An advanced dual labeled gold nanoparticles probe to detect Cryptosporidium parvum using rapid immuno-dot blot assay.

Abstract

The zoonotic protozoan parasite Cryptosporidium parvum poses a significant risk to public health. Due to the low infectious dose of C. parvum, remarkably sensitive detection methods are required for water and food industries analysis. However PCR affirmed sensing method of the causative nucleic acid has numerous advantages, still criterion demands for simple techniques and expertise understanding to extinguish its routine use. In contrast, protein based immuno detecting techniques are simpler to perform by a commoner, but lack of sensitivity due to inadequate signal amplification. In this paper, we focused on the development of a mere sensitive immuno detection method by coupling anti-cyst antibody and alkaline phosphatase on gold nanoparticle for C. parvum is described. Outcome of the sensitivity in an immuno-dot blot assay detection is enhanced by 500 fold (using conventional method) and visually be able to detect up to 10 oocysts/mL with minimal processing period. Techniques reported in this paper substantiate the convenience of immuno-dot blot assay for the routine screening of C. parvum in water/environmental examines and most importantly, demonstrates the potential of a prototype development of simple and inexpensive diagnostic technique.

Full text

Page 1

Biosensors
and
Bioelectronics
26 (2011) 4624–
4627
Contents
lists
available
at ScienceDirect
Biosensors
and
Bioelectronics
jou
rn al
h om
epa
ge:
www.elsevier.com/locate/bios
Short
An
parvum
communication
advanced
dual
labeled
gold
nanoparticles
probe
to
detect
Cryptosporidium
using
rapid
immuno-dot
blot
assay
Chinnasamy
Periyakaruppan
Thiruppathirajaa,
Senthilkumar
Kamatchiammalb,
Adaikkappanc,
Muthukaruppan
Alagara,∗
aNanocomposites
bNational
cCenter
Research
group,
Department
of
Chemical
Engineering,
Anna
University,
Chennai,
TN 600025,
India
Environmental
Engineering
Research
Institute,
Chennai
Zonal
Laboratory,
Chennai,
TN 600113,
India
for
Nano-science
and
Nanotechnology,
NASA
Ames
Research
center,
Moffett
Field,
CA
94035,
USA
a
r
t
i
c
l
e

i
n
f
o
Article
Received
Received
Accepted
Available online 11 May 2011
history:
16 February
2011
in revised
form
22
April
2011
4 May
2011
Keywords:
Gold
Cryptosporidium
Drinking
Antibody
Alkaline
Immuno-dot
nanoparticles
parvum
water
phosphatase
blot
assay
a
b
s
t
r
a
c
t
The
the
food
ous
its
moner,
development
phosphatase
blot
up
convenience
examines
inexpensive
zoonotic
protozoan
parasite
Cryptosporidium
parvum
poses
a
significant
risk
to
public
health.
Due
to
low
infectious
dose
of
C.
parvum,
remarkably
sensitive
detection
methods
are
required
for
water
and
industries
analysis.
However
PCR
affirmed
sensing
method
of
the
causative
nucleic
acid
has
numer-
advantages,
still
criterion
demands
for
simple
techniques
and
expertise
understanding
to
extinguish
routine
use.
In
contrast,
protein
based
immuno
detecting
techniques
are
simpler
to
perform
by
a
com-
but
lack
of
sensitivity
due
to
inadequate
signal
amplification.
In
this
paper,
we
focused
on
the
of
a
mere
sensitive
immuno
detection
method
by
coupling
anti-cyst
antibody
and
alkaline
on
gold
nanoparticle
for
C.
parvum
is
described.
Outcome
of
the
sensitivity
in
an
immuno-dot
assay
detection
is
enhanced
by
500
fold
(using
conventional
method)
and
visually
be
able
to
detect
to
10
oocysts/mL
with
minimal
processing
period.
