Assessment of a semi-automated protocol for multiplex analysis of sepsis-causing bacteria with spiked whole blood samples
Version of Record online: 18 FEB 2013
© 2013 The Authors. MicrobiologyOpen published by Blackwell Publishing Ltd.
This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited.
Volume 2, Issue 2, pages 284–292, April 2013
How to Cite
Laakso, S. and Mäki, M. (2013), Assessment of a semi-automated protocol for multiplex analysis of sepsis-causing bacteria with spiked whole blood samples. MicrobiologyOpen, 2: 284–292. doi: 10.1002/mbo3.69
- Issue online: 8 APR 2013
- Version of Record online: 18 FEB 2013
- Manuscript Accepted: 7 JAN 2013
- Manuscript Revised: 1 JAN 2013
- Manuscript Received: 29 SEP 2012
- Molecular diagnostics;
- MRSA ;
- whole blood
Sepsis is associated with high morbidity and mortality rates worldwide. Rapid and reliable diagnostic methods are needed for efficient and evidence-based treatment of septic patients. Recently, new molecular tools have emerged to complement the conventional culture-based diagnostic methods. In this study, we used spiked whole blood samples to evaluate together two ready-to-use molecular solutions for the detection of sepsis-causing bacteria. We spiked whole blood with bacterial species relevant in sepsis and extracted bacterial DNA with the NorDiag Arrow device, using the SelectNA Blood pathogen DNA isolation kit. DNA extracts were analyzed by the polymerase chain reaction (PCR)- and microarray-based Prove-it™ Bone and Joint assay, resulting in correctly identified bacterial species with detection limits of 11–600 colony-forming unit/mL (CFU/mL). To understand the recovery losses of bacterial DNA during the sample preparation step and the capability of the PCR- and microarray-based platform to respond to the sensitivity requirements, we also determined the analytical sensitivity of the PCR and microarray platform to be 1–21 genome equivalents for the tested bacterial species. In addition, the inclusivity of the Prove-it™ Bone and Joint assay was demonstrated with methicillin-resistant Staphylococcus aureus (MRSA) clones carrying SCCmec types I, II, IV, or V and a nontypable SCCmec type. The proof-of-concept for accurate multiplex pathogen and antibacterial resistance marker detection from spiked whole blood samples was demonstrated by the selective bacterial DNA extraction method combined with the high-throughput PCR- and microarray-based platform. Further investigations are needed to study the promising potential of the concept for sensitive, semi-automated identification of sepsis-causing pathogens directly from whole blood.