In order to generate a standard curve for realtime PCR, we transcribed standard RNA in vitro using T7 RiboMAX Express Large-Scale RNA Production System (Promega, Madison, WI). Briefly, RNA extraction was done by using Trizol reagent (Invitrogen, Carlsbad, CA) according to the manufacturer. The coding region of the M gene from the LPAI subtype H5N2 was amplified using previously described universal primers [26] . RT-PCR was performed using a Qiagen One-Step RT-PCR kit (Qiagen, Valencia, CA) according to the standard manufacturer's protocol. PCR products were visualized by electrophoresis through ethidium-bromide-stained (0.5 μg/mL) 1.5% (40 mM Tris-Acetate pH 7.8, 0.1 mM EDTA) agarose gels under UV light.