As Aspergillus flavus is the most common agent isolated from paranasal sinus (PNS) mycoses in north India, a need was felt to develop DNA based typing methods. Twenty A. flavus strains (10 isolated from PNS mycoses, 5 from other invasive infections and 5 from environment) were analyzed by a) PCR-RFLP of two specific protein encoding genes (acetamidase, O-methyl transferase); b) PCR-RFLP of internal transcribed spacer (ITS) region or rDNA; c) Sequence analysis of ITS region; (d) PCR-RFLP of non transcribed spacer (NTS) region of rDNA; e) Microsatellite finger printing using primer pMSI (GTG)5 and pMS2 (GAC)5; f) RAPD using three primers (R-108, R-151, OPQ-6). No polymorphism was detected by the first three methods. PCR-RFLP of the NTS region divided the strains into three groups. Microsatellite fingerprinting had seven types using pMS1 and six types with pMS2. RAPD with primer R-108 had nine types and with other two primers 10 types each. Although discrimination appeared to be maximum using the RAPD method, the lack of reproducibility could pose problems. The PCR-RFLP method of NTS region and the microsatellite fingerprinting methods seems promising. Sequence analysis of NTS region may predict better restriction sites. But we found it difficult to sequence whole NTS region due to absence of 5s rRNA gene in A. flavus.
Full conference title:
The 15 th Congress of the International Society for Human and Animal Mycology
- ISHAM 15th (2003)