The first publication on the use of digital PCR to quantify a target
of interest, in this case HIV, was that by Simmonds et al. [5]
in 1990 and I am indebted to Professor Simmonds for information
on the background. The group was interested in determining
the genetic diversity of HIV populations infecting lymphocytes in
blood samples from HIV-positive individuals but recognised that
study of bulk samples would prevent study of sequence differences
between individual proviral molecules. Limiting dilution followed
by PCR of replicates and sequencing of positives was performed
(another early example of single molecule PCR). It soon became
evident that the frequency of positive amplifications followed the
Poisson distribution and that, conversely, the number of target HIV
provirus molecules in the original sample could be calculated from
the degree of dilution and the frequency of negative (or positive)
amplifications. The original publication described the limiting dilution
of both mononuclear cells and provirus molecules and in this
way documented the number of cells carrying HIV provirus and
the number of provirus molecules per infected cell. In subsequent years the group continued to use limiting dilution PCR in a number
of follow-up studies of HIV and HCV.