Analysis of Random Amplicied Polyorphic DNA (RAPD) for Sporothrix schenckii and Related Fungi

Lee, Jee Bum; Kim, Min; Lee, Seung Chul; Won, Young Ho; Kim, Young Pio

Abstract

BACKGROUND: Sporotrichosis is a common deep cutaneous fungal disease caused by Sporothtix (S.) schenckii. The recent development of polymerase chain reaction (PCR) technology, in particular, arbitrarily primed PCR (AP-PCR) or random amplified polymorphic DNA (RAPD), has greatly enhanced the molecular detection and identification of various pathogenic agents, including fungi.
OBJECTIVE
This study was aimed to differentiate Sporothrix schenckii, and related fungi such as S. schenckii var. luriei, S. flocculosa, S. nivea, Ophiostoma stenoceras, and clinical isolates on the basis of distinct DNA band patterns in the RAPD.
METHODS
S. schendcii, S. schenckii var. luriei, S. flocculosa, S. nivea, Ophiostoma stenoceras from ATCC and KCCM, and clinical 10 isolates from Chonnam University Hospital were used for RAPD analysis. For RAPD, 3 random primers were used. Genomic DNA was extracted by Liu method. Amplification reactions were performed in volumes of 50 microL containing 10 mM Tris-HCI (pH 8.3), 50 mM KCl, 1.5 mM MgCl2, 0.1% Triton X-100, 200microM dNTP mixture, 40 pM primer, 1 U of Taq polymerase, DNA 20 ng.
RESULTS
3 decamers (5'-TGCCGAGCTG-3', 5'-AGTCAGCCAC-3', 5'-AATCGGGCTG-3') are generated in the RAPD, distinct DNA products from S. schenckii forming characteristic band patterns upon gel electrophoresis. Each random primer amplified characteristic same band patterns in DNA from clinical 8 isolates among 10 isolates, 2 isolates have different DNA band patterns. These results suggest of being a Sporothrix anamorph different from S. schenckii in Korea.
CONCLUSION
With 2 random primers (5'-TGCCGAGCTG-3', 5'-AGTCAGCCAC-3') S. schenckii and related fungi investigated produced distinct DNA band patterns on gel electrophoresis. The RAPD was a very valuable laboratory method for identification of S. schenckii isolates.