Techniques
reported
in
this
paper
substantiate
the
of
immuno-dot
blot
assay
for
the
routine
screening
of
C.
parvum
in
water/environmental
and
most
importantly,
demonstrates
the
potential
of
a
prototype
development
of
simple
and
diagnostic
technique.
© 2011 Elsevier B.V. All rights reserved.
1.
Introduction
Cryptosporidium
parasitic
2004).
of all
prior
very
infectious
the
tion
At
ronmental
methods
C.
et
2002)
sis
could
PCR
parvum
is a key
significant
diarrhoea-causing
protozoan
found
both
in humans
and
animals
(Fayer,
Approx
30%
adults
of
eminent-rich
countries
and
virtually
adults
in resource-poor
countries
have
serologic
evidence
of
infection
with
this
organism
(David
et
al.,
2002).
In general
less
number
of
oocysts
found
in environmental
water,
but
an
dose
suggested
to exist
is as
low
as
30 oocysts.
Due
to
low
infectious
dose
of
C. parvum, extremely
sensitive
detec-
methods
are
required
for
water
and
food
industries
analysis.
present
DNA
based
methods
in use
to detect
oocysts
in the
envi-
samples
like
water
etc.
Where
as
PCR-based
detection
demoed
to be sensitive
and
specific
for
the
detection
of
parvum
in clinical
specimens
and
environmental
samples
(Xiao
al.,
2000;
Amar
et
al.,
2001;
Sturbaum
et al.,
2001;
Limor
et al.,
which
developed
a real-time
PCR
and
melting
curve
analy-
for
five
major
Cryptosporidium
species
pathogenic
to humans
detect
only
up to five
oocysts.
Quenching
probe
(QProbe)
and
nested
PCR
on QProbe
PCR
products
were
used
for
quan-
∗Corresponding
E-mail
author.
Tel.:
+91
44 22203543;
fax:
+91
44 22203543.
address:
mkalagar@yahoo.com
(M.
Alagar).
titative
and
oocysts
Assorted
technologies
detection
(Arrowood
linked
magnetic
bination
assay
and
2003),
detection
(Tomoyuki
(rCPV40)
2005)
C.
detection
materials
oocyst
1997).
resonance
identifies
detection
of
oocysts
in water
using
the
modified
forward
reverse
primers
with
a detection
limit
ranging
from
2.5
to 59
100−1(Morgan
et
al.,
2000;
Masago
et al.,
2006).
choice
of methods
based
on
the
immunological
investigated
in efforts
to provide
more
effective
of
waterborne
Cryptosporidium
includes,
flow
cytometry
and
Donaldson,
1996;
Vesey
et al.,
1998),
enzyme-
immuno-sorbent
assays
(Lindergard
et
al.,
2001),
immuno-
separation
(IMS)
(McCuin
et
al.,
2001),
Color–PAC
com-
rapid
solid-phase
qualitative
immunochromotographic
(Garcia
and
Shimizu,
2000),
immuno-fluorescence
assay
(IFA)
immuno
magnetic
chemiluminescence’s
(Kuczynska
et al.,
fluorescence
in situ
hybridization
(FISH)
membrane
filter
methods
to find
the
oocytes
in direct
water
samples
et al.,
2006) and
anti
recombinant
capsid
protein
antibody
of
immuno
dot
blot
assay
(Kniel
and
Jenkins,
method
to detect
oocytes
in food
samples.
The
exposure
of
parvum
oocysts
in soil
easily
adapted
by
ELISA
protocol,
but
the
limit
merely
exists
in 10,000
oocysts
per
gallon.
Nano
based
detection
procedures
also
performed
to spot
the
in water/environmental
samples
(Dykman
and
Bogatyrev,
Rule
and
Vikesland
(2009)
accounted
that
surface
enhanced
spectroscopy
(SERS)
immuno-gold
labeling
detection
up to 20
cysts
of water
born
pathogen
C. parvum
and
0956-5663/$
doi:10.1016/j.bios.2011.05.006
– see
front
matter ©

2011 Elsevier B.V. All rights reserved.

Page 2

C. Thiruppathiraja
et
al.
