 clinical specimens and 100 Candida isolates.Results DNA frag-ments of 302～441 bp were amplified from all the standard strains of 6 species of Candida.The products could specifically hybridize with their corresponding probes.The sensitivity of PCR-RLB was 10 cfu/mL for Candida.The identification results were consistent between this method and culture in 97 isolates.DNA sequencing confirmed the identification results from PCR-RLB of the remaining 3 isolates.Of the 200 clinical specimens,49% were positive for Candida by PCR-RLB,27% by microscopic examination,39% by culture; the positivity rate by PCR-RLB was significantly higher than that by the latter two methods (both P<0.05).Conclusion PCR-RLB is a rapid,specific and sensitive method for the identification of common species of Candida.

Key words: Nucleic acid hybridization, Candida, Oligonucleotide probes, Polymerase chain reaction