Optimization of a 6-plex Crystal Digital PCR® assay and its application to simultaneous surveillance of enteric and respiratory viruses in wastewater
The Science of The Total Environment,
Journal Year:
2025,
Volume and Issue:
970, P. 178939 - 178939
Published: March 1, 2025
Multiplex
digital
PCR
(dPCR)
approaches
are
commonly
employed
in
wastewater-based
epidemiology
(WBE)
studies.
However,
optimizing
the
dPCR
workflow
is
a
critical
step
to
ensure
its
reliability
and
accuracy
before
application.
In
this
study,
6-plex
Crystal
Digital
PCR®
(cdPCR)
was
optimized
for
simultaneous
detection
of
six
epidemiologically
important
pathogens,
including
three
enteric
viruses,
noroviruses
genogroups
I
II
(NoV-GI
GII)
enteroviruses
(EnV),
respiratory
severe
acute
syndrome
coronavirus
2
(SARS-CoV-2),
which
causative
agent
disease
2019
(COVID-19),
influenza
A
virus
(InfA),
syncytial
B
(RSVB),
wastewater.
Four
cDNA
input
ratios
(20
%-70
%)
two
extraction
kits
were
evaluated
optimization,
with
30
%
AllPrep
PowerViral
DNA/RNA
Kit
(Qiagen)
exhibiting
optimal
performance.
The
cdPCR
assay
applied
year-long
wastewater
surveillance
study
Japan
(n
=
52),
revealing
distinct
trends
prevalence
viruses.
NoV-GII
detected
96
samples
highest
mean
concentration
(6.1
±
0.6
log10
copies/L),
while
SARS-CoV-2
InfA
60
50
samples,
respectively,
reflected
circulation
these
pathogens
within
community.
Notably,
RSVB
less
frequently
(25
%),
line
fewer
cases
reported
during
period.
concentrations
EnV
showed
significant
positive
correlations
hand
foot
mouth
herpangina
cases,
respectively.
no
observed
RSV
COVID-19,
possibly
due
testing
RSVA
more
prevalent
also
cluster
outbreaks.
These
findings
demonstrated
utility
detecting
provided
insights
into
community
trends,
representing
an
advancement
WBE.
Language: Английский
Persistence of human Aichi virus infectivity from raw surface water to drinking water
Applied and Environmental Microbiology,
Journal Year:
2024,
Volume and Issue:
91(1)
Published: Dec. 31, 2024
Human
Aichi
virus
1
(AiV-1)
is
a
water-
and
food-borne
infection-associated
picornavirus
that
causes
gastroenteritis
in
humans.
Recent
studies
on
environmental
waters
showed
high
frequency
abundance
of
AiV-1,
suggesting
it
might
be
an
appropriate
indicator
fecal
contamination.
We
screened
450
surface
drinking
water
samples
from
Tunisian
treatment
plant
(DWTP)
the
Sidi
Salem
dam
for
AiV-1
by
real
time
reverse
transcriptase
PCR
(RT-qPCR).
The
persistence
infectious
particles
was
evaluated
using
integrated
cell
culture
approach
coupled
with
quantitative
molecular
detection
(ICC-RT-qPCR).
In
all,
85
(18.9%)
were
positive
viral
loads
ranging
0.47
to
11.62
log10
cp/L
median
4.97
cp/L,
including
30/100
raw,
18/50
decanted,
14/50
flocculated,
9/100
treated,
1/50
tap,
13/100
samples.
Of
these,
15
(17.6%)
contained
genotype
A
particles,
five
four
one
surface,
three
two
treated
Our
data
suggest
represent
potential
threat
public
health.
This
study
also
indicates
ICC-RT-qPCR
practical
tool
monitoring
human
waterborne
risk
aquatic
environments.IMPORTANCEHuman
Its
would
analysis
(ICC-RT-qPCR)
confirmed
at
all
stages
process,
except
tap
water.
suggests
infectivity
environments.
Language: Английский