/ Biosensors
and
Bioelectronics
26 (2011) 4624–
4627
4625
Giardia
reported
ples
millimeter-sized
to
(Xu
The
since
provides:
ular
amplification
Fang
reported
et
et
lococcus
(Hwa
luminescence’s
assay
labeled
reported
detection
cal
various
overrun,
To overcome
recent
tion
of
cyst
and
C.
new
offers
tions
concern
lamblia. Likewise
quantum
dots
based
finding
of cyst
also
and
detected
up
to 20–50
cysts
in environmental
sam-
(Ferrari
and
Bergquist,
2007).
Similarly,
piezoelectric-excited
cantilever
(PEMC)
based
biosensor
method
used
detect
G.
lamblia
in water
samples
limit
approx
1–10
cells/mL
and
Mutharasan,
2010).
highly
sensitive
biomolecule
detection
strategies
developed
using
nanoparticles
for
at
any
rate
of two
major
advantages
(i)
a huge
surface
area
to facilitate
efficient
macromolec-
interactions
compared
to bulk
solvent;
and
(ii)
effective
signal
(Li
et
al.,
2006;
Peng
et
al.,
2007;
Feng
et
al.,
2008;
et
al.,
2009).
Few
sensing
methods
based
on
nanoparticles
for
the
detection
of
Hepatitis
B and
C viruses
(Wang
al.,
2003;
Lianlian
et
al.,
2005),
Respiratory
Syncytial
Virus
(Tripp
al.,
2007),
Escherichia
coli
in water
(Temur
et al.,
2010),
Staphy-
aureus
(Huang,
2007) and
Staphylococcal
enterotoxin
B
et
al.,
2010) in culture
medium
using
non-stripping
chemi-
detection
procedures
like
immuno-gold
filtration
and
immuno-gold
chromatographic
assay.
In addition
to dual
gold
nanoparticles
bio-probe
facilitated
immuno
assay
also
by Yin
et al.
(2011)
and
Zhou
et
al.
(2011)
for
ultra
sensitive
of
the
biomolecules.
Though
all
the
existing
analyti-
methods
could
detect
a small
number
of
biomolecules,
it has
difficulties
including
time
consumption
of
the
assay,
cost
complicated
equipment
and
lacking
expertise
knowledge.
such
difficulties,
enormous
efforts
have
been
made
in
years
to develop
more
sensitive
and
specific
visual
detec-
methods
for
related
issues.
Hither
we
report
the
development
dual
functional
gold
nanoparticle
(AuNP)
coupled
with
both
anti-
monoclonal
antibody
and
enzyme
alkaline
phosphatase
(ALP)
its
subsequent
utilization
in an
immuno-dot
blot
assay
to detect
parvum. Our
results
evidenced
the
eminence
sensitivity
of this
approach
as
compared
to the
customary
method.
This
strategy
a simple
instructive
(visually
monitored),
low-cost
utiliza-
and
robust
reagents
which
shows
promising
potential
aim
of
for
pathogen
diagnostics.
2. Experimental
work
2.1.
Preparation
of
dual
labeled
AuNP
probe
The
dual
labeled
AuNP
probe
was
synthesized
under
nor-
mal
AuNP
cryptosporidium
Followed
and
gate
supernatant
with
10,000
and
the
aliquot
dilutions
optimized
condition.
In brief,
1 mL
of
(0.75
A520units/mL)
was
added
and
stirred
gently
with
100
?L of
10
?g/mL
anti
antibody
kept
at room
temperature
for
60 min.
by 100
?L of
0.2
mg/mL
alkaline
phosphatase
was
added
the
reactant
kept
at room
temperature
for
60 min.
The
conju-
was
centrifuged
at 10,000
rpm
under
4◦C for
10 min
and
the
was
discarded.
The
pellet
was
dispersed
and
mixed
1 mL
of
0.1
M Tris
buffer,
pH 7.6
with
1% BSA
centrifuged
at
rpm
under
4◦C for
10 min.
