Nucleotide polymorphisms associated with Internal Transcribed Spacer (ITS) regions of ocular isolates of Aspergillus flavus R. Bagyalakshmi, K. Lily Therese ⁎ , H.N. Madhavan Larsen and Toubro Microbiology Research Centre, Vision Research Foundation, No. 18, College road, Sankara Nethralaya, Chennai-600006, India Received 16 February 2006; accepted 31 May 2006 Available online 7 September 2006 Abstract Purpose: To analyse the genetic similarity among ocular isolates of Aspergillus flavus by Polymerase chain reaction based Restriction Fragment Length Polymorphism (PCR-RFLP) and DNA sequencing. Materials and methods: Seven ocular isolates of A. flavus from 5 patients (3 from paient 1, and four isolates from patients no. 2, 3, 4, and 5 respectively) consisting of 2 Aqueous Humor (AH), 2 Vitreous fluid (VF), 1 eviscerated material, 1 corneal button were included in the study. The three specimens from 1 were one each of AH, VF and corneal button. The fungal isolates were amplified using primers targeting ITS region and the amplicons were subjected to PCR-RFLP using Hae-III enzyme and DNA sequencing to analyse the genetic similarity. Results: A. flavus isolates yielded a specific product of 595 bp after amplification. All the seven A. flavus isolates showed similar pattern of digestion with Hae-III . However, DNA sequencing of ITS amplicons revealed 97.7% genetic similarity and 2.3% dissimilarity with nucleotide polymorphisms — single, double and multiple pertaining to inversion, substitution, insertion and deletion in comparison with that of standard strain of A. flavus ATCC 16883 [Accession Number AB008415]. A. flavus isolated from AH, VF and corneal button from the same patient showed similar nucleotide polymorphisms as against other isolates which exhibited distinct polymorphisms. This pattern of nucleotide polymorphisms in A. flavus isolates is novel and first time reported in literature to the best of our knowledge. Conclusion: DNA sequencing proves to be a useful molecular tool in screening for nucleotide polymorphisms among fungal isolates. © 2006 Elsevier B.V. All rights reserved. Keywords: ITS region; Nucleotide polymorphism; PCR-RFLP 1. Introduction Aspergillus species are ubiquitous, commonly occurring in soil, water and decaying vegetation (Teresa et al., 2000). Members of the genus Aspergillus are opportunistic pathogens and most infections occur in immuno-compromised patients (Buffington et al., 1994). Target for the genus level detection of Aspergillus species have included the 18S rRNA gene (Jagger et al., 2000) mitochondrial DNA (Bretagne et al., 1995), the intergenic spacer region, the Internal Transcribed Spacer (ITS) region (Einsle et al., 1997) and 28S rRNA region (Anand et al., 2001). The ITS region is located between the 18S and 28S rRNA genes and offer distinct advantages over other molecular targets including high sensitivity of detection due to the existence of 100 copies per genome. The sequence variation of ITS region has led to their use in phylogenetic studies of different fungal organisms (Yamakami et al., 1998; Guarro et al., 1999; White et al., 1990). Determining genetic sequence variation at the molecular level is an alternative to culturing Aspergillus flavus. The ribosomal genes demonstrate conserved sequence regions ideal for primer targeting as well as regions of variability useful for species identification. Various molecular techniques like DNA finger printing, RFLP analysis with repetitive probes, poly- morphic microsatellite marker analysis and microsatellite length polymorphism have been used to assess the strain variations within A. flavus (Healy et al., 2004).In this report, we have standardized DNA sequencing technique to analyse the Journal of Microbiological Methods 68 (2007) 1 – 10 www.elsevier.com/locate/jmicmeth ⁎ Corresponding author. Tel.: +91 4428171616; fax: +91 4428254180. E-mail address: lilyirudayam@gmail.com (K. Lily Therese). 0167-7012/$ - see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.mimet.2006.05.021