This document discusses molecular techniques that are used in food microbiology. It describes several applications of molecular methods including detecting genetically modified foods, food authenticity testing, and microbial contamination detection. Common molecular methods described are PCR, RFLP, PFGE, and DNA microarrays. Specific techniques covered in more depth include real-time PCR, multiplex PCR, plasmid profiling, ribotyping, AFLP, LAMP, FISH, and biosensors. The document provides details on the basic principles and procedures for several of these important molecular analysis methods.

1. MOLECULAR
TECHNIQUES IN FOOD
MICROBIOLOGY
Presented by :
Najiya K V
MSc Biotechnology

2. 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

3. 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 Gene Chip,
FISH

4. COMMON MOLECULAR METHODS
Random Amplified Polymorphic DNA Analysis (RAPD)
Amplified Fragment Length Polymorphism (AFLP)
Loop Mediated Isothermal Amplification (LAMP)
‘Biosensors
• Although there are many nucleic acid molecular
detection methods, only DNA probe and PCR has
been developed commercially for detection of food
pathogens.

5. PCR STEPS
1. isolation of DNA from the food (CTAB method is
common)
2. amplification of the target sequences by PCR
separation of the amplification products by agarose gel
electrophoresis
3. estimation of their fragment size by comparison with a
DNA molecular mass marker after staining with ethidium
bromide
4. 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 hybridization
with a DNA probe specific for the target sequence

7. REAL TIME PCR
• Real Time PCR is a technique in which fluoroprobes bind
to specific target regions of amplicons to produce
fluorescence during PCR.
• The fluorescence, measured in Real Time, is detected in
a PCR cycler with an inbuilt filter flurometer.

9. 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 should have same melting temperature
• Must not interact with each other.
• The amplified fragments of same length cannot be
detected

10. 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

11. a multiplex PCR assay for the efficient detection of chicken, turkey and duck meat. Three species-
specific primer sets targeting 12S rRNA gene of turkey and mitochondrial cytochrome b (CYTB)
gene of chicken and duck were selected. A universal primer pair targeting 18S rRNA gene was
additionally used as a positive control. PCR product sizes for endogenous control, chicken, turkey
and duck were confirmed to be 99 bp, 133 bp, 163 bp and 204 bp respectively. The specificity of
each primer pairs was evaluated in 20 animal species. The limit of detection of this multiplex PCR
assay was 1 pg for all species. Furthermore, this method was successfully applied to 31
commercial meat samples and validated at intra-laboratory

12. 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.

13. To authenticate these species, we developed heptaplex polymerase chain reaction-restriction
fragment length polymorphism (PCR-RFLP) assay using species-specific primer sets which amplified
short-length amplicons (73–263 bp) targeting mitochondrial cytochrome b (cytb) and NADH
dehydrogenase subunit 5 (ND5) genes. Target specificity was confirmed through cross-amplification
reaction with 25 non-target species and PCR products were authenticated by restriction digestion
with FatI, BfaI, and HPY188I enzymes followed by separation and visualization of fragments in an
automated electrophoretic system

14. SSCP (single strand confirmation
polymorphism)

15. 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 labour 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.

17. 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).

18. • Plasmids are extrachromosomal, circular DNA molecules that
are located in the bacterial cytoplasm, that contain at least one
origin of replication
• Isolation of plasmid DNA released under alkaline and high
temperature conditions that denature the chromosomal DNA
• Phenol: chloroform mixture to precipitate the plasmid DNA.
• Separated by gel electrophoresis, stained with a dye and
viewed.
• Typically, supercoiled molecular size standards from E. coli
R861 (NCTC 50192) and V517 (NCTC 50193), to determine the
sizes of the isolated plasmids
• The number and size of plasmid bands are analyzed to define
the plasmid profile for a particular isolate

19. 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

21. MACRORESTRICTION
• The macrorestriction analysis of bacterial genomes is a
method that is generally applicable to the typing of
bacteria.
• The chromosome is cleaved with a restriction
endonuclease that cuts infrequently and subsequently
separated by pulsed-field gel electrophoresis.
• The fragment pattern defines the genotype of the strain.
• The relatedness of strains is evaluated from the similarity
of the fragment patterns.
• Macrorestriction analysis is a sensitive and specific
method to trace the origin and spread of infections and to
analyse the clonal structure of bacterial populations.

22. Pulse Field Gel Electrophoresis
• Pulsed-field gel electrophoresis is based on the digestion of bacterial
DNA with restriction endonucleases that recognize few sites along the
chromosome, generating large DNA fragments (30-800 Kb) that cannot be
effectively separated by conventional electrophoresis.
• The basis for PFGE separation is the size-dependent time-associated
reorientation of DNA migration achieved by periodic switching of the
electric field in different directions.
• The DNA fragments will form a distinctive pattern of bands in the gel,
which can be analyzed visually and electronically.
• Bacterial isolates with identical or very similar band patterns are more
likely to be related genetically than bacterial isolates with more divergent
band patterns.

24. 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
• Self-contained integrated device that is capable of
providing specific qualitative or semi qualitative analytical
information using a biological recognition element which is
in direct spatial contact with a transduction elemen

27. classification

28. • Applications in Food
Industry
• Detection of heavy metals
• Detection of residual
agrochemicals
• Detection of toxic
metabolites
• Food borne pathogen
detection
• Assure food safety
• As indicators of product
acceptability
• Detection of unpermitted
chemicals
• Sensory analysis
• Features of biosensors
used in the food industry
• Target specificity
• Electronic integration
• Small size
• Fast response time
• Stability
• Mass producible
• Continuous signal

30. 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

33. AFLP ( Amplified Fragment Length
Polymorphism)
• AFLP is a technique based on the principle of selectively
amplifying the subset of restriction fragments from a
complex mixture of DNA fragments obtained after the
digestion of genomic DNA with restriction endonucleases.
• Steps :1. Digestion 2. Adaptor ligation 3. Amplification 4.
Electrophoresis

34. LAMP
• LAMP is a rapid DNA amplification method that relies on
a DNA polymerase that is able to amplify DNA at constant
temperature (typically 60oC-65oC).
• A minimum set of four primers (short DNA strands that
specifically determine which DNA fragment will be
amplified) is required which recognise six independent
regions in the target DNA sequence, achieving high
specificity.
• The amplification products are a variety of DNA molecules
with a characteristic loop structure containing copies of
the target sequence.

35. Workflow of a carcass swab testing procedure using LAMP. Results are
obtained within one hour

37. 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.

40. REFERANCE
• Murugaboopathi G., Parthasarathy V., Chellaram C., Anand T.P., and
Vinurajkumar s. (2013) Applications of Biosensors in Food Biosciences
Biotechnology Research Asia. 10(2), 707-711 Industry
• 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
• Sharma H., Agarwal M., Goswami M., Sharma A., Roy S.K., Rai S., and
Murugan M.S.(2013) Biosensors: tool for food borne pathogen detection.
Veterinary World. 6(12), 968-973. 18
• http://www.worldfoodscience.org/cms/
• Naravaneni R, Jamil K. J Med Microbiol. 2005 Jan;54(Pt 1):51-4
• www.researchgate.net
• https://www.newfoodmagazine.com/article/74029/loop-mediated-isothermal-
nucleic-acid-amplification-lamp-for-food-microbiology-testing/
• foodsafety.merieuxnutrisciences.com
• https://pubmed.ncbi.nlm.nih.gov/7698814/