The
supernatant
was
removed
the
final
volume
adjusted
to 100
?L of
preservative
buffer
and
conjugate
kept
at 4◦C prior
to use.
The
suspensions
were
then
separately
and
added
with
PBS
to yield
3,
2,
1,
and
0.6
?g/mL
respectively.
2.2.
Preparation
of
AuNP
based
immuno
dot
blot
assay
Various
lulose
with
serum
was
oclonal
and
mixtures
of
C. parvum
were
spotted
directly
on
nitrocel-
membrane
with
a blotting
manifold,
allowed
to dry,
washed
phosphate
buffer
saline
(PBS)
and
blocked
with
3% of bovine
albumin
(BSA)
at
37◦C for
60
min.
The
saturated
membrane
incubated
at room
temperature
for
60 min
with
anti
cyst
mon-
antibody
in 5 mL
of
PBS
buffer
for
conventional
method
dual
labeled
AuNP
in 5 mL
of
PBS
buffer
for
enhanced
method.
The
for
with
buffer
by
zolium
chromogenic
was
membrane
was
then
washed
thrice
with
excess
of
PBS
buffer
2 min,
followed
by
incubated
at room
temperature
for
60 min
alkaline
phosphatase
conjugated
anti-mouse
antibody
in PBS
for
conventional
method.
And
in enhanced
method,
followed
wash,
the
membrane
was
developed
with
Nitro
Blue
Tetra-
(NBT)
and
5-Bromo-4-Chloro-3-Indolyl
Phosphate
(BCIP)
substrate.
The
color
intensity
of
the
immuno-dot
blot
measured
using
Molecular
Imager®GelDocTMXR.
2.3.
Water
sample
collections
for
study
evaluation
Drinking
rately
samples
out
extracted
the
of
which
The
agarose
gel
order
assay,
assay
water
of
10
L samples
were
randomly
collected
sepa-
from
five
different
zones
of
Chennai,
India.
Subsequently
the
were
concentrated
using
U-APB
method
and
used
through-
this
study
(Anbazhagi
et al.,
2007).
The
total
genomic
DNA
was
by
using
CTAB
method
subjected
to PCR
analysis
to detect
C.
parvum
occurrence
with
the
reverse
and
forward
primer
CGTTAACGGAATTAACCAGAC
and
AGTGCTTAAAGCAGGCAACTG
designed
for
18S
rRNA
gene
used
for
Cryptosporidium.
amplification
yielded
556
bp products
were
resolved
using
1%
gel
with
ethidium
bromide
and
visualized
under
Bio-Rad
documentation
system
(Molecular
Imager®GelDocTMXR).
In
to evaluate
the
efficiency
of
the
enhanced
immuno-dot
blot
the
processed
water
samples
were
subjected
to immuno-blot
by both
conventional
and
enhanced
method.
3.
Results
and
discussion
3.1.
assay
Principle
of
the
dual
labeled
AuNP
based
immuno-dot
blot
The
principle
of
enhanced
method
for dual
labeled
AuNP
(ALP–AuNP-anti-cysts
dot
intensity
bound
The
anchorage
shown
Ab)
based
on
direct
sandwich
of
immuno-
blot
assay
and
an
enzymatic
catalytic
reaction.
The
color
was
amplified
by manifolds
since
each
nanoparticles
by
anti-cysts
antibody
and
many
number
of ALP
molecules.
characteristics
nature
of
AuNP
acted
as a platform
for
the
of
both
the
antibody
as
well
enzyme
ALP,
which
clearly
in the
schematic
Diagram
1.
3.2.
Characterization
of
dual
labeled
AuNP
probe
Spectrophotometry
with
S1) revealed
of the
upon
used
analysis
pled
Microscopy
It
shown
and
eter
supporting
observed
biomolecules
et
analysis
of
AuNP
before
and
after
coupled
antibody
and
ALP
(data
shown
in supporting
information
Fig.
that
the
absorption
maximum
shifted
from
518
nm
typical
plasmon
resonance
band
nanoscale
gold
to 524
nm
coupling,
similar
criterion
of
plasmon
resonance
peak
shift
for
linking
evidenced
by
Kumar
et al.
