Sources of GM food

The process of GM produces combinations of genetic material that is never made previously, including genes sequences that are completely synthesized in the laboratory, differing significantly from their the original DNA. This process alters the recombination sequences of the DNA and therefore induces positive changes like, tolerance to crops, better sensory attributes, to crops.

The sources of GM foods can be used for:

Consumption:A crop, such as a fruit or vegetable, that is genetically modified.
An ingredient for a food product:Food, take example, flour that comes from a GM crop, such as maize, and the GM DNA is still present in the food and can be identified.

GM processing aid:In cheese production, the gene for producing chymosin is inserted in bacteria, so the bacteria can produce chymosin. Only the bacteria are genetically modified and so the cheese is not GM-ed.

Animal feed:GM crops, such as maize, are used to feed animals which are later eaten, such as chickens. The GM material is not in the meat that we eat. There are also animal products, such as eggs and milk that come from animals fed on GM crops.

http://www.food.gov.uk/gmdebate/aboutgm/108106?view=GM%20Microsite

Analytical techniques of toxins

Test kits

Rapid results. .g. BTA test strip for C. botulism
BTA test strip for Staphylococcal Enterotoxin
High performance Liquid Chromatography (HPLC)
-Detection of very small amt. of toxins by using fluorescence detector.
-Gives better results when coupled with Atmospheric Pressure/ Electrospray Ionisation (API/ESI)

Detection of toxins of Bacillus cereus

Gene detection techniques can be applied including PCR.PCR primers for the detection of these genes were used to detect the genes. A commercial immunoassay (Tecra visual immunoassay [VIA]) is used for identification of enterotoxic strains of B. cereus. The gene was cloned and sequenced from B. cereus. For DNA preparation, bacteria were plated on agar and incubated overnight at 30°C. An amount of bacteria corresponding to a colony 1 to 2 mm in diameter was transferred to Tris-EDTA buffer. Bacteria were lysed by incubation, and debris was removed by centrifugation. The DNA-containing supernatant was transferred to a new Microfuge tube and stored. Primers for detection of gene were given. PCR was performed essentially. PCR products were analyzed by agarose gel electrophoresis.

http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=92543

Detection of staphylococcal enterotoxin

For detecting trace amounts of staphylococcal enterotoxin in foods, the toxin must be separated from food constituents and concentrated before identification by specific precipitation with antiserum. The principles are used for the purpose: the selective adsorption of the enterotoxin from an extract of the food onto ion exchange resins and the use of physical and chemical procedures for the selective removal of food constituents from the extract, leaving the enterotoxin(s) in solution.

Developed rapid methods based on monoclonal antibodies like the ELISA and Reverse Passive Latex Agglutination are used for detection of enterotoxins. The principle of using ELISA is by binding an immunosorbent substrate onto either the enzyme or the antibody. They both retain their biologic activity; the change in enzyme activity as a result of the enzyme-antibody-antigen reaction is proportional to the concentration of the antigen and can be measured spectrophotometrically.

http://www.cfsan.fda.gov/~mow/chap3.html

Analytical techniques for isolation and identification of food borne pathogens

Immunoassays

Fluorescent enzyme immunoassay

The assay utilizes a beta-galactosidase-murine myeloma monoclonal antibody (M467) conjugate prepared with the heterobifunctional coupling reagent, N-succinimidyl 3-(2-pyridyldithio) propionate (SPDP) and uses 4-methyl umbelliferyl beta-D-galactoside as a fluorogenic substrate for the enzyme.

-To test for Salmonella, Listeria, Staphylococcus Aureus

Visual Immunoassay (VIA)

Visual immunoassay is used based on two monoclonal antibodies for the semi-quantitative determination of plasma elastase levels. VIA is considered a reliable ELISA (enzyme linked immunosorbent assay) for the detection of Salmonella spp. E. coli, Campylobacter, Staphylococcus Aureus and Listeria in food samples.

High affinity "capture" antibodies specific for the pathogen tested have been adsorbed onto the surface of wells. If antigens are present in the sample, they are captured by the antibodies. All other material in the sample is washed away before the addition of enzyme labelled antibodies (conjugate) specific for the pathogen. Presence is indicated when the bound conjugate converts the substrate to a green color, detected by a colourimetric detection system. Alternatively, if the result shows absence, no green color develops.

Results can be out within 42 hours, and it is AOAC Official Methods Validated and other international approvals.

http://www.hc-sc.gc.ca/fn-an/res-rech/analy-meth/microbio/volume3/mflp35_e.html

Staphylococcus Aureus Visual Immunoassay

This test employs S aureus-specific DNA probes and a colourimetric detection system for the detection of S. aureus in food samples following broth culture enrichment. A sample is considered non-reactive for the presence of S. aureus if the absorbance value obtained is less than or equal to the established cutoff value for the assay and vice versa.

Detection using DNA probe

In fluorescence in situ hybridization (FISH), a DNA probe is labeled with a fluorescent dye or a haptene. The labeled DNA is purified, concentrated, resuspended in hybridization buffer and is hybridized onto chromosomes and nuclei on slides. After overnight hybridization, the slides are rinsed in fluorescent-labeled antibodies to detect haptene-labeled DNA. The slides are mounted with antifade solution and are visualized at the fluorescent microscope, using appropriate filters. This procedure is illustrated in the figure.

This method identifies the pathogens by extracting a small amount of DNA and amplifies the target sequences without cultivation. The DNA probe allows the simultaneous analysis of thousands of gene in a short assay time providing high accuracy by using the species-specific probes. Probes made of peptide nucleic acids (PNA), which have very strong affinity for complementary DNA sequence, can further improve the specificity. Therefore, using PNA probes can more effectively discriminate the pathogens.

To detect the specific microbes, a small amount of the specific sample is necessary, with the scale-down of a DNA chip.

http://info.med.yale.edu/genetics/ward/tavi/fi16.html
http://www.biotrace.co.uk/content.php?hID=2&nhID=211

Bead immunocapture

A test using immunomagnetic beads with an antibody to test for Listeria. These beads are mixed with a sample to isolate Listeria. In this test, Listeria is bound to antibodies on microscopic magnetic beads. These beads are then isolated from the rest of food or environmental sample. Listeria is