Control RNA products derive from in vitro transcription of the matrix (M) gene RNA of A/Anhui/1/2005(H5N1), human house keeping gene beta-actin and human cytokines/chemokines (including IP-10, RANTES, TRAIL, MIP-3b and TNF-a) genes were used as positive controls and to establish the detection limit of the assay. Recombinant plasmids with entire M gene, beta-actin gene segment and cytokines/chemokines gene segment were linearised by restriction enzyme, and then purified using a DNA clean-up kit. DNA concentration was measured as OD units at 260 nm. One mg of linearized plasmid DNA was transcribed using Riboprobe in vitro transcription system kit (Promega, USA) according to the manufacturer's instructions. The transcribed RNA was purified using phenyl/chloroform solution and was quantified by spectrophotometer. RNA copy number was then determined following the method of Fronhoffs [49] .