eBook: Cell and Gene Therapy - 24

detection, verification or comparison of test methods,
and other molecular applications. Here, we
demonstrate the use of these products in evaluating
the sensitivity of two different molecular-based
detection systems.
Establishing the Sensitivity of Molecular-Based
Detection Systems
The limit of detection was established for two
molecular-based detection systems comprising
a quantitative mycoplasma DNA control (Acholeplasma
laidlawii, Mycoplasma orale, or Mycoplasma
hominis), a proprietary reaction mixture containing
pre-evaluated 16S rRNA-specific primers and
probes, and either the Bio-Rad® QX100 Droplet
DigitalTM PCR (ddPCR) or CFX96 Touch™ Real-Time
PCR (QPCR) platform. For each control, predicted
input quantities of 1, 10, 100, 1000, 10000, and
100000 were measured using both systems, and
the observed sample copy number was plotted
(Figure 1). Each assay was performed twice (N=2),
and the individual input quantity for each sample
was tested in triplicate (n=6). The total mean and
standard deviation of the combined samples were
calculated at a 95% confidence interval using JMP®
Software. (Table 1).
At each of the predicted input copy numbers, the
mean observed copy number for the combined
samples fell within the 95% confidence interval.
In particular, for predicted input copy numbers of
1 and 10, the mean observed copy number was
statistically closer in value to the predicted copy
Figure 1. Observed sample copy number relative to predicted input copy number by ddPCR and QPCR for three quantitative mycoplasma
DNA controls
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