(2008).
Thus,
UV-spectral
used
as
initial
stage
of
confirmation
of antibody
cou-
with
nanoparticles.
High
Resolution
Transmission
Electron
image
displays
the
AuNP
and
nanoparticles
conjugate.
revealed
that
the
AuNP
has
an average
diameter
of
16
±
0.2
(data
in supporting
information
Fig.
S2(a)),
after
the
antibody
ALP
coupled
on
nanoparticles
surface
have
an average
diam-
of
18
±
0.2
nm and
were
dispersed
uniformly
(data
shown
in
information
Fig.
S2(b)).
Under
higher
magnification,
we
grayish
halos
around
the
modified
AuNP,
indication
of
coupled
on
the
AuNP
surface
(Yang
et al.,
2009;
Li
al.,
2010).
3.3.
Effect
of
antibody
concentration
for
immuno
dot
blot
assay
The
effect
of
concentration
of
anti-cyst
antibody
coupled
AuNP
were
anti-cyst
parvum.
established
as
follows.
A wide
range
of
concentrations
of
antibody
coupled-nanoparticles
was
applied
to detect
C.
Each
row
of
the
strips
indicated
the
choice
of amount
of

Page 3

4626
C.
Thiruppathiraja
et
al.
/ Biosensors
and
Bioelectronics
26 (2011) 4624–
4627
Scheme
on
and
amplification.
1. The
schematic
diagram
represents
the
newly
developed
immuno-dot
blot
assay
for
C.
parvum
detection
using
dual
labeled
AuNP
probe.
C. parvum
was
immobilized
the
nitrocellulose
membrane,
followed
by the
exposure
of
dual
labeled
(anti-cyst
antibody
and
ALP)
AuNP.
Finally
the
blot
was
developed
with
the
buffer
containing
NBT
BCIP
and
the
color
intensity
of
the
immuno-dot
blot
was
measured
by Molecular
Imager®GelDocTMXR.
In enhanced
system
the
color
intensity
is higher
due
to the
signal
C. parvum
tion
assay,
of
body
effectively
in 2 and
method;
low
consequence,
of
lowest
and
each
column
represented
the
particular
concentra-
of
the
antibody-coupled
nanoparticles.
In the
immuno-dot
blot
the
color
appearance
was
noticed
in all
the
concentration
antibody-coupled
nanoparticles.
Though
as
expected,
the
anti-
concentration
was
correlated
with
the
intensities.
Outcome
detected
the
lowest
number
of
corresponding
10
oocysts
3 ?g/mL
of
antibody
using
enhanced
immuno-dot
blot
simultaneously
40–50
oocysts
were
detected
using
very
concentration
of
0.6
and
1 ?g/mL
respectively
(Fig.
S3(A)).
As
the
determined
optimum
concentration
of
2 ?g/mL
anti-cyst
antibody
coupled
AuNP
was
adequate
to detect
the
number
of
oocysts
spotted
in the
immuno
assay
(Fig.
S3(B)).
3.4.
Specificity
and
detection
limit
of
AuNP
based
immuno
assay
The
efficiency,
specificity
and
minimal
detection
limit
were
analyzed
assay
7.0,
of
100,
immuno-blot.
negative
conditions
assay
gen
intensity
In
any
In
validated
enhanced
using
freshly
developed
enhanced
immuno-dot
blot
performed
under
the
optimum
reaction
conditions
of pH
2 ?g/mL
anti-cyst
antibody
coupled
nanoparticles
at 90
min
duration.
A series
of
oocysts
were
quantified
from
200,
150,
50,
40,
30,
20,
10,
5 and
3 oocysts/mL
of
cells
spotted
in the
A non-specific
antigen
E. coli
and
PBS
employed
as
control.
