Droplet digital PCR for rapid enumeration of viral genomes and particles from cells and animals infected with orthopoxviruses

Droplet digital polymerase chain reaction (ddPCR) was adapted for quantifying the number of orthopoxviral genomes in purified virus samples, infected cell lysates and tissues of infected animals. In contrast to the more commonly used qPCR, the newer ddPCR provides absolute numbers of DNA copies in samples without need for standard curves and has the ability to detect rare mutants in a population. The genome/infectious unit ratio for several sucrose gradient-purified orthopoxviruses varied from 5 to 10, which correlated well with values obtained using the Virocyt, a dedicated fluorescence flow cytometer. By employing a nuclease step to digest unencapsulated DNA, the genome/infectious unit ratios of virus in crude cell lysates approached that of purified virus particles. The speed, accuracy, sensitivity, and dynamic range of less than one to millions of infectious units in a sample make this semi-automated method well suited to a variety of laboratory, animal and clinical studies.