Introduction: Detecting target DNA at low concentrations poses a significant challenge in molecular diagnostics. Achieving high sensitivity is crucial for accurate detection, especially in cases of clinically manifest sepsis, where bacterial concentrations in the blood can be extremely low (one bacterial cell/CFU ml blood). The sample matrix, especially blood, adversely affects accuracy and sensitivity due to the presence of residual background DNA. To overcome this problem, we developed a targeted pre-amplification of marker sequences to enhance the diagnostic sensitivity down to a level of single molecules.

Material and methods: For this purpose, a sample preparation of whole blood samples directly followed by a downstream pre-amplification was developed, which amplifies species-specific and resistance markers in a multiplex procedure. A panel of 24 markers was defined. The pre-amplification was performed in a multiplex approach and quantified by qPCR. The method was tested with blood samples that were spiked with several Gram-positive and Gram-negative bacterial pathogens.

Results: When applying pre-amplification techniques, the sensitivity of the pathogen detection in whole blood samples was up to 100 times higher than in non-pre-amplified samples. By applying this method to artificially spiked blood samples, it was possible to demonstrate a sensitivity of 1 colony-forming unit (CFU) per millilitre of blood for S. aureus and E. faecium. A detection limit of 28 and 383 CFU per ml of blood was achieved for E. coli and K. pneumoniae, respectively.

Summary: In the present study, we have established a method that bridges the analytical gap between low concentrations of molecular markers and the minimum requirements for molecular testing. If the sensitivity is also confirmed for real clinical blood samples from septic patients, the novel technique can be used for pathogen detection without cultivation, which might help to accelerate diagnostics and, thus, to decrease sepsis mortality rates.