Development and optimization of a biopreparedness protocol for extracting and detecting avian influenza virus in broiler chicken meat.

Abstract

Detection of avian influenza virus (AIV) in poultry meat is hampered by the lack of an efficient analytical method able to extract and concentrate viral RNA prior to PCR. In this study we developed a method for extracting and detecting AIV from poultry meat by a previously standardized 1-step real-time reverse transcriptase PCR (RRT-PCR) assay. In addition, a new process control, represented by feline calicivirus (FCV), was included in the original protocol, to evaluate all analytical steps from sample preparation to the detection phase. The detection limit was below 1×10(-1) TCID50 of AIV per sample, and the quantification limit corresponded to 1×10(1) TCID50 of AIV per sample. Moreover, the addition of 1×10(2) TCID50/sample of FCV did not affect the quantification and detection limit of the reaction. These results show that the developed assay is suitable for detecting small amounts of AIV in poultry meat. In addition, the developed biopreparedness protocol can be applied to detect AIV in legal or illegal imported broiler chicken meat. The availability of a rapid and sensitive diagnostic method based on molecular identification of AIV in poultry meat provides an important tool in the prevention of AIV circulation.