Pyrosequencing to Reduce Time for Detection in Human Sepsis

Sepsis results from systemic presence of infectious agents, and it involves dysregulated immune response to such infections. Because sepsis can progress to varying degrees of tissue and organ failure, with nearly 40 percent mortality rate, it is imperative to implement effective treatment modalities as early as possible. To do that, rapid and precise identification of the sepsis-causing infectious agents is important. Although the traditional microbiological methods can identify the causative agents, they may require days to do so. Pyrosequencing is a technique suited for sequencing relatively short DNA molecules, which could be generated by PCR. This projects aim was to 1 assess the utility of the BioMatrix muSeq Sepsis Diagnostic Sequencing kit, 2 coupled with pyrosequencing using Qiagens PyroMark Q24 Advanced System. The PyroMark Q24 Advanced was selected for this project based on its small footprint, user-friendly software and easy-to-use pyrosequencing protocol. The kit utilizes three primer sets, each specific for amplifying a bacterial 16S rRNA gene segment encompassing the hypervariable region V1, V2, or V3. The segments thus amplified are then sequenced using PyroMark Q24 Advanced. The combined approach is reputed to be useful for expedited identification of bacteria. Further, the approach is said to require only hours to identify the bacteria, unlike the traditional microbiological approaches that require culturing bacteria, and thus take days. To assess this combined approach, we used commercially available purified genomic DNAs of 31 bacteria. The DNAs were used singly or in combinations to assess the technique.