Detection and classification of microorganisms by combination of qPCR and pyrosequencing

Abstract
Microbial and viral typing is usually performed by Sanger DNA sequencing. Pyrosequencing (PSQ) is a new sequencing-by-synthesis method allowing a fast and reliable detection of polymorphisms and sequence variations between different species. Performed in combination with quantitative PCR, PSQ can verify positive qPCR results and concurrently identify the pathogen species immediately after the PCR run is finished. To combine qPCR with PSQ, target regions within the respective pathogen genome were amplified in parallel by qPCR with one biotinylated primer for each assay. Positive qPCR results indicated the presence of a pathogen. Additionally, biotinylated DNA strands of PCR products were separated and purified on Streptavidin-coated beads and hybridized to a sequencing primer. Subsequent PSQ generated a pyrogram with a specific sequence that allowed classification of the pathogen by local BLAST search. As different qPCR and corresponding PSQ assays can be easily performed in parallel, a broad range of pathogens can be detected rapidly allowing accurate and reliable microbial and viral diagnostics in samples of unknown infectious origin.