Molecular Identification of Human Fungal Pathogens

The focus of the work during this funding period mainly centered around Task 2, which consisted of developing standardized protocols for PCR and sequencing template preparation. The two major subtasks, development of a universal DNA extraction strategy and development of a universal PCR reaction, were initiated during the first funding period and have been completed. These accomplishments now allow us to utilize a standard DNA extraction method and a standard PCR reaction that uses the same primer set, for any unknown fungus. Confirmation of these conclusions has been obtained using a broad range of clinical specimens from both humans and animals, and demonstrates that our standard protocol works across all fungal phyla. In addition to the technical developments, we have now completed the design of the database and have been uploading sequences throughout this second year. During the course of uploading sequences, we have been testing the database since it must perform a number of tasks upon data entry. Debugging issues have been minor and although we will continue to revise the database, we now have a working version that will be used to generate output after searches.