Assessment of a PCR technique for the detection and identification of Cryptococcus neoformans

Abstract

The 18S ribosomal RNA gene of Cryptococcus neoformans was amplified by polymerase chain reaction (PCR). The primers CPL1 and CPR4 were tested for their ability to amplify DNA from 30 strains of C. neoformans and 27 specimens of cerebrospinal fluid (CSF) from patients with cryptococcal meningitis. A 343 bp product was obtained and its specificity confirmed by Southern hybridization with an internal sequence (INSR4) probe. The sensitivity was 100 fg by Southern analysis and 1 pg using the PCR. Neither human nor a variety of other fungal and bacterial strains (n = 78) gave an amplified product. This PCR method can detect as few as 5 cells ml-1 of C. neoformans in spiked-CSF following a simple processing procedure. The developed system of PCR was more sensitive than the culture method and revealed a very high specificity. The PCR was easy to perform and needed only 4 h for all processes from receiving the CSF to detection of a specific DNA band after agarose gel electrophoresis. This would provide another rapid laboratory method for the diagnosis of cryptococcal meningitis.