Abstract
Contamination of cell cultures can result in a significant loss of precious biological material, particularly in long-term processes including amplification of chimeric antigen receptors (CAR)-T cells and differentiation of patient-derived stem cells, for therapeutic purposes. Bacterial contamination can also lead to more complex conditions such as sepsis which can cause morbidity and mortality, despite strict controls and good laboratory/manufacturing practices in the manipulation of complex biological samples such as blood used in autologous and allogeneic stem cells transplantation. The current standard method to identify biological risk is the set-up of microbial cultures, which can be time consuming with the likelihood of wasting large amounts of reagents in the event of contamination. Real-Time Polymerase Chain Reaction (qPCR) is a molecular method able to detect biological agents in a highly sensitive and specific way and in a short time. However, qPCR assays require complex DNA/RNA purification steps and expensive benchtop instruments, which may not always be available. This paper reports an extraction-free and low-volume protocol for qPCR in a standard instrument, which has been demonstrated to be effective on both Gram-positive (Gram+) and Gram-negative (Gram-) bacteria. Detection has been obtained from spiked cell culture samples, reaching a limit of detection (LOD) of 1 colony forming unit (CFU)/ml. To demonstrate the high potential of this optimized procedure, the same samples were also tested on a Point-Of-Care platform, which includes a cartridge with micro-chambers and a compact instrument, capable of performing qPCR with the same efficiency. Staphylococcus aureus (Gram+) was selected as the target for a proof of concept, achieving a LOD of 1 CFU/ml also on the portable device. The availability of these results paves the way for a simplified protocol for DNA extraction and amplification.