Natan Gollop Elango mathavan The Volcani Center, ARO Israel

Natan Gollop Elango mathavan The Volcani Center, ARO Israel

Natan
Gollop
Elango
mathavan
The
Volcani
Center,
ARO
Israel
Contents
(Microbiological
quantification
methods)
1.
Conventional
microbiological
test
methods
2.
Biochemical
characterization
methods
3.
Immunoassays
4.
Molecular
methods
5.
Development
of
rapid
methods
in
food
microbiology
Why
to
develop
new
detec1on
methods
• Foodborne
pathogens
are
causing
food
poisoning;
• Foodborne
pathogens
are
necessary
to
be
examined
in
farms,
food
industries
and
markets;
• Rapid
and
dependable
methods
are
needed
for
the
detection
and
identification
of
the
foodborne
pathogens
• Food
spoilage
bacteria
cause
economical
lost
Conven1onal
microbiological
test
methods
Advantages:
reliable,
official,
cheap
facilities
Disadvantages:
time‐consuming,
labor
consuming
etc
Examples:
Plate
count
methods
and
the
most‐probable‐
number
(MPN)
methods
Rapid
detec1on
methods
in
Microbiology
Types:
• Biochemical
characterization
methods;
• Immunoassays;
• Molecular
methods
Advantages:
real‐time
detection,
labor‐saving,
easy
operation
Disadvantages:
non‐official
(mostly),
highly
skilled
labor,
expansive
Biochemical
characteriza1on
methods
 Detection
systems
based
on
automatic
analysis
of
carbon
utilization
and
other
bio‐reactions
 Detection
systems
based
on
whole
cellular
fatty
acid
analysis
Systems
based
on
carbon
u1liza1on
and
other
bio‐reac1ons
 Kits
based
on
bio‐reactions
 Examples:
API
System；EnterotubeⅡ；Micro‐IDR；MinitekTM；
CrystalTM
Identification
System:
RapID
One
System:
RapIDTM
ANA
Ⅱ
System
 Characterization
systems
based
on
carbon
utilization
or
sensitivities
of
pathogens
to
antibiotics
 Examples：
 BiologTMATBR
Identification:
API:
Vitek
Example
I
for
bio‐reac1on
systems
 API
20ER
System
 Organisms:
G‐
bacteria
 Types
of
bio‐reactions:
23
 Time
for
tests:
18‐24hror
38‐48hr
 Advantages:
reliable,
portable
 Limitations:
professional
workers
are
needed
Biolog
Example
for
bio‐reac1on
systems
 Type
of
carbon
sources:
95
 Biolog
database
for
2000
species
of
microorganisms,
including:
‐
 Aerobic
G :
526
 Aerobic
G+:
339
 Anaerobes:
361
 Yeasts:
26
 Filamentus
fungi:
618
 Accuracy:
>95%
Systems
based
on
whole
cellular
faCy
acid
analysis
Example：Sherlock®Microbial
Identification
System
Disadvantage:
Highly
expensive
instrument,
highly
skilled
operators
The
Aerobe
library
contains
over
695
Environmental
species
and
430
Clinical
species.
The
Anaerobe
library
contains
725
species.
This
library
contains
190
Yeast
species.
Immunoassays
 TECRA
system:
based
on
Enzyme‐linked
immuno
sorbent
assay
(ELISA)
 VIDAS
system:
based
on
Enzyme‐linked
Fluorescent
immuno‐Assay
(ELFA)
 Transia
 Biocontrol
1‐2
Test
Molecular
methods
DNA
banding
pattern‐based
methods
Multiplex
PCR
Real‐time
PCR
Restriction
fragment
length
polymorphis(RFLP)
Randomly‐amplified
polymorphic
DNA
(RAPD)
Pulsed‐field
gel
electrophoresis
(PFGE)
Ribotyping
(RT)
DNA
sequence‐based
methods
rRNA
sequence
typing
Multilocus
sequence
typing
(MLST)!
DNA‐DNAmicroarray
Ribotyping
(RT)
system
Pulsed‐field
gel
electrophoresis
(PFGE)
Pulsed‐field
Gel
Electrophoresis
(PFGE)
Mul1locus
sequence
typing
(MLST)
Mul1locus
sequence
typing
(MLST)
DNA
sequence‐based
methods
 Multiplex
PCR
 Real
time
PCR
 DNA
detection
chips
 Foodborne
pathogens
focused:
—
Salmonella
—
Vibrio
parahaemolyticus
—
Staphylococcus
aureus
—
Listeria
monocytogenes
—
Enterobactercea
—
E.coli
—
Spoilage
bacteria
(pseudomonas)
Mul$plex
PCR
 Two
sets
of
primers
 Identification
based
on
two
or
more
genes
 Confidently
in
identification
 Interaction
ad
Interference
between
the
two
sets
of
primers
 Only
one
bacteria
per
reaction
Real
$me
PCR
 High
sensitivity
 Fast
 Only
one
gene
 Only
one
bacteria
Biotecon
GmbH
 Expensive
(instrument
and
kits)
Q
–Bioanalytivc
GmbH
Real
$me
PCR
Beacon
TaqMan
Syber
green
Real
$me
PCR
DNA‐DNA
Microarray
Prove‐it™
Bacteria
Strategy
—DNA
amplification
+
DNA
chips
Bacteria
—more
than
10
species
Very
High
Accuracy
Key
point—number
of
target
genes
MicroArray strategy
www.chill-on.com
Whole
genome
Direct
amplification hybridization
Multiplex
PCR
PCR
Sampling
DNA
isola$on
Whole
genome
amplifica$on
Shearing
Labeling
Labeling
PCR
+
labeling
Hybridiza$on
Mul$plex
PCR
+
Labeling
Detec$on
Direct
hybridization
–
Simple,
straight
forward,
low
sensitivity.
