Toxins found in corn

• Alfatoxin
• Fumonisins
• Zearalenone
• Trichothescenes

Genetic techniques: Nucleic acid sequence-based amplification (NASBA)

· Amplify nucleic acid at a constant temperature of 4oC.
· Involve the reaction of three enzymes:
1. Avian Myoblastosis Virus-Reverse Transcriptase (AMV-RT)
2. Ribonuclease H
3. T7 RNA polymerase
· Used to selectively amplify RNA sequences in the presence of DNA strands.
· Two assays:
1. Hybridisation (Northern blotting and southern blotting, depending on whether DNA or RNA is being analyed)
- Joining of single stranded nucleotides
- Correspondence of two sequences: determines their degree if hybridization.
- Procedure:
1) Nucleic acid separate by electrophoresis.
2) Transferred to and fixed on a membrane probed with a labeled probe to detect the sequence of interest.
2. Microarrays (gene chips)
- Provide a miniaturized system for the simultaneous analysis of hybridization to an array of oligonucleotide probes immoblised on a support.
- Enables the analysis of large number of genetic features in a single hybridization experiment.
- Quantitative study of gene expression.

References: K. Sanderson and D. Nichols, University of Tasmania, Australia. 2003. Genetic techniques: PCR, NASBA, Hybridisation and microarrays. In: Thomas A. McMeekin. Detecting pathogens in food. North America: CRC Press LLC.261-262.

Genetic techniques: polymerase Chain Reaction (PCR)

• Used to amplify number of copies of a specific region of DNA.
• Region to amplified is specified by the choice of primers (Short chain DNA with sequence matches the ends of the region of interest.) used.

Cyclic process, Procedure:
1. In the first cycle, double stranded DNA templates are denatured by heating to a temperature of 95oC to single strands
2. Reaction mixture is cooled and allow primers to bind to the single-stranded DNA; providing an active site for DNA polymerase (thermostable polymerase: allows the cycling to continue with minimal loss of enzymatic activity.) which synthesis the complementary strand.
3. Subsequent cycle, primers blind to both the original DNA and the newly synthesised DNA: resulting in an exponential increase in the number of copies which are visualized using agarose gel electrophoresis and spectometrically.

Types of PCR:

1. Nested PCR
2. RT-PCR
3. Real-time PCR

References: K. Sanderson and D. Nichols, University of Tasmania, Australia. 2003. Genetic techniques: PCR, NASBA, Hybridisation and microarrays. In: Thomas A. McMeekin. Detecting pathogens in food. North America: CRC Press LLC.259-260.

Immunological techniques: Enzyme-linked Immunosorbent assay (ELISA)

Uses the highly specific binding reaction between antibodies and antigens.

Twp types of antibodies used:
1. Monoclonal
2. Polyclonal

Replace the detection or isolation stage on agar.

Positive result obtained is presumptive and must be confirmed using conventional tests.

Advantages:
- Easy to perform
- Can be applied to range of pathogens
- Can be semi-automated
- Give rapid result

ELISA formats:

- Sandwich ELISA:
Ø Uses two antibodies which trap the target antigens
Ø Test well is coated with antibody.
Ø Procedure:
1) Antibody is coated to the test well
2) Enriched sample added to the test well; presence of any target antigen will bind to the antibodies.
3) Washing procedure; remove food debris and unbound materials
4) Second antibodies added to the well with an enzyme label attached; presence of any target antigen will bind to the antibodies creating the ‘antibody sandwich.
5) Washing procedure
6) Addition of colourless substrate; convert by enzyme to coloured product.
7) Stop solution added; preventing further enzyme activity and any change in colour measured.

- Indirect Sandwich ELISA:
Ø Antibody does not carry enzyme label, instead it carry marker molecule which specifically bind to other molecule.
Ø Biotin is often used a s a marker with aviding as the protein binding site; resulting in a biotin-aviding, enzyme labeled antibody which catalyze conversion of colourless substrate to a coloured product.

- Competitive ELISA:
Ø Test well is coated with antigen
Ø Procedure:
1) Test well is coated with antigen
2) Sample and labeled antibodies are added to the test well simultaneously. Target antigen:
v Not present in sample; antibodies blind to the antigen coated on the wells.
v Present in sample; antibodies blind to the target antigen + antibodies coated on the wells.
3) Washing stage: antibody-antigen complexes in solution are removed; change in colour is due solely to the antibody-antigen complex on the test well.
4) Addition of colourless substrate; convert by enzyme to coloured product
v Coloured product: negative result
v Colourless (or weak colour): positive result.

Other Immunological techniques include:
1. Immunochromatography
2. Enzyme-linked immunofluorescent assays
3. Agglutination techniques.


References:

C.L. Baylis, Campden and Chorleywood Food Research Association, UK. 2003. Immunological techniques: Immunochromatography, Enzyme-linked immunofluorescent assays and Agglutination techniques. In: Thomas A. McMeekin. Detecting pathogens in food. North America: CRC Press LLC.217.

