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.


 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.


 Saponin combined with DNase-1 allowed the efficient recovery of microbial DNA from blood and pus samples in sepsis using qPCR assay.


 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. 
qPCR
Real-time PCR versus MALDI-TOF MS and culture-based techniques for diagnosis of bloodstream and pyogenic infections in humans and animals.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.


 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.  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. 
MALDI-TOF MS
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.  DNase-1 was efficient for removing the DNAs from the host cells as well as dead cells with damaged cell membranes.  Saponin combined with DNase-1 allowed the efficient recovery of microbial DNA from blood and pus samples in sepsis using qPCR assay.



DNase-1