Taqman-MGB probe one step quantitative real time PCR with internal positive control for influenza virus RNA detection.

The specificity of the RRT-PCR assay was assessed with respect to a panel of representative strains of different subtypes of influenza A virus, two influenza B viruses and six viral pathogens of avian origin (PMV2, PMV3, PMV4, TRTV, IBV, IBDV) (tab.1). No false positive signals were observed with influenza B viruses or avian pathogens; only the influenza A viruses were amplified by RRT-PCR method (fig.3) The assay sensitivity was determined by amplification of RNA extracted from 10-fold dilutions of TCID50/ml titrated stock of Ty/214845 virus. The detection limit of the assay was found to be equal to 0.005 TCID50/ml of Ty/214845 virus (fig. 4-5). The suitability of RRT-PCR was tested analysing two positive (previously confirmed with RT-PCR) pools of five samples each. Also a negative control, consisting of transport medium, was inserted to check for contamination during processing of the samples. The reference pools were positive confirming the presence of influenza A virus in the samples, while the negative control gave no signal of fluorescence