Transcript Polymerase Chain Reaction (PCR)

MOLECULAR DETECTION TECHNIQUES IN
FOOD QUALITY CONTROL: AN OVERVIEW
By: Yakindra Prasad Timilsena
ID- 111332
BACKGROUND
 Food
Quality control is the multidisciplinary
approaches of maintaining physical, chemical,
microbiological, technological and sensory
wholesomeness in foods
 Method
of detection of food adulteration is the
core of food quality control program.
 Traceability and quality assurance in the food
and feed industry through detection technique
at every step of the manufacturing chain 'from
farm to fork’ are essential for regulatory
agencies.
PROBLEM
 Chemistry
STATEMENT
alone can’t solve all the problems of
detection
 Chemical methods of analysis are time
consuming and costly. Need of rapid and
reliable methods
 Methods based on molecular biology and
immunology approaches- better alternatives
 Knowledge on molecular organization of the cell
has led to the development of powerful new
techniques that bring greater accuracy, rapid,
cost effective
 Molecular methods-more superior than
immunological methods.
COMMON MOLECULAR
 PCR
METHODS
(RT-PCR, Multiplex), RFLP, SSCP and
sequencing
 Plasmid profiling, ribotyping, macrorestriction
analysis by pulsed-field gel electrophoresis
(PFGE)
 Newer techniques which use fluorescent dyes,
DNA microarrays, protein chemistry and mass
spectrometry.
 DNA chip, the GeneChip,
COMMON MOLECULAR TECHNIQUES
Random Amplified Polymorphic DNA Analysis
(RAPD)
 Amplified Fragment Length Polymorphism (AFLP)
 Loop Mediated Isothermal Amplification (LAMP)


Biosensors
Gold Nanoparticle-based Biosensor
 Fiber Optic Biosensor
 Electrochemical Biosensor

 Although
there are many nucleic acid molecular
detection methods, only DNA probe and PCR has
been developed commercially for detection of food
pathogens.
APPLICATIONS
 Detecting
and identifying specific genes (GM foods)
 Application
 Detection
OF MOLECULAR METHOD
to Food Authenticity and Legislation
of microbial contamination of foods
 Species
Identification
 Detection of Food Constituents (Ingredients or
Contaminants)
 Detection of antibiotics, pesticides residues etc.
 Halal
and Kosher certification
What is PCR?
DNA replication in a tube (in vitro). Xeroxing (copying) of DNA.
The Components of PCR
The basic components of a PCR reaction are
- one or more molecules of target DNA
- two oligonucleotide primers
- thermostable DNA polymerase
- dNTPs
The Process of PCR
Each PCR cycle requires three temperature steps to complete a round of DNA
synthesis:
MINIMUM
CRITERIA FOR
PCR
 The
sample must contain at least one
intact DNA strand comprising the region
to be amplified
 impurities must be sufficiently diluted so
as not to inhibit the polymerization step of
the PCR reaction.
DNA samples for PCR, regardless of preparation method, are
generally run in duplicate in order to provide a control for the
relative quality and purity of the original sample.
PCR
STEPS
isolation of DNA from the food (CTAB method is common)
amplification of the target sequences by PCR
separation of the amplification products by agarose gel
electrophoresis
estimation of their fragment size by comparison with a DNA
molecular mass marker after staining with ethidium bromide
verification of the PCR results by specific cleavage of the
amplification products by restriction endonuclease, transfer of
separated amplification products onto membranes (Southern Blot)
followed by hybridisation with a DNA probe specific for the target
sequence
Gel electrophoresis for detecting PCR products
Agarose Gels:
•
NuSieve agarose separates short products better
than the regular agarose. More expensive but use
less for the same gel strength as regular agarose.
Real Time detection of PCR products
• No gels required. Recent method. Relies on the
ability of a dye, SYBR Green, to interact with
double stranded amplicons produced during PCR,
to produce fluorescence which is detected in a
flurometer.
MULTIPLEX PCR
 Several
primers pairs with similar annealing
requirements can be added to a PCR mixture to
simultaneously detect several target sequences
 saves time and minimize the expense on
detection of food borne pathogens
 primers shoud have same melting temperature
 must not interact with each other.
 the amplified fragments of same length cannot
be detected
MULTIPLEX PCR
 Standard
PCR- unable to differentiate viable
and non-viable microorganisms
 Ethidium monoazide can be used to separate
dead and viable bacteria
 Real-time PCR using RNA as template is more
authentic since the RNA is present only in viable
microbes.
 RNA is first reverse transcribed to cDNA and
then used for amplification.
POLYMERASE CHAIN REACTION – RESTRICTION
FRAGMENT LENGTH POLYMORPHISM
(PCR-RFLP)
 The
method includes amplification of a known
DNA sequence using two specific primers,
subsequent digestion of an amplicon with
restriction endonucleases and separation and
comparison of DNA restriction fragments.
 The disadvantage of RFLP analysis of PCR
product is that incomplete digestion may
occasionally occur and intra-specific variation
could delete or create additional restriction sites
(Lockley and Bardsley, 2000).
RAPD-PCR
 Random
amplified polymorphic DNA PCR
uses a random primer (10-mer) to
generate a DNA profile.
 The primer anneals to several places on
the DNA template and generate a DNA
profile which is used for microbe
identification.
 RAPD has many advantages:
 Pure DNA is not needed
 Less labor intensive than
 There is no need for prior
 RAPD
RFLP.
DNA sequence data.
has been used to fingerprint the
outbreak of Listeria monocytogenes from
milk.
RIBOTYPING
 Ribotyping
is a method that can identify and
classify bacteria based upon differences in
rRNA. It generates a highly reproducible and
precise fingerprint that can be used to classify
bacteria from the genus through and beyond the
species level.
 Databases for Listeria (80 pattern types),
Salmonella (97 pattern types), Escherichia (65
pattern types) and Staphylococcus(252 pattern
types) have been established.
PLASMID PROFILING
 Plasmid
profile analysis involves extraction of
plasmid DNA and separation by electrophoresis.
The plasmids are visualized under UV light and
sized in relation to plasmids of known molecular
mass carried in a reference strain of E. coli.
 Plasmid analysis of over 120 strains of Cl.
perfringens, isolated during food-poisoning
incidents was carried out by Jones et al., 1989.
 A high proportion (71%) of fresh and wellcharacterized food-poisoning strains possessed
plasmids of 6.2 kb in size (compared with 19% of
non-food-poisoning strains).
LAB-ON-A-CHIP
 An
TECHNOLOGY
alternative approach for the visualization of
the PCR products by the CE on a card-sized
device.
 Can be used to replace the gel-electrophoretic
step in the PCR end-point detection,
 DNA fragments were detected using laserinduced fluorescence, which enables accurate
sizing and quantification of DNA fragments.
 Higher speed, simplicity and safety.
 This approach allowed identification of 5% fish
species admixed into a product containing two
fish species.
DIRECT EPIFLOURESCENT TECHNIQUE
(DEFT)
Direct method used for enumeration of microbe based on
binding properties of flurochrome acridine orange dye.
 Food samples are pretreated with detergents and
proteolytic enzymes, filtered on to a polycarbonate
membrane stained with acridine orange and examined
under fluorescent microscope