PCR
–
High
sensitivity,
identification
relay
on
one
gene
only.
Many
bacteria
can
be
detect
in
one
reaction
Multiplex
PCR
‐
High
sensitivity,
identification
relay
two
2
to
3
genes
only.
Many
bacteria
can
be
detect
in
one
reaction,
complexity.
Whole
genome
amplification
–
Simple,
very
high
sensitivity,
,
identification
relay
may
relay
on
several
genes
no
limitation,
.
Many
bacteria
viruses
and
molds
can
be
detect
in
one
single
reaction
Oligo
MicroArray
Genomic
DNA
extraction
DNA
amplification
(labeling)
Design
the
oligo
nucleotides
probes
Printing
the
microarray
Hybridization
Detection
identification
MicroArray
for
six
bacteria
www.chill‐on.com
16S
rRNA
MicroArray
PCL
–
Positive
control
GPB
–
Gram
+
Bacteria
GNB‐
Gram
–Bacteria
PSU
–
Pseudomonas
ESC
–
E.coli
SAL
–
Salmonella
STP
–
Staphylococcus
auerus
VIB
–
Vibrio
cholerae
Lis
–
Listeria
monocytoge
Microarray
of
16
S
ribosomal
RNA
gene
www.chill‐on.com
Fish
homogenate
sample
prepara1on
Filter
with
5µM
pore
size
paper
(2
1mes)
Filter
with
0.45µM
pore
size
paper
Inculca1ng
six
pathogenic
and
spoilage
bacteria
(102
CFU/ml)
(Control
no
Bacteria)
NO
PMA
Control
Propidium
monoazide
(PMA)
Whole
genome
amplifica1on
and
DNA
labeling
Whole
genome
amplifica1on
and
DNA
labeling
DNA
Isola1on
and
cleaning
DNA‐
DNA
hybridiza1on
against
16S
and
gyrB
and
Unique
genes
based
probes
Propidium
monoazide
(PMA)
Whole
genome
amplifica1on
and
DNA
labeling
www.chill‐on.com
Whole
Genome
Amplification
1
2
3
4
1. DNA
Marker 2. Control
DNA
5
ng
supplied
with
–
WGA
kit
3. Fish
homogenate
+
E.coli
WGA
(20
CFU/ml)
4. Fish
homogenate
+
Staphylococcus
WGA
(
10
CFU/ml)
www.chill‐on.com
16S
rRNA
PCR
based
(678F
+
888R)
confirmation
of
WGA
product
1
2
3
4
5
1. DNA
Marker
2. Fish
homogenate
+
E.coli
–
WGA
3.
Fish
homogenate
+
Staphylococcus
–
WGA
4.
Pure
E.coli
DNA
–
5.
Pure
Staphylococcus
DNA
www.chill‐on.com
Salmonella
Pseudomonas
Staphylococcus
Fish
Sample
E.Coli
PSU
PCL
ESC
LIS
SAL
STA
VIB
GPB
PCL
PCL
+
GNB
GYR
S
PSU
S
ESC
S
LIS
S
SAL
S
STA
S
VIB
G2
PB
G1
PSU
G1
ESC
G2
LIS
G1
SAL
G2
STA
G1
VIB
PCL
GYR
G1
PCL
ureC
algT
rfbE
rfA
ssaT
sarZ
trh
www.chill‐on.com
16S
rRNA
PCR
based
(678F
+
888R)
confirmation
of
WGA
product
1
2
3
4
5
1. DNA
Marker
2. Fish
homogenate
+
E.coli
–
WGA
3.
Fish
homogenate
+
Staphylococcus
–
WGA
4.
Pure
E.coli
DNA
–
5.
Pure
Staphylococcus
DNA
www.chill‐on.com
Whole
Genome
Amplification
1
2
3
4
1. DNA
Marker 2. Control
DNA
5
ng
supplied
with
–
WGA
kit
3. Fish
homogenate
+
E.coli
WGA
(20
CFU/ml)
4. Fish
homogenate
+
Staphylococcus
WGA
(
10
CFU/ml)
HOW LONG IT’S TAKE?!
www.chill‐on.com
Sampling
0
WGA
1
Hybridization
6
Hours
Scanning
Bacterial
11 12identification