Results
noticeably
found
that
under
optimized
the
color
intensity
of
the
enhanced
immuno-dot
blot
decreased
linearly
with
the
decreasing
number
of anti-
and
could
detected
visually
up to 10 oocysts
and
the
color
of
the
dot
was
saturated
from
150
oocysts/mL
(Fig.
1).
contrast,
the
negative
control
E.
coli
and
PBS
were
not
found
changes
while
using
the
enhanced
immuno-dot
blot
assay.
addition
to that
the
enhanced
immuno-dot
blot
assay
was
also
with
other
water
borne
pathogens.
It revealed
that
the
immuno-dot
blot
was
more
specific
to the
C. parvum
and
in
with
could
conditions
oocysts/mL
(Ferrari
2009).
for
10
sophisticated
there
was
no
false
positive
or non-specific
detection
found
the
assay.
Moreover,
the
obtained
result
was
good
coherence
PCR
(Fig.S6). Conversely,
the
conventional
immuno-dot
blot
detect
visually
up to 5000
oocysts/mL
using
similar
assay
(data
not
shown).
Results
of
minimal
detection
10
comparably
lower
than
the
assays
reported
earlier
and
Bergquist,
2007;
Kang
et
al.,
2008;
Rule
and
Vikesland,
The
similar
observation
also
found
in PEMC
based
biosensor
G.
lamblia
detection
and
the
limit
of
detection
was
about
1 to
cyst/mL
(Xu
and
Mutharasan,
2010),
which
carried
out
using
instrument
and
expertise
knowledge.
Since,
we
Fig.
parvum
than
1. Specificity
and
sensitivity
of
the
enhanced
immuno-dot
blot
assay
for
C.
detection.
The
relative
standard
deviation
(RSD)
of
the
assay
was
less
5%.

Page 4

C. Thiruppathiraja
et
al.
/ Biosensors
and
Bioelectronics
26 (2011) 4624–
4627
4627
Fig.
analysis
M—marker;
lane
2. Field
level
analysis
of
C.
parvum
occurred
in drinking
water
samples
PCR
(A),
enhanced
immuno-dot
blot
assay
(B)
and
conventional
method
(C).
1—negative
control
E.coli; 2—positive
control
(1000
oocysts/mL);
from
3 to 7—zonal
1 to 5.
made
detection
minimum
an attempt
that
the
AuNP
based
biosensor
for
visually
lowest
of
C. parvum
(10
oocysts/mL)
in real
time
samples
at
assay
period,
cost
effective
and
fleeceable
instruments.
3.5.
Field
level
evaluation
of enhanced
immuno-dot
blot
assay
The
major
aim of
this
study
comprises
of
monitoring
the
C.
parvum
and
evaluation
ples
blot
analysis
to be
shown
rably
2
tional
very
based
in
occurrences
in drinking
water
samples.
In order
to assess
validate
the
efficiency
of
enhanced
immuno-dot
blot,
a
field
study
was
carried
out
collecting
random
water
sam-
from
different
zones
examined
by
PCR,
enhanced
immuno-dot
and
conventional
immuno-dot
blot
methods.
In effects
of
PCR
of
C. parvum, samples
from
zone
1,
2 and
4 were
found
occurred
whereas
devoid
from
zone
3 and
5 (Fig.
2A).
As
in Fig.
2B,
the
enhanced
immuno-dot
blot
method
compa-
detected
as
the
PCR
confirmed
water
samples
from
zone
1,
and
4.
However,
similar
furtherance
was
lacked
in the
conven-
method
and
C. parvum
occurred
in only
sample
zone
2 with
low
color
intensity
(Fig.
2C).
It clearly
ascribed
that
the
AuNP
immuno
assay
method
successfully
detected
the
C. parvum
drinking
water
samples
which
identical
to PCR
analysis.
4.
