An optimized protocol for isolating Rhinosporidium seeberi sporangia from rhinosporidiosis tissue and extracting genomic DNA for next-generation sequencing.

BACKGROUND

Rhinosporidium seeberi is the causative agent of rhinosporidiosis, a chronic granulomatous disease. Progress in the comprehensive genomic characterization of this organism, which is indispensable for elucidating its pathogenic mechanisms, has been hindered due to challenges in isolating high-quality genomic DNA from infected tissues without host genome carryover. Here, we described an optimized protocol for isolating intact R. seeberi sporangia from infected tissues and extracting high-quality DNA, suitable for downstream molecular biology applications, such as high-throughput genomics studies.

METHODS AND

RESULTS

Sporangia were manually isolated from infected tissue and subjected to serial phosphate-buffered saline washes to minimize host cell contamination. Genomic DNA was extracted using a combination of manual and column-based methods, and DNA concentration was determined with a fluorometric assay. DNA quality and integrity were assessed via agarose gel electrophoresis and DNA Integrity Number (DIN) analysis. To evaluate potential host DNA contamination, PCR amplification was performed using R. seeberi-specific and human GAPDH primers.The protocol consistently yielded high concentrations of gDNA, with a total of 4.05 µg(∼6.76 ng/µL) obtained from 1548 sporangia.Agarose gel electrophoresis revealed intact, high-molecular-weight DNA, and DNA Integrity Number (DIN) values ranged from 8.1 to 8.3, indicating excellent DNA integrity. PCR assays confirmed specific amplification of R. seeberi DNA, with negligible human DNA contamination. Sequencing and BLASTn analysis of the 18SrRNA gene further confirmed R. seeberi identity.

CONCLUSIONS

Taken together, this study presents a robust and reproducible protocol for isolating unruptured sporangia of R. seeberi from rhinosporidiosis tissue for high-quality genomic DNA extraction, thus enabling high-throughput genomic, molecular, and diagnostic investigations.