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Efficiency of RNA isolation from respiratory samples using the NucliSENS miniMAG and easyMAG extraction procedures

Abstract number: 1733_1226

Manji R., Zhang F., Ginocchio C.

Objectives: The semi-automated NucliSENS miniMAG (MM) and the automated NucliSENS easyMAG (EM) (bioMérieux, Durham, NC) are platforms for the extraction of total nucleic acids (NAs) from clinical samples. The method used by both is based on the established Boom chemistry but utilises magnetic silica particles. The purpose of this study was to evaluate both systems for the removal of inhibitors and the efficiency of isolation of respiratory syncytial virus (RSV) and human metapneumovirus (hMPV) RNA from paediatric respiratory samples.

Methods: NAs were extracted from 656 samples (nasopharyngeal [NP] washes, NP aspirates, NP swabs in VTM) using either MM (n = 390) or EM (n = 181) or both systems (n = 85). Samples (0.2 ml) were pretreated with DNAse, transferred to a NucliSENS lysis buffer tube, and target specific Inhibition Controls for RSV and hMPV were added to assess the efficiency of extraction, amplification and the presence of inhibitors. Each NA extract was tested for RSV and hMPV using a laboratory validated protocol and NucliSENS analyte specific reagents (bioMérieux). Sensitivity of each method was compared using aliquots of serially diluted in vitro transcribed RSV RNA.

Results: For RSV testing, initial inhibition rates for samples extracted with EM was 0% (0/266) and for MM 2.11% (10/475). Inhibition was resolved for all samples after repeat MM extraction from a new aliquot. For hMPV testing, initial inhibition rates for samples extracted with EM was 1.88% (5/266) and for MM 2.95% (14/475). Inhibition was resolved for all EM samples and for 10/14 MM samples after repeat extraction from a new sample aliquot for a final inhibition rate of 0% for EM and 0.84% for MM. RSV assay sensitivity using MM and EM extraction was 66.7% and 87.5%, respectively, for detecting 10 RSV RNA copies/isolation. RSV and hMPV were detected in 22.5% and 5.36% of the samples, respectively.

Conclusions: MM and EM provided highly purified NAs and efficiently removed inhibitory substances from difficult respiratory samples. A slightly better performance seen with EM may relate to the fuller extent of the automation and less operator error as compared to the semi-automated MM. Other benefits of the EM were better throughput per run (EM:24 samples vs MM:12 samples) and less hands on time (EM:15 min vs MM:45 min). In addition, better assay sensitivity was achieved with EM extraction. Both systems provide a generic extraction method for a variety of analytes and sample types.

Session Details

Date: 31/03/2007
Time: 00:00-00:00
Session name: European Society of Clinical Microbiology and Infectious Diseases
Subject:
Location: ICC, Munich, Germany
Presentation type:
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