Computational protocol: Molecular characterization, biofilm analysis and experimental biofouling study of Fusarium isolates from recent cases of fungal keratitis in New York State

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Protocol publication

[…] Specific identifications and typing of Fusarium isolates were done by PCR and nucleotide sequencing. Fungal DNA extraction involved grinding mycelia in a pestle and mortar under liquid nitrogen, suspending the slurry in lysis buffer (200 mM Tris-HCl, pH 8.5, 250 mM NaCl, 25 mM EDTA, 0.5%SDS), extraction with phenol: chloroform: isoamyl alcohol, and ethanol precipitation []. Fungal ribosomal DNA internal transcribed spacers 1 and 2 (ITS1, ITS2) and nuclear 28S rRNA [], and Fusarium-specific partial β-tubulin and elongation factor (EF-1α) genes were amplified with published primers []. Nucleotide sequencing of the PCR amplicons was done on both strands according to the standard methods []. Nucleotide sequences were manually edited and compared against the NCBI database and Fusarium database at the Pennsylvania State University []. These sequences have been deposited in the GenBank under Accession Numbers DQ852626 – DQ852630 and DQ813505 – DQ813508. Percentage of nucleotide identity among various genes was compared by ClustalW (v1.4) multiple alignment, using MacVector 7.1.1 software (Accelrys, San Diego, CA). Phylogenetic analyses of nucleotide sequences were done with the PAUP v4 program using a bootstrap method with a Neighbor-Joining or Maximum Parsimony search [].Molecular typing of Fusarium isolates was carried out using two random probes: a 15-bp minisatellite probe from M13 bacteriophage [] and a simple DNA repeat (GACA)4 probe []. These probes were chosen given that they have been widely used to discriminate between related strains of a variety of pathogenic microorganisms. Three laboratory isolates- F. oxysporum 163-05 (corneal ulcer), F. oxysporum 974-05 (finger nail) and F. solani 1064-05 (corneal ulcer), unrelated to this outbreak, were used as outliers to check robustness of the two genotyping methods. Single M13 (5' GAGGGTGGCGGTTCT 3') and (GACA)4 primers were used in PCR reaction. PCR reaction volume of 50 μl included 5 μl of 10 × PCR buffer with 15 mM MgCl2, 3.0 μl dNTP mix (10 μmol/L each), 30 ng primer, 2.5 U Taq DNA polymerase (Perkin Elmer, Foster City, CA, USA). Initial denaturation was at 94°C for 20 sec, followed by 35 cycles of denaturation at 94°C for 20 sec, annealing at 50°C for 1 min, amplification at 72°C for 20 sec and final extension 72°C for 4 min, in a GenAmp PCR System 2400 (Perkin Elmer). PCR products were concentrated to 20 μl and resolved by electrophoresis on 1.4% agarose gels in Tris-borate-EDTA (TBE) buffer, and were detected by ethidium bromide staining []. […]

Pipeline specifications

Software tools Clustal W, MacVector, PAUP*
Application Phylogenetics
Organisms Fusarium solani, Fusarium oxysporum, Homo sapiens
Diseases Keratitis, Mycoses