J. McCarthy, Unilever R&D Colworth, UK. 2003. Immunological techniques: ELISA. In: Thomas A. McMeekin. Detecting pathogens in food. North America: CRC Press LLC.241-245.

ATP bioluminescene:

Principles:

Make use of adenosine triphosphate, an energy donar for metabolic reactions in cell to measure cell varbility.
ATP level in a cells remain relatively constant and the content decrease sharply when cell death, thus allow the measure of biomass and cell viability.
Luciferase-luciferin (enzyme-substrate complex) catalyzes the conversion of ATP into light.
Amount of light emitted is directly proportional to the concentration of ATP which in term measure the number of variable cell.
Quantified using light detection devices.
Adenylate kinase (AK)
- An intracellular enzyme
- Equilibrates AMP, ADP and ATP by the reaction:
MgATP + AMP <=> MgADP + ADP
- When excess ADP is used as substrate, ATP will be produced in an amount proportional to the concentration of AK present which is then assayed using the luciferin-luciferase system.
- From the turnover number and the cellular ratio of AK to ATPit was predicted that, with 1 minute incubation, 40-50 times more ATP would be present for bioluminescent detection.

Assays to detect particular pathogens:
- Antibody-based bioluminescent assays
- Phage-based bioluminescent assays

Reference: M.Griffiths and L.Brovo. 2003. ATP bioluminescene. In: Thomas A. McMeekin. Detecting pathogens in food. North America: CRC Press LLC.166 and 174-178.

Pulse-field gel electrophoresis (PFGE):

Molecular typing method

Uses: Epidemiological analysis to identify the spread of a clone of Salmonella.

Advantages:

• Interpretation of the entire bacterial genome in a single gel, high discrimination, reproducibility, and typability.
• Guidelines for interpretation of PFGE results prepared by Tenover et al., offering a distinct advantage over other genotyping methods.

Principle:

• PFGE is originated from conventional gel electrophoresis.
• Conventional gel electrophoresis applied that the DNA molecules pass through an agarose gel matrix in an electric field.
• The problem of this is that its migration is inversely proportional to the log of its size whereby small fragments move through the gel matrix faster than larger ones, and very large molecules express the same mobility, resulting in poor resolution of bands.
• PFGE overcome the problem encounter by conventional gel electrophoresis by applying the idea that once the electric field has been removed the DNA returns to its relaxed state, thus charging the orientation of the electric field at regular intervals.
• This forces the DNA molecules ion the gel to relax on removal of the first field and elongate to align with a new field, a process that is size-dependent.
• This technique of PFGE involves embedding the organism in an agarose plug, thus reducing shearing of the DNA, lysing the organism in situ, and digesting the chromosomal DNA with an appropriate restriction enzyme,

Type of electrophoresis systems commercially available:

• contour-clamped homoge-nous electric field (CHEF),
• transverse alternating field electrophoresis (TAFE)
• field-inversion gel electrophoresis (FIGE)
• orthogonal-field alternation gel electrophoresis.

Interpretation of PFGE Gel:

Chromosomal DNA restriction patterns (Banding pattern) produced by PFGE are interpreted based on the Tenover et al. criteria for bacterial strain typing. The banding pattern is affected by the genetic events. By comparing the banding pattern to that of the outbreak strain, each isolate is assigned to one of four categories:

- Indistinguishable: Restriction patterns had the same number of bands and the corresponding bands were the same apparent size from the outbreak strain.
- Closely related: Restriction patterns differed from the outbreak strain by a single genetic event that explains by a point mutation or an insertion or delete of DNA.
- Possibly related: Restriction patterns differed from the outbreak strain by two independent genetic events giving rise to 4-6 band differences that explains by a point mutation or simple insertions or deletions of DNA.
- Unrelated: Restriction patterns differed from the outbreak strain by three or more independent genetic events giving rise to seven or more band differences.
Statistical Analysis of PFGE Gel interpretation:
- Uses the ‘Dice coefficient’,
- Expressed algebraically as: 2* n/ (a +b).
Where n is the number of restriction fragments that both isolates have ion common;
a is the number of restriction fragments observed for the outbreak strain;
b is the number of restriction fragments observed for the isolates being compared to the outbreak strain.
- The closer the Dice coefficient is to 1, the greater the similarity between the Salmonella strain.


Reference: Rachel Gorman and Catherine C. Adley. 2006. Pulsed-Field Gel Electrophoresis As a Molecular Technique in Salmonella Epidemiological Studies. In: Catherine C. Adley. 21. Place of publication: Human Press Inc. 81 – 90.

Definition of novel food or food ingredient

Novel food or food ingredient is deemed to be no longer equivalent if scientific, based upon an appropriate analysis of existing data, can demonstrate that the characteristics assessed are different in comparison with a conventional food or food ingredient, having regard to the accepted limits of natural variations for such characteristics.


References: Rudolf Streinz. The Legal Situation for Genetically Engineered Food in the Europe. In: Knut J. Heller. Genetically Engineered Food. Germany: WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim. Pages 122