Clinical specimens consisted of a nasopharyngeal aspirate (NPA) taken from each patient at admission. All clinical specimens were sent for virological investigation to the Respiratory Virus and Influenza Unit at the National Microbiology Center (ISCIII, Madrid, Spain), NPAs were processed within 24 hours after collection. Upon receipt, three aliquots were prepared and stored at -70°C.
RNA and DNA from 200 μl-aliquots of NPA were extracted by using the QIAamp Mini Elute Virus spin kit in an automated extractor (QIAcube, Qiagen, Valencia, CA). From 2005 to 2010, three conventional multiplex RT-nested-PCR assays were performed to detect a total of sixteen respiratory viruses [9 (link),10 (link),11 (link)]. From 2011 to 2014, detection of HMPV and the other respiratory virus were performed by real time multiplex RT-PCR assays, not published yet, but based on the same equivalent conventional methods (9, 10, 11).
RNA and DNA from 200 μl-aliquots of NPA were extracted by using the QIAamp Mini Elute Virus spin kit in an automated extractor (QIAcube, Qiagen, Valencia, CA). From 2005 to 2010, three conventional multiplex RT-nested-PCR assays were performed to detect a total of sixteen respiratory viruses [9 (link),10 (link),11 (link)]. From 2011 to 2014, detection of HMPV and the other respiratory virus were performed by real time multiplex RT-PCR assays, not published yet, but based on the same equivalent conventional methods (9, 10, 11).
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