File:Geomyces destructans molecular analysis.png

English: Molecular analysis of bat tissues and fungal cultures.

(A) ITS PCR analysis of bat tissues and fungal cultures from DNA extracted from bat tissues and from pure Geomyces destructans isolates. PCR amplification was carried out with primer set V47/V50. PCR amplicons were electrophoresed on 2 % agarose gel, stained with ethidium bromide and photographed with a imaging software. Four bat tissues and respective fungal isolates showed perfect matches (blue connectors); one tissue DNA amplicon did not match with G. destructans amplicon obtained from pure culture (green connector). Also shown are amplicons from two additional G. destructans isolates (MYC80280, MYC80282) where corresponding tissues samples were not processed. (B) ITS PCR analysis of bat tissue samples positive for G. destructans. Ten bat tissues including five untreated samples and five paraffin-fixed samples were positive for G. destructans DNA (details in Table 1). (C-D) Molecular typing of G. destructans was performed with RAPD primers. (C) Results shown were obtained by PCR of fungal genomic DNA with M-13 and (GACA)4 primers, amplicons were run on 2 % agarose gels and band patterns were used to construct dendrograms with Applied Math software. Geomyces pannorum (UAMH 1062 and UAMH 2586) were used as outgroup. (D) Results shown were obtained by PCR of genomic DNA with Operon Technology 10-mer primers OPA1, OPA2 and OPA3; outgroup strains are similar to panel in C. Genotyping with five different primers showed that all six G. destructans culture isolates obtained from two sites, approximately 200-km apart, had indistinguishable band patterns. These preliminary results raised the possibility of involvement of a single strain of G. destructans in the outbreak of WNS in bats in upstate NY.