Conclusion
A
novel
enhanced
immuno-dot
blot
assay
was
developed
using
dual
asite
be
assay
detect
total
view
30.
to any
labeled
AuNP
assay
to detect
a minimal
concentration
of
par-
C. parvum. It demonstrated
and
confirmed
that
AuNP
could
successfully
enhanced
the
performance
of the
immuno-dot
blot
by achieving
higher
sensitivity,
specificity
and
above
all
the
limit
of
10 oocysts/mL
with
a very
short
period
study
of
90 min
assay
duration.
This
is a significant
consideration
in
that
the
infectious
dose
ranges
from
1 number
of
oocyst
up to
The
developed
AuNP
based
immuno
assay
can
also
be
applicable
proteins
and
whole
cell
detection
for
early
diagnostics.
Acknowledgement
Authors
and
study
thank
to Supra
Institutional
Project,
Council
of
Scientific
Industrial
Research,
New
Delhi,
India
for
funding
to execute
this
successfully.
Appendix
A.
Supplementary
data
Supplementary
the
data
associated
with
this
article
can
be
found,
in
online
version,
at
doi:10.1016/j.bios.2011.05.006.
References
Amar,
Anbazhagi,
Kumar,
Arrowood,
David,
2002.
Dykman,
356.
Fang,
Methods
Fayer,
Feng,
Ferrari,
Garcia,
Huang,
Hwa, S.R.,
Kang,
1693–1699.
Kniel, K.E.,
Kuczynska,
Kumar,
Li,
Li,
Lianlian,
Limor,
Lindergard,
Parasitol.
Masago,
119–126.
McCuin,
69–76.
Morgan,
Moore,
Peng,
40–44.
Rule,
Sturbaum,
Appl. Environ.
Temur,
397, 1595–1604.
Tomoyuki,
2006. J. Microbiol.
Tripp,
Nanomed.
Vesey,
1998.
Wang,
205–211.
Xiao,
(12), 5492–5498.
Xu,
Yang,
2853–2857.
Yin,
Zhou, Y.,
S.Y., Li,
C.,
Pedraza
Diaz,
S.,
McLauchlin,
J.,
2001.
J.
Clin.
Microbiol.
39,
401–403.
M.,
Loganathan,
D.,
Tamilselvan,
S.,
Jayabalou,
R.,
Kamatchiammal,
S.,
R.,
2007.
Clean
35,
167–171.
M.J.,
Donaldson,
K.,
1996.
J. Eukaryot.
Microbiol.
43,
89S.
J.T.,
Alejandro,
C.G.,
Shannon,
E.W.A.,
Clinton
White
Jr.,
Rebecca
Richards,
K.,
J.
Clin.
Microbiol.
47,
4060–4066.
L.A.,
Bogatyrev,
V.A.,
1997.
Annu.
Rev.
Biochem.
(Moscow)
62,
350–
S.B.,
Tseng,
W.Y.,
Lee,
H.C.,
Tsai,
C.K.,
Huang,
J.T.,
Hou,
S.Y.,
2009.
J. Microbiol.
77
(1),
225–228.
R.,
2004.
Vet.
Parasitol.
126
(1–2),
37–56.
D.C.,
Yu,
Y.,
Cui,
H.,
2008.
Analyst
133,
1250–1255.
B.C.,
Bergquist,
P.L.,
2007.
Cytometry
Part
A 71,
265–271.
L.S.,
Shimizu,
R.Y.,
2000.
J. Clin.
Microbiol.
38,
1267–1268.
S.,
2007.
Sens.
Actuators
B 127,
335–340.
Shek,
T.S.,
Jiang,
C.D.,
Wen,
H.Y.,
2010.
Food
Chem.
118,
462–466.
C.D.,
Cao,
C.,
Lee,
J.,
Choi,
I.S.,
Kim,
B.W.,
Sim,
S.J.,
2008.
Water
Res.
42,
Jenkins,
M.C.,
2005.
J. Food.
Prot.
68,
1093–1096.
E.,
Boyera,
D.G.,
Shelton,
D.R.,
2003.
J. Micobiol.
Methods
53,
17–26.
