In summary, the RT-PCR/ESI-MS method has the capacity to provide rapid diagnosis of human influenza in individual patients with respiratory symptoms, as well as public health surveillance of emerging, potentially pandemic strains, including novel reassortants. The use of RT-PCR/ESI-MS for human as well as avian/ animal surveillance offers the potential for new insights into viral evolution on a scale and at a cost previously not possible. Benchtop mass spectrometers are capable of analyzing complex PCR products at a rate of approximately one reaction product/minute, making the RT-PCR/ESI-MS technology practical for large-scale analysis of clinical specimens or for animal surveillance. Further, as we have demonstrated with influenza viruses, this method provides sensitive detection directly from patient specimens with specificity approximating sequence-level resolution. The ability to quantitate virus shedding, detect low-abundance, mixed infections, and identify new genetic variants without prior knowledge of viral sequence also make this technology ideally suited for monitoring the emergence of drug-resistance mutations during therapy or for identifying newly emerging antigenic variants. Sample processing Viral stock samples consisting of cultured virus or stocks obtained from ATCC were prepared for analysis using the Qiagen QiaAmp Virus kit (Valencia, CA). Both manual (mini spin) kits and BioRobot kits were used. Robotic-based isolations were done on both the Qiagen MDx and Qiagen BioRobot 8000 platforms. Clinical swab samples were stored in Viral Transport Media (VTM). VTM solution (1 ml) was passed over a 0.2 micron filter, which was then subjected to bead beating in a small amount of lysis buffer. The resulting viral lysate was then used following the same protocol as above.