eBook: New Frontiers in Infectious Disease Research - 16

reaction) and lowest concentration (5 copies per
reaction), respectively.
Discussion
Figure 3. Triplex detection of MPXV targets from synthetic
cDNA using fast cycling conditions. See main text for experimental
details.
ed in the NTCs. Efficiency for the three targets was
94-95%, with an R2
value above 99% in all cases.
The mean ± SD target Ct values for all targets were
5.40 ± 0.03 and 29.97 ± 0.21 (Generic MPXV), 7.19
± 0.07 and 31.81 ± 0.24 (MPXV_CB), and 9.51 ±
0.06 and 33.78 ± 0.30 (MPXV_WA), at their highest
(50,000,000 copies per reaction) and lowest concentration
(5 copies per reaction), respectively.
In regular cycling conditions, amplification of
MPXV could be achieved in about 70 minutes. We
therefore repeated the experiment shown in figure
2 with a fast-cycling program, allowing the entire
detection in about half of the time. Also under
these conditions, all 3 targets were successfully
detected before 40 cycles (Figure 3), even at the
lowest concentration (5 copies per 20 μL reaction),
with no product detected in the NTCs. Efficiency
for the three targets was between 95-105%, with
an R2
value above 99% in all cases. The mean ± SD
target Ct values for all targets were 8.21 ± 0.09 and
32.07 ± 0.16 (Generic MPXV), 8.07 ± 0.17 and 32.07
± 0.19 (MPXV_CB), and 8.68 ± 0.14 and 31.40 ± 0.19
(MPXV_WA), at their highest (50,000,000 copies per
We report here a fast and reliable detection protocol
for the monkeypox disease. Our approach
has proven to be successful in detecting very low
amounts of viral targets in a multiplex qPCR reaction
(less than 10 copies per 20 μL reaction). Using
the BMS MIC qPCR instrument, detection can be
achieved in under 40 minutes. This offers a rapid
means of detecting MPXV and of identifying
the clade in a single test tube, and therefore constitutes
a valid tool to limit the spreading of this
pathogen. These results also highlight the power
and efficiency of multiplex qPCR reactions. In this
context, it is crucial to state that additional targets
can be included in this panel, to confirm or exclude
pathologies with a similar aetiology.
At the time of writing, due to the limitation of available
vaccines, the spread of monkeypox is still ongoing.
Therefore, a fast-screening program could
help in identifying and isolating cases even before
symptoms have developed.
Our Clara™ Probe Mix allows the rapid setup of new
multiplex assays and is designed to be reliable under
a range of template amounts, sample types, and target
nucleotide compositions, eliminating the need
for laborious multiplex assay optimisation.
Product use
PCR Biosystems products, including Clara™ Probe
Mix, alone do not provide diagnostic results and are
supplied for research use only. However, all products
are manufactured under an ISO 13485-compliant
management system and are suitable for use as
components in molecular diagnostic assays, where
16
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