High-throughput detection of bacterial, fungal and viral nucleic acids in routine microbiological sample types using one generic Pathogen Universal Protocol on the MagNA Pure 96 System

The objective of this study was to compare the MagNA Pure 96 Pathogen Universal protocol to the MagNA Pure LC DNA Isolation Kit III that is well established in our microbiological laboratory. For this we isolated nucleic acids from ten different sample materials: body fl uids (CSF, urine, sputum), feces, swabs, as well as whole blood, EDTA-, Citrate-plasma and serum. This spectrum is typical for a microbiology laboratory and includes easy and diffi cult-toprocess samples, as sputum and stool are. Into these sample types we spiked classifi ed pathogen stocks from in-house cultures. Subsequently real-time PCR analysis of fungal, bacterial and viral targets was done using the LightCycler® 480 Instrument. The 200 μl protocol performed effi cient and sensitive nucleic acid extraction from up to 96 samples requiring less than 60 minutes runtime. It yielded superior or similar results compared to the respective MagNA Pure LC protocol. Higher input volumes, 500 or 1000 μl, available only for the MagNA Pure 96 System, generally increased sensitivity of detection. Direct use of some routine microbiological samples without dilution by pretreatment reagents, resulted in improved Crossing Point (CP) values. For normal sample types including whole blood this made pretreatment dispensable. A pretreatment step did however improve pathogen detection in the more diffi cult-to-process samples, such as sputum, swabs and stool. Our results demonstrate that both methods allow efficient and convenient isolation of high quality nucleic acid.Compared to the Viral NA Universal protocol, the Pathogen Universal protocol is further optimized for diverse sample materials, including diffi cult samples and for the detection of various pathogen types. The MagNA Pure 96 Instrument thus enables high throughput sample preparation applications in microbiology laboratories. March 2015 Michael Kirchgesser*, Agnes Aschenbrenner * Markus Bollwein**, Udo Reischl ** * Roche Diagnostics GmbH, Penzberg, Germany ** Institute of Medical Microbiology and Hygiene, University Hospital of Regensburg, Regensburg, Germany High-throughput detection of bacterial, fungal and viral nucleic acids in routine microbiological sample types using one generic Pathogen Universal Protocol on the MagNA Pure 96 System