Total RNA was extracted from 20–50 mg of tissue, using the PureLink Micro-to-Midi total RNA purification kit (Invitrogen, San Diego, CA). cDNA, synthesized using the SuperScript III First-Strand Synthesis Systems (Invitrogen), were analyzed for hantavirus RNA by RT-PCR, using oligonucleotide primers designed from highly conserved regions of hantavirus genomes (Table 2 ) (Song et al., 2007b (link), 2007c (link), 2009 (link); Kang et al., 2009a (link), 2009b (link); Gu et al., 2013 (link), 2014a (link), 2014b (link)).
Nested or hemi-nested PCR was performed in 20-μL reaction mixtures, containing 250 μM dNTP, 2.5 mM MgCl2, 1 U of Takara LA Taq polymerase (Takara, Shiga, Japan) and 0.25 μM of each primer (Table 2 ). Initial denaturation at 94°C for 2 min was followed by two cycles each of denaturation at 94°C for 30 sec, two-degree step-down annealing from 46°C to 38°C for 40 sec, and elongation at 72°C for 1 min, then 30 cycles of denaturation at 94°C for 30 sec, annealing at 42°C for 40 sec, and elongation at 72°C for 1 min, in a GeneAmp PCR 9700 thermal cycler (Perkin-Elmer, Waltham, MA) (Arai et al., 2008b (link)). PCR products were separated, using MobiSpin S-400 spin columns (MoBiTec, Goettingen, Germany), and amplicons were sequenced directly using an ABI Prism 3130 Genetic Analyzer (Applied Biosystems, Foster City, CA).
Nested or hemi-nested PCR was performed in 20-μL reaction mixtures, containing 250 μM dNTP, 2.5 mM MgCl2, 1 U of Takara LA Taq polymerase (Takara, Shiga, Japan) and 0.25 μM of each primer (