Aims: This study was applied to evaluate the usefulness of a high-throughput sample preparation protocol prior to the application of quantitative real-time PCR (qPCR) for the early diagnosis of bloodstream and pyogenic infections in humans and animals compared to matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF MS) and classical culture. Methods and Results: Saponin-mediated selective host cell lysis combined with DNase-1 was applied for processing of whole blood and pus clinical samples collected from suspected cases of septicaemia and pyogenic infections in humans and animals. The pre-PCR processing strategy enabled the recovery of microbial cells with no changes in their colony forming units immediately after the addition of saponin. DNase-1 was efficient for removing the DNAs from the host cells as well as dead cells with damaged cell membranes. The metagenomic qPCR and MALDI-TOF MS could identify the bacterial community of sepsis at species level with a concordance of 97
37% unlike the conventional culture. According to qPCR results, Staphylococcus aureus (24
24%) was predominated in animal pyogenic infections, whereas Klebsiella pneumonia (31
81%) was commonly detected in neonatal sepsis. Conclusions: Saponin combined with DNase-1 allowed the efficient recovery of microbial DNA from blood and pus samples in sepsis using qPCR assay. Significance and Impact of the Study: Metagenomic qPCR could identify a broad range of bacteria directly from blood and pus with more sensitivity, higher discriminatory power and shorter turnaround time than those using MALDI-TOF MS and conventional culture. This might allow a timely administration of a prompt treatment.