Prospective use of RFLP analysis on amplified Cryptococcus neoformans URA5gene sequences for rapid identification of varieties and serotypes in clinical samples

Clinical isolates of Cryptococcus neoformans, whole blood, cerebrospinal fl uid, bronchoalveolar lavage fluid from patients with positive cryptococcal antigen latex-agglutination test, and spiked clinical material from healthy individuals, were tested by polymerase chain reaction (PCR) with primers a mplifying C. neoformans URA5 gene sequences. To test compatibility of diffe rent DNA extraction protocols with the PCR-restriction fragment length poly morphism (RFLP) assay, a commercial DNA extraction kit (XTRAX(TM); Gull Lab oratories, UT, USA) was used alongside with the hexadecyltrimethylammonium bromide (CTAB) method on spiked biological fluids. Both methods extracted D NA from spiked clinical samples containing C neoformans (8 +/-2 cells ml(-1 )) and generated amplification products suitable for restriction enzyme ana lysis. Alu I digestion differentiated the two varieties of C neoformans. Th ree distinct R-FLP patterns were obtained upon restriction with MspI corres ponding to serotypes A, AD and B, C and D. URA5 PCR followed by RFLP analys is, coupled with a sensitive in-house or commercially available DNA extract ion method from clinical samples, could be successfully incorporated into r apid routine diagnostic strategies. It could also provide an expeditious to ol for epidemiology-based population genetics studies.