This document provides an overview of molecular detection techniques used in food quality control. It discusses how chemistry alone cannot solve all detection problems and that molecular biology methods like PCR, RFLP, and sequencing are better alternatives as they are more accurate, rapid and cost-effective. It describes several common molecular detection methods and their applications in detecting food pathogens, adulterants, allergens and GM ingredients. The document emphasizes that molecular methods can identify microbes at the strain level and detect viable cells, but may not be able to find non-authorized GMOs due to lack of molecular information.

1. MOLECULAR DETECTION TECHNIQUES IN
FOOD QUALITY CONTROL: AN OVERVIEW
By: Yakindra Prasad Timilsena
ID- 111332

2. BACKGROUND
 FoodQuality 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.

3. PROBLEM STATEMENT
 Chemistry 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.

4. COMMON MOLECULAR METHODS
 PCR (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,

5. 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.

6. APPLICATIONS OF MOLECULAR METHOD
 Detecting and identifying specific genes (GM foods)
 Application to Food Authenticity and Legislation
 Detection of microbial contamination of foods
 Species Identification
 Detection of Food Constituents (Ingredients or
Contaminants)
 Detection of antibiotics, pesticides residues etc.
 Halal and Kosher certification

7. 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:

8. 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.

9. 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

10. 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.

11. 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

12. 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.

13. 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).

14. 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 RFLP.
 There is no need for prior DNA sequence data.
 RAPD has been used to fingerprint the
outbreak of Listeria monocytogenes from milk.

15. 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.

16. 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 well-
characterized food-poisoning strains possessed
plasmids of 6.2 kb in size (compared with 19% of
non-food-poisoning strains).

17. LAB-ON-A-CHIP TECHNOLOGY
 An 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 laser-
induced 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.

18. 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 - Acridine-
orange leucocyte cytospin test

19. ELECTROPHORETIC METHODS
 Electrophoretic 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:

20. ELECTROPHORETIC METHODS
 isoelectric focusing (IEF)
 urea isoelectric focusing (urea-IEF)
 sodium dodecyl sulphate – polyacrylamide gel
electrophoresis (SDS-PAGE)
 two dimensional electrophoresis (2DE)
 capillary electrophoresis (CE)

21. GM-PLANTS AND DERIVED FOODS DETECTION
PROCEDURE
Samples
Sampling
Tested Samples Saved Samples
Protein Detection Nucleic Acids
Methods Detection Methods
DNA Extraction
ELISA Lateral Flow Strip
Conventional PCR
Negative Positive
No GM contents Contained
GM contents
Quantitative PCR
GM Contents (xx%)

22. 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.

23. 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.

24. 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)

25. 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).

26. BIOSENSOR
Majority of the Biosensors are based on immunological methods,
Ritcher 1993

27. 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

28. 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.

29. 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

30. 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.

31. FLUORESCENT IN SITU HYBRIDIZATION (FISH)

32. 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

33. The Karnali Bridge, Near My Home Town
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