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We demonstrate a high-throughput molecular technique for H5N1 avian influenza detection that is far more rapid and cost-effective than cell-culture-based methods and requires less training, equipment, and risk. Furthermore, mutations in template DNA do not disrupt this method’s efficacy (a potential problem for RT-PCR-based diagnostic methods). Nine H5N1 DNA samples were obtained from the Center for Disease Control. PCR primers were designed to amplify a portion of the hemagglutinin gene and an entropy-based analysis of variance among several hundred strains was used to design pyrosequencing primers flanking clade markers and biologically significant active sites. Pyrograms were easily matched with Sanger sequencing information manually. It was also possible to identify discrepancies between pyrosequencing data and available GenBank data. Pre-programmed nucleotide dispensation orders were also designed to permit sample preparation and sequencing in less than an hour. The bioluminescence regenerative cycle (BRC) correlates amount of inorganic pyrophosphate (PPi) released during DNA polymerization to DNA copy number following specific primer-target hybridization. The enzymatic cascade utilized consists of (1) PPi conversion to ATP by ATP-sulfurylase in the presence of APS and (2) ATP consumption by luciferase to generate detectable photons and PPi, as a byproduct. This positive feedback increases the generated photons several orders of magnitude higher than available chemiluminescent methods. Adenylate kinase and pyruvate kinase with PEP allow substrate cycling, further increasing ATP quantity. We are currently working on the development of a hand-held tool that could use the BRC to directly detect avian flu with a low risk of false negatives. Faced with the continued spread of the virus, this tool could be conducive to safe travel and minimized transmission.