eBook: New Frontiers in Infectious Disease Research - 15

Table 2. Reaction setup and composition
Cycling conditions
Thermocycling was carried out on a Bio-Rad CFX96
Touch™ qPCR machine (for regular cycling) or on a
BMS MIC qPCR machine (for fast cycling), with the
cycling parameters outlined in Table 3. Fluorescence
measurements were acquired at the end of
each cycle.
Table 3. Cycling conditions
Figure 1. Triplex detection of MPXV targets in a clinical extract
sample. See main text for experimental details.
copies per reaction) and lowest concentration (7
copies per reaction), respectively.
On the other hand, with synthetic DNA target, all
3 targets were successfully detected before 40 cycles
(Figure 2), even at the lowest concentration (5
copies per 20 μL reaction), with no product detectResults
In
the clinical extract, as expected, only the generic
and the West African targets were successfully detected
before 40 cycles (Figure 1), even at the lowest
concentration (7 copies per 20 μL reaction) with
no product detected in the NTCs. The clade-specific
oligonucleotides for the Congo basin MPXV type
did not result in any amplification as desired. Efficiency
for the two targets was 94-96%, with an R2
value above 99% in all cases. The mean ± SD target
Ct values for the 2 targets were 26.99 ± 0.03 and
37.21 ± 0.26 (Generic MPXV) and 28.05 ± 0.05 and
38.02 ± 0.44 (MPXV_WA), at their highest (7,000
Figure 2. Triplex detection of MPXV targets from synthetic cDNA.
See main text for experimental details.
15

eBook: New Frontiers in Infectious Disease Research

Table of Contents for the Digital Edition of eBook: New Frontiers in Infectious Disease Research