S.,
Aaron,
J.,
Sokolov,
K.,
2008.
Nat.
Protoc.
3,
314–320.
X.M.,
Fu,
P.Y.,
Liu,
J.M.,
Zhang,
S.S.,
2010.
Anal.
Chim.
Acta
673,
133–138.
Z.P.,
Liu,
C.H.,
Fan,
Y.S.,
Wang,
Y.C.,
Duan,
X.R.,
2006.
Anal.
Biochem.
359,
247–252.
D.,
Yefu,
W.,
Chen
Li,
S.S.,
Wan,
Z.,
2005.
BMC
Infect.
Dis.
5,
53–60.
J.R.,
Lal,
A.A.,
Xiao,
L.,
2002.
J. Clin.
Microbiol.
40,
2335–2338.
G.,
Wade,
S.E.,
Schaaf,
S.,
Barwick,
S.R.,
Mohammeda,
M.C.,
2001.
Vet.
94,
163–176.
Y.,
Oguma,
K.,
Katayama,
H.,
Ohgaki,
S.,
2006.
Water
Sci.
Technol.
54,
R.M.,
Bukhari,
Z.,
Sobrinho,
J., Clancy,
J.L.,
2001.
J. Microbiol.
Methods
45,
U.,
Weber,
R.,
Xiao,
L.,
Sulaiman,
I.,
Thompson,
R.C.,
Ndiritu,
W.,
Lal,
A.,
A.,
Deplazes,
P.,
2000.
J. Clin.
Microbiol.
38,
1180–1183.
Z.,
Chen,
Z.,
Jiang,
J., Zhang,
X.,
Shen,
G.,
Yu,
R.,
2007.
Anal.
Chim.
Acta
583
(1),
K.L.,
Vikesland,
P.J.,
2009.
Environ.
Sci.
Technol.
43,
1147–1152.
G.D.,
Reed,
C.,
Hoover,
P.J.,
Jost,
B.H.,
Marshall,
M.M.,
Sterling,
C.R.,
2001.
Microbiol.
67,
2665–2668.
E.,
Boyaci,
I.H.,
Tamer,
U.,
Unsal,
H.,
Aydogan,
N.,
2010.
Anal.
Bioanal.
Chem.
T.,
Youhei,
S.,
Haruko,
T.,
Satoshi,
H.,
Masato,
Y.,
Masao,
K.,
Tadashi,
M.,
Methods
67,
373–380.
R.A.,
Alvarez,
R.,
Anderson,
B.,
Jones,
L.,
Weeks,
C.,
Chen,
W.,
2007.
Int.
J.
2,
117–124.
G.,
Ashbolt,
N.,
Fricker,
E.J.,
Deere,
D.,
Williams,
K.L.,
Veal,
D.A.,
Dorsch,
M.,
J. Appl.
Microbiol.
85,
429–440.
Y.F.,
Pang,
D.W.,
Zhang,
H.Z.,
Cao,
J.P.,
Shen,
J.T.,
2003.
J. Med.
Virol.
70,
L.,
Alderisio,
K.,
Limor,
J.,
Royer,
M.,
Lal,
A.A.,
2000.
Appl.
Environ.
Microbiol.
66
S.,
Mutharasan,
R.,
2010.
Environ.
Sci.
Technol.
44
(5),
1736–1741.
L.,
Yi,
L.,
Raymond,
R.L.,
Xiangqun,
Z.,
2009.
Biosens.
Bioelectron.
24,
Z.,
Liu,
Y, Jiang,
L.,
Zhu,
J.,
2011.
Biosens.
Bioelectron.
26,
1890–1894.
Tian,
X.,
Li,
Y.,
Pan,
F.,
Zhang,
Y.Y.,
Zhang,
J.H.,
Yang,
L.,
Wang,
X.,
Ren,
H.,
Lu,
Z.,
Chen,
Q.J.,
Liu,
Z.S.,
Liu,
J.,
2011.
Biosens.
Bioelecron.
26,
3700–3704.