title: Nucleotide polymorphisms associated with Internal Transcribed Spacer (ITS) regions of ocular isolates of Aspergillus flavus.
abstract: OBJECTIVE
  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.
  
  
  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 ]. 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.
  
  
  CONCLUSIONS
  DNA sequencing proves to be a useful molecular tool in screening for nucleotide polymorphisms among fungal isolates.
authors.affiliation: Larsen and Toubro Microbiology Research Centre, Vision Research Foundation, No. 18, College road, Sankara Nethralaya, Chennai-600006, India.
authors.forename: R
authors.initials: R
authors.lastname: Bagyalakshmi
authors.forename: K Lily
authors.initials: KL
authors.lastname: Therese
authors.forename: H N
authors.initials: HN
authors.lastname: Madhavan
mesh_terms: D001082:Aqueous Humor / Q000382:microbiology*; D001228:Aspergillosis / Q000382:microbiology*; D001231:Aspergillus flavus / Q000235:genetics* / Q000302:isolation & purification; D001483:Base Sequence; D003315:Cornea / Q000382:microbiology*; D004271:DNA, Fungal / Q000737:chemistry / Q000235:genetics; D021903:DNA, Ribosomal Spacer / Q000737:chemistry / Q000235:genetics; D005128:Eye Diseases / Q000382:microbiology*; D006801:Humans; D008969:Molecular Sequence Data; D010802:Phylogeny; D016133:Polymerase Chain Reaction; D011110:Polymorphism, Genetic; D012150:Polymorphism, Restriction Fragment Length; D016415:Sequence Alignment
chemical_list: D004271:DNA, Fungal; D021903:DNA, Ribosomal Spacer