Magmax pathogen rna kit

Total nucleic acids were extracted from all rectal swabs using the MagMax™ Pathogen RNA Kit (Applied Biosystems, Life Technologies, Carlsbad, CA, USA) on an automated nucleic acid extraction system (Thermo Scientific Kingfisher^® Flex, Thermo Fisher Scientific, Pittsburgh, PA, USA) according to the instructions of the manufacturer. All RNA extracts were tested for the presence of PEDV RNA by a quantitative real-time PCR [19 (link)]. Samples were considered negative when no signal was observed within 40 amplification cycles.

Total nucleic acids were extracted from all serum samples using the MagMax™ Pathogen RNA Kit (Applied Biosystems, Life Technologies, Carlsbad, CA, USA) on an automated nucleic acid extraction system (Thermo Scientific Kingfisher® Flex, Thermo Fisher Scientific, Pittsburgh, PA, USA) according to the instructions of the manufacturer. All DNA extracts were tested for the presence of PCV2 DNA by a quantitative real-time PCR assay targeting a conserved region in ORF1 as described previously [25] (link), [26] (link). Samples were considered negative when no signal was observed within the 40 amplification cycles. A differential real-time PCR assay targeting open-reading frame 2 (ORF2) and capable of detecting and differentiating PCV2a, PCV2b and PCV2d was done on all PCV2 PCR-positive pigs at dpc 21 [27] (link). The differential PCR assay does not react with PCV2c due to a primer mismatch. Selected PCV2 PCR-positive samples were sequenced by using a conventional PCR covering the entire ORF2 as described previously [18] (link) at the Iowa State University DNA Facility, Ames, IA, USA.