Streptococcus and Staphylococcus
can be detectedd by this method
Fig. Staphylococcus aureus - Acridineorange leucocyte cytospin test
ELECTROPHORETIC
 Electrophoretic
METHODS
methods are based on the ability of
molecules to migrate according to their molecular
weight (Mw) in the electric field due to the effect of
electrostatic forces attracting them to reversely
charged electrode.
 The
migration is performed on agarose or
polyacrylamide gel. Various modifications of
electrophoretic methods are used depending on a type
of the analysed product:
ELECTROPHORETIC
 isoelectric
 urea
METHODS
focusing (IEF)
isoelectric focusing (urea-IEF)
 sodium
dodecyl sulphate – polyacrylamide gel
electrophoresis (SDS-PAGE)
 two
dimensional electrophoresis (2DE)
 capillary
electrophoresis (CE)
GM-PLANTS AND DERIVED FOODS DETECTION
PROCEDURE
Samples
Sampling
Tested Samples
Protein Detection
Methods
ELISA
Lateral Flow Strip
Saved Samples
Nucleic Acids
Detection Methods
DNA Extraction
Conventional PCR
Negative
No GM contents
Positive
Contained
GM contents
Quantitative PCR
GM Contents (xx%)
GM-PLANTS AND DERIVED FOODS DETECTION
PROCEDURE
Commercial GMO contain the 35S promoter of
Cauliflower Mosaic Virus and/or the NOS terminator of
Agrobacterium, these genetic elements are used as target
sequences for a general screening
 Since primer selection has to be based on target
sequences that are characteristic for the individual
transgenic organism. Therefore, a prerequisite for
designing specific primers for the identification of GMOs
by PCR is the availability of detailed information on
their molecular make-up.
 Molecular make-up of non-authorized GMOs is generally
not available and so impossible to detect the presence of
non-authorized GMOs.

DETECTION
OF
FOOD-BORNE PATHOGENS
A
short cultural enrichment followed by physical
separation of the organisms from the culture
medium is required for food samples prior to
analysis. Enrichment prior to DNA extraction and
PCR analysis results in a dilution of PCR inhibitors
and an increased number of target cells and
therefore in a higher sensitivity. Only viable cells
are detected.
 RNA
based methods more preferred since mRNAs
are short living molecules and can be amplified in
the PCR system only in case of viable cells. Cultural
enrichment step is not required.
DETECTION
OF
FOOD-BORNE PATHOGENS
In a study the development of a PCR-based technique
for the rapid identification of the food-borne
pathogens Salmonella and Escherichia coli was
undertaken. Suitable primers were designed based on
specific gene fimA of Salmonella and gene afa of
pathogenic E. coli for amplification.
 Agarose gel electrophoresis and subsequent staining
with ethidium bromide were used for the
identification of PCR products. The size of the
amplified product was 120 bp as shown by
comparison with marker DNA. These studies have
established that fimA and afa primers were specific
for detecting Salmonella and pathogenic E. coli,
respectively, in the food samples (Naravaneni &
Jamil, 2005)

DNA MICROARRAY
DNA microarray (DNA chip) is rapid and provides
simultaneous DNA screening of hundreds of species at
once.
 The chip is a glass or nylon membrane with spots of
probes oligonucleotides that are complementary to the
specific target DNA sequence. The targets hybridize
with the captured oligonucleotides on the chip and the
fluorescent label, which is attached to the target during
the PCR, is detected.
 The oligonucleotide microarray analysis of the PCR
product from the mt cyt b gene was applied to identify
different animal species in food samples (Peter et al.,
2004).

BIOSENSOR
Majority of the Biosensors are based on immunological methods,
Ritcher 1993
IMPEDANCE-BASED
BIOCHIP SENSOR
 Based
on the changes in conductance in a medium
due to microbial breakdown of inert substances
into electrically charged ionic compounds.
 Allows the detection of only the viable cells
PIEZOELECTRIC
BIOSENSOR
Very attractive and offers real time output, simplicity
of use and cost effectiveness
 Based on coating the surface of piezoelectric sensor
with a selective binding substance e.g. antibodies,
placing it in a solution containing bacteria, the
bacteria/antigen will bind to the antibodies and the
mass of the crystal increase while the resonance
frequency will decrease

FOURIER
TRANSFORM INFRARED
(FT-IR)
SPECTROSCOPY TECHNIQUES
FT-IR spectroscopy enables rapid and non-invasive
characterization of molecular structures in a sample
 Can be used to provide compositional and quantitative
information.
 Can be used for discriminating and classifying intact
microbial cells down to the strain level in pure culture
 With help of chemometric there has been improvement in
the sensitivity of FT-IR to identify, discriminate, and
quantify bacteria
 Peaks in bacterial spectra are assigned to specific chemical
bonds, which may be correlated to bacterial concentrations
 Spectral libraries may be created for bacteria in foods and
based on comparison between spectra of artificially
contaminated samples with these libraries; the extent of
contamination may be quantifiable.

DETECTION
OF VIRUSES IN FOODS
Virus has been identified in food by Ligase
Chain Reaction (LCR) Nucleic Acid Sequence
,
Based Amplification (NASBA), Self sustaining
sequence replication (3SR), Strand Displacement
Amplification (SDA), situ hybridization
(FISH), development of gene probes and PCR
amplification techniques are used to detect
the virus in food samples
FLUORESCENT IN SITU HYBRIDIZATION (FISH)
A
molecular technique often used to identify and
enumerate specific microbial groups.
 The FISH technique is dependent upon
hybridizing a probe with a fluorescent tag,
complementary in sequence, to a short section of
DNA on a target gene.
 The tag and probe are applied to a sample of
interest under conditions that allow for the
probe to attach itself to the complementary
sequence in the specimen
 After sample treatment, excess fluorophore is
washed away and the sample can be visualized
under a fluorescent microscope.
FLUORESCENT IN SITU HYBRIDIZATION (FISH)
REFERENCES


Mandal, P.K., A.K. Biswas, K. Choi and U.K.
Pal, 2011. Methods of Rapid Detection of
Foodborne Pathogens: An Overview. Am. J.
Food Tech. 6(2): 87-102
http://www.fda.gov/food/scienceresearch/Laboratory
methods/bacteriologicalanalyticalmanualbam/ucm1096
52.htm#ref4

http://www.worldfoodscience.org/cms/
 Naravaneni
R, Jamil K. J Med Microbiol. 2005
Jan;54(Pt 1):51-4

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