ABSTRACT
One of the greatest challenges to the use of molecular methods for diagnostic purposes is the detection of target DNA that is present only in low concentrations. One major factor that negatively impacts accuracy, diagnostic sensitivity, and specificity is the sample matrix, which hinders the attainment of the required detection limit due to the presence of residual background DNA. To address this issue, various methods have been developed to enhance sensitivity through targeted pre-amplification of marker sequences. Diagnostic sensitivity to the single molecular level is critical, particularly when identifying bloodstream infections. In cases of clinically manifest sepsis, the concentration of bacteria in the blood may reach as low as one bacterial cell/CFU per mL of blood. Therefore, it is crucial to achieve the highest level of sensitivity for accurate detection. In the present study, we have established a method that fills the analytical gap between low concentrations of molecular markers and the minimum requirements for molecular testing. For this purpose, a sample preparation of whole blood samples with a directly downstream pre-amplification was developed, which amplifies specific species and resistance markers in a multiplex procedure. 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. The method was tested with blood samples that were spiked with several Gram-positive and Gram-negative bacterial pathogens. By applying this method to artificial 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. 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.
ABSTRACT
Digital PCR (dPCR) is based on the separation of target amplification reactions into many compartments with randomly distributed template molecules. Here, we present a novel digital PCR format based on DNA binding magnetic nanoreactor beads (mNRBs). Our approach relies on the binding of all nucleic acids present in a sample to the mNRBs, which both provide a high-capacity binding matrix for capturing nucleic acids from a sample and define the space available for PCR amplification by the internal volume of their hydrogel core. Unlike conventional dPCR, this approach does not require a precise determination of the volume of the compartments used but only their number to calculate the number of amplified targets. We present a procedure in which genomic DNA is bound, the nanoreactors are loaded with PCR reagents in an aqueous medium, and amplification and detection are performed in the space provided by the nanoreactor suspended in fluorocarbon oil. mNRBs exhibit a high DNA binding capacity of 1.1 ng DNA/mNRB (95% CI 1.0-1.2) and fast binding kinetics with ka = 0.21 s-1 (95% CI 0.20-0.23). The dissociation constant KD was determined to be 0.0011 µg/µL (95% CI 0.0007-0.0015). A simple disposable chamber plate is used to accommodate the nanoreactor beads in a monolayer formation for rapid thermocycling and fluorescence detection. The performance of the new method was compared with conventional digital droplet PCR and found to be equivalent in terms of the precision and linearity of quantification. In addition, we demonstrated that mNRBs provide quantitative capture and loss-free analysis of nucleic acids contained in samples in different volumes.
