Detection of Avian Influenza A Virus Using Pyrosequencing

A pyrosequencing method was developed for rapidly detecting avian influenza A virus and predicting the pathogenicity. The avian influenza A virus and its subtype were preliminarily determined by PCR on a species-specific sequence of the M gene and a subtype-specific sequence of the HA gene containing a cleaving site, respectively. The results obtained by PCR were further validated via the pyrosequencing method. As apyrase and Klenow play an important role in pyrosequencing, their concentrations were optimized. The results indicate that the nonspecific signals were effectively suppressed using 1.6 U ml−1 apyrase, and a readable sequence length of 33 bases was obtained with 90 U ml−1 Klenow. On the basis of the optimum pyrosequencing system, four specimens including an H5N1 subtype with high pathogenicity and three specimens of avian influenza A H9N2 subtype with low pathogenicity were confirmed. This method is accurate, fast, and can be used efficiently for identifying the pathogenicity of the avian influenza A virus.