MOLECULAR DETECTION TECHNIQUES IN FOOD QUALITY

CONTROL: AN OVERVIEW

By: Yakindra Prasad TimilsenaID- 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 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.

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,

COMMON MOLECULAR TECHNIQUES

Random Amplified Polymorphic DNA Analysis (RAPD)

Amplified Fragment Length Polymorphism (AFLP)Loop Mediated Isothermal Amplification (LAMP)

BiosensorsGold Nanoparticle-based BiosensorFiber Optic BiosensorElectrochemical 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 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

What is PCR?DNA replication in a tube (in vitro). Xeroxing (copying) of DNA.

The Components of PCRThe basic components of a PCR reaction are

- one or more molecules of target DNA- two oligonucleotide primers

- thermostable DNA polymerase- dNTPs

The Process of PCREach 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

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.

Gel electrophoresis for detecting PCR products

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 neededLess 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.

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

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.

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

DIRECT EPIFLOURESCENT TECHNIQUE (DEFT)

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:

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)

GM-PLANTS AND DERIVED FOODS DETECTION PROCEDURE

Samples

Tested Samples Saved Samples

Protein Detection Methods

Nucleic Acids Detection Methods

ELISA Lateral Flow StripDNA Extraction

Conventional PCR

Contained GM contents

No GM contents

Quantitative PCR

GM Contents (xx%)

Sampling

Negative Positive

Samples

Tested Samples Saved Samples

Protein Detection Methods

Nucleic Acids Detection Methods

ELISA Lateral Flow StripDNA Extraction

Conventional PCR

Contained GM contents

No GM contents

Quantitative PCR

GM Contents (xx%)

Sampling

Negative Positive

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

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

PIEZOELECTRIC BIOSENSOR

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/ucm1

096 52.htm#ref4

http://www.worldfoodscience.org/cms/

Naravaneni R, Jamil K. J Med Microbiol. 2005

Jan;54(Pt 1):51-4

THANK YOU for your kind attention!

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