Marwa M. Hassan, Andrea Ranzoni, Matthew A. Cooper

Point-of-care (POC) diagnostics are one of the quick and sensitive detection approaches used in current clinical applications, but always face a performance tradeoff between time-to-result and assay sensitivity. One critical setting where these limitations are evident is the detection of sepsis, where 6-10mL of whole blood may contain as little as one bacterial colony forming unit (cfu). The large sample volume, complex nature of the sample and low analyte concentration necessitates signal enhancement using culture-based or molecular amplification techniques. In the time-critical diagnosis of sepsis, waiting for up to 24h to produce sufficient DNA for analysis is not possible. As a consequence, there is a need for integrated sample preparation methods that could enable shorter detection times, whilst maintaining high analytical performance. We report the development of a culture-free bacterial enrichment method to concentrate bacteria from whole blood in less than 3h. The method relies on triple-enrichment steps to magnetically concentrate bacterial cells and their DNA with a 500-fold reduction in sample volume (from 10 to 0.02mL). Using this sample preparation method, sensitive qPCR detection of the extracted S. aureus bacterial DNA was achieved with a detection limit of 5±0.58cfu/mL within a total elapsed time of 4h; much faster than conventional culture-based approaches. The method could be fully automated for integration into clinical practice for point-of-care or molecular detection of bacterial DNA from whole blood.