Qualitative and quantitaive

assay demonstrating presence

and characterization of
Microorgenism

Monika Mathur
2014FST29D
 Constraints in food detection
Bacteria are not uniformly distributed in the food
Heterogeneity of food matrices
ingredients such as proteins, carbohydrates, fats, oil, chemicals,
preservatives etc.
physical form of food (powder, liquid, gel, semisolid or other forms)
difference in viscosity due to fats and oils, which may interfere in
proper mixing
Presence of indigenous microbes which do not cause health risk but
their presence often interferes with the selective identification and
isolation of specific pathogens, which are usually found in low numbers
 Detection of Micro organism
Must be rapid and sensitive
Methods include:
Culture techniques may be too slow
Immunological techniques- very sensitive
Molecular techniques
Probes used to detect specific DNA or RNA
Sensitive and specific
 Conventional Methods

Slow Results
Delays release finished products and ingredients
Delays response to data from environmental monitoring programs
Aerobic Count = 72 hours
MPN of Coliform Bacteria = 72 hours
Listeria Neg= 4-5 days Pos= 5-7 days
Inefficient

Laborious.
Numerous supplies.
High human cost.

Necessity for well-trained operators.

Very subjective results, depending on each operators competence.
Many yield false positive and false negative results
Large measurement uncertainty
 Definition of methods
Methods for microbial analysis comprise methods for enumeration
(quantitative methods) and methods for detection (qualitative methods).
The classical (conventional) culture methods (based upon multiplication of
target organisms in broth or agar media to numbers visual by the naked eye
as turbidity, colour change or colonies) are referred to as the reference
methods for microbial analysis.
rapid methods , a subjective term used loosely to describe a vast
array of tests that includes miniaturized biochemical kits, antibody-
and DNA based tests, and assays that are modifications of
conventional tests to speed up analysis.
 Need for Rapid detection
To provide immediate information on the possible presence of pathogen in
raw material and finished products
Low numbers of pathogenic bacteria are often present in complex biological
environment along with many other non pathogenic organisms
To presence of even a single pathogenic organism in the food may be a
infectious dose
For monitoring of process control, cleaning and hygienic practices during
manufacture
To reduce human errors and to safe time and labor cost
 Rapid method can be defined as any method or
system that reduces the time taken to obtain a
microbiological test result. Rapid may be
interpreted as a shorter time to detection, but
may also refer to a better flow through/handling of
multiple samples and thus refer to convenience
and automation in work in the lab.
Traditional method
Plate counts
Membrane filtration
Most probable number
Direct microscopic count
Dye reduction tests
Indicator

Rapid Method
Direct epifluorescent filter technique (DEFT)
Electrical impedance
Polymerase chain reaction (PCR)
Multiple PCR
Real-time PCR
 Continued..

Nucleic acid sequence-based amplification

(NASBA)
Loop-mediated isothermal amplification (LAMP)
Oligonucleotide DNA microarray which classified
as nucleic acid-based methods
Biosensors -Optical, electrochemical and mass-
based
Enzyme-linked immunosorbent assay (ELISA)
Flow immunoassay
 Rapid methods
First made available in the early 1980s for several
groups of bacteria
Alternative approach besides convenient methods,
less time, labor and set up costs
Extensively evaluated
Now accepted by most microbiologists
 Rapid methods
Nucleic Acid-Based Detection Method
PCR-based detection
Multiplex PCR
Quantitative real-time PCR
Quantitative reverse transcription PCR
Non-PCR-based nucleic acid detection
Loop-mediated isothermal amplification
Nucleic acid sequence-based amplification
DNA microarray
Micro fluidic chip
Immunological-Based Methods
ELISA
Lateral flow assays
Biosensors
Optical
Electrochemical biosensors
Mass based biosensors
 PCR
PCR stands for Polymerase Chain Reaction
First described only 10 years ago, in its short life
PCR has transformed the life sciences utterly.
It is far simpler and less expensive than previous
techniques for duplicating DNA, PCR has
democratized genetic research, putting it within reach
of all biologists, even those with no training in
molecular biology.
 PCRs requirement
DNA is double-stranded, consisting of two such
nucleotide chains that wind around each other in the
famous shape known as the double helix
Primers are single-stranded
Primers must be duplicates of nucleotide sequences
on either side of the piece of DNA of interest, which
means that the exact order of the primers' nucleotides
must already be known
 PCRs three steps
First, the target genetic material must be denatured-that is, the
strands of its helix must be unwound and separated-by heating to
90-96C.
The second step is hybridization or annealing, in which the
primers bind to their complementary bases on the now single-
stranded DNA.
The third is DNA synthesis by a polymerase. Starting from the
primer, the polymerase can read a template strand and match it
with complementary nucleotides very quickly. The result is two
new helixes in place of the first, each composed of one of the
original strands plus its newly assembled complementary strand.
The PCR amplification products are visualized on
electrophoresis gel as bands by staining with ethidium bromide.
Used in detection of like Listeria monocytogenes,
Escherichia coli
Commercialized PCR equipment
The key to PCR's automation has been Taq
polymerase. Taq is a nickname for Thermus
aquaticus, a bacterium that happily survives and
reproduces in an environment that is lethal to other
organisms: hot springs
So that it can stand rapidly fluctuating temperatures
of automated PCR
 Multiple PCR
A more rapid detection as compared to simple
PCR through the simultaneous amplification of
multiple gene targets.
Basic principle of mPCR is similar to conventional
PCR
Several sets of specific primers are used
 Primer design is very important for the development of mPCR,
as the primer sets should have similar annealing
temperature in order to produce a successful mPCR assay
Besides, the concentration of primers is also important in
mPCR. This is because interaction may occur between the
multiple primer sets in mPCR that results in primer dimers,
thus, the concentration of primers may need to be adjusted
to ensure the production of reliable PCR Products.
Other important factors for a successful mPCR assay
include the PCR buffer concentrations
The balance between magnesium chloride and
deoxynucleotide concentrations, the quantities of DNA
template, cycling temperatures and Taq DNA polymerase
 mPCR is more advanced it can detect up to five
or more pathogens simultaneously.
improvements of mPCR include the development
of a novel GeXP-PCR for the simultaneous
detection of six foodborne bacterial pathogens.
The analytical procedure involves primer design, PCR
amplification and capillary electrophoretic separation of
PCR products instead of agarose gel electrophoresis.
REAL-TIME OR QUANTITATIVE
PCR (qPCR)
Real-time PCR or quantitative PCR is different from simple PCR
does not require agarose gel electrophoresis for the detection of
PCR products.
This method is able to monitor the PCR products formation
continuously in the entire reaction by measuring the fluorescent
signal produced by specific dual labeled probes or intercalating
dyes.
The fluorescence intensity is proportional to the amount of PCR
amplicons.
most commonly used fluorescent systems include SYBR green,
TaqMan probes and molecular beacons.
This non-sequence-specific intercalating dye emits little
fluorescence and the fluorescence signal is enhanced when
bound to the minor groove of the DNA double helix
 Quantitative reverse transcription
PCR
Quantitative reverse transcription PCR (RT-qPCR)
can simultaneously detect and measure the quantity of target-amplified DNA.
uses RNA instead of DNA as starting material in the reaction. The RNA is reverse transcribed
into complementary DNA (cDNA) using a reverse transcriptase and specific primers .
The cDNA used as template in a general PCR assay.
RT-qPCR performed in 1 step in a single reaction or in 2 sequential steps. Single-step
protocols are preferred due to the minimized risk for contamination.
RT-qPCR is also highly sensitive, so small differences in sample preparation and
amplification conditions can impact the results.
Since RNA degrades faster when outside of a cell, an advantage of RT-qPCR is that
generally only RNA from live cells is extracted .
Another advantage of this technique is that the relative or quantitative mRNA levels of an
organism under certain conditions can be deduced. This could apply to the food matrix
composition, the temperature, the pH or water activity, or the amount of oxygen
 Non-PCR-based nucleic acid detection
Loop-mediated isothermal amplification
Nucleic acid sequence-based amplification
DNA microarray.
Microfluidic chip

Loop-mediated isothermal amplification. Loop-mediated isothermal

amplification (LAMP) is a relatively novel method developed in 2000 and has
been used for the specific detection of foodborne pathogens. LAMP uses the
Bst DNA polymerase large fragment under isothermal conditions to amplify
target DNA by an autocycling strand displacement DNA synthesis method
 Nucleic acid sequence-based amplification (NASBA) is a primer-
dependent technology which amplifies nucleic acids in a single
reaction at isothermal temperature. The method is used for the
detection of specific RNA sequences in vitro.

DNA microarray. A microarray is a small device consisting of short single-

stranded DNA oligonucleotide probes attached to glass slides or chips During
a microarray assay, the DNA (or RNA) from a target organism is extracted
and labeled with a fluorescent dye. The DNA is then denatured to generate
single-stranded molecules which bind to their corresponding complementary
probes on the array. Results are obtained through visualization of the
fluorescence signal when double-stranded DNA is formed.
 IMMUNOLOGICAL-BASED
METHODS
based on antibody-antigen interactions
2 major categories of immunological methods
reported for the culture-independent detection of
foodborne pathogens are :
ELISA
lateral flow immunoassay (LFI).
Many biosensors are also based on antibody
antigen interactions
 ELISA
ELISA - commonly used immunological methods for the detection of
foodborne pathogens.
Sandwich ELISA is the most effective form of ELISA whereby it involves two
antibodies.
The primary antibody is usually immobilized onto the walls of the microtiter
plate wells.
The target antigen like bacterial cells or bacterial toxins from the food sample
binds to the immobilized primary antibody and the remaining unbound
antigens are removed.
an enzyme conjugated secondary antibody is added and it will bind to the
antigen and the remaining unbound antibodies are removed.
The complex consisting antigen sandwiched between two antibodies is
formed and it can be detected by adding a colorless substrate which will be
converted into a colored form in the presence of the enzyme.
There are different types of enzymes can be used in ELISA, some of the most
commonly used enzymes include horseradish peroxidase (HRP), alkaline
phosphatase andbeta-galactosidase
 Lateral flow assays

Lateral flow along the membrane strip provides a

simple and rapid form of detection assay.
Usually, a test result is indicated by color change,
which is provided by AuNPs or enzymatic
reactions
The assays have advantages of low cost, long-
term stability, minimum skill required, short
detection time, and are suitable for in-field
screening of large numbers of samples, even
though they are mostly for qualitative detection
 Biosensor
A compact analytical device incorporating a biological or
biologically-derived sensing element(such as enzyme,
antibody, microbe or DNA) either integrated with a
physicochemical transducer.
Transducer:
Electrochemical
Optical
Piezoelectric
Thermal
 Biosensors
BIOSENSOR-BASED METHODS
Biosensor is an analytical device that consists of two main elements:a
bioreceptor and a transducer. The bioreceptor responsible for recognizing
the target analyte can either be a:
(1) Biological material: enzymes, antibodies, nucleic acids and cell
receptors, or
(2) Biologically derived material: aptamers and recombinant antibodies, or
(3) Biomimic: imprinted polymers and synthetic catalysts.
 The transducer that converts the biological interactions into a measurable
electrical signal can be optical, electrochemical,massbased, thermometric,
micromechanical or magnetic.
Biosensors are easy to operate and they do not require sample pre-
enrichment, unlike nucleic-acid based methods and immunological methods
which require sample pre-enrichment for concentrating the pathogens
before detection.
The recent biosensors that commonly used for the detection of foodborne
pathogens are optical, electrochemical and mass-based biosensors.
 OPTICAL BIOSENSORS

OPTICAL BIOSENSORS
The most commonly used optical biosensor for the detection of foodborne
pathogen is surface plasmon resonance (SPR) biosensor due to their
sensitivity.
SPR employs reflectance spectroscopy for the pathogen detection .
In SPR, bioreceptors are immobilized on the surface of a thin metal.
The electromagnetic radiation of a certain wavelength interacts with the
electron cloud of the thin metal and produces a strong resonance.
When the pathogen binds to the metal surface, this interaction alters its
refractive index which results in the change of wavelength required for
electron resonance.
 ELECTROCHEMICAL
BIOSENSORS
Electrochemical biosensors are further classified into several types such as
amperometric, impedimetric, potentiometric, and conductometric
Measurement of changes in current, impedance, voltage and conductance respectively,
which caused by antigen-bioreceptor interactions
An electrochemical measurement can be applied to liquid, solid, or gaseous analytes,
but the latter 2 are not common for food sample analysis.
Electrochemical biosensors have good sensitivity, fast response, and simplicity
They can be used in turbid media, and can be miniaturized and are disposable such as
screen-printed carbon electrodes (SPCEs).
Enzymatic reactions and electroactive labels, including nanomaterials are commonly
used in culture-independent amperometric/ voltammetric biosensors.
Piezoelectric quartz crystal microbalance (QCM) is one major type of piezoelectric
biosensor which has been investigated for pathogen detection. QCM biosensors have
the advantage of realtime monitoring, ease of use, biocompatible electrodes (such as
Au) for ligand immobilization and label-free detection.
Magnetoelastic (ME) sensors are made of amorphous ferromagnetic alloys. When
excited by an external time-varying magnetic field, the materials exhibit a ME resonance
which can be detected by using a noncontacting pickup coil
s
 Limitation for rapid methods
A positive result by a rapid method is only regarded
as presumptive and must be confirmed by standard
methods
Most rapid methods lack of sufficient sensitivity and
specificity for director testing, foods still need to be
culture-enriched before analysis
Rapid methods are food dependent
Can detect cell but cant detect the toxin occurrence
 Future trend
DNA biochip
A miniature silicon surface containing thousands of gene
probes in a thumbnail size area
 CONCLUSION
Conventional methods are selective, but they are time-consuming and
laborious. Hence, various rapid detection methods have been developed in
order to overcome the limitations of conventional detection methods. Rapid
methods are important for the rapid detection of food borne pathogens in
food products to prevent outbreaks of food borne diseases and the spread
of foodborne pathogens. Rapid detection methods are generally more
sensitive, specific, time-efficient, labor-saving, and reliable than
conventional methods. Nucleic acid-based methods such as PCR, mPCR,
qPCR, and DNA microarray have high sensitivity and they are widely used
for the detection of foodborne pathogens, but these methods require trained
personnel and specialized instruments. Alternative nucleic acid-based
methods such as NASBA and LAMP are available for the detection of
foodborne pathogens and their toxins. NASBA and LAMP are relatively
sensitive, specific and cost efficient