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The PCR amplicons were obtained using three rounds of PCR.

Sequences of PCR amplicons were obtained using an ABI 3730 DNA Analyzer (Life Technologies, Taipei, Taiwan).

Target VMG amplicons were obtained using multiplex polymerase chain reaction (PCR), labeled with biotin, and hybridized to the microarray.

The complete 26S gene of one subtype IIID strain (human isolate FSL190) was amplified and sequenced for further comparison with other VEE complex subtypes; two overlapping PCR amplicons were obtained by using primer pairs E/V 7514/VIIID 10471, and α10247A/Mlu-T25, and sequenced by gene walking.

For the genotyping, two polymerase chain reaction (PCR) amplicons were obtained from genomic DNA for each sample using GoldTaq polymerase (PE Applied Biosystems, Foster City, CA) in the presence of 5%dimethyl sulfoxide.

PCR amplicons were analyzed by agarose gel electrophoresis and images were obtained using the GelDoc system (Biorad, Hercules, CA).

The optimal melting temperature for each PCR amplicon was obtained by analysis of the wild-type sequence, using an algorithm at the Stanford Denaturing High-Performance Liquid Chromatography website (http://insertion.stanford.edu/melt.html).html

Because amplicons were obtained by multiplex PCR, sensitivity of this method is similar to PCR.

Most 18S and 28S rDNA amplicons were obtained by nested PCR with different combinations of primers.

The expected amplicons were obtained only by RT-PCR from the DNase-treated RNA extract.

PCR products were electrophoresed on an agarose gel to ensure that correctly sized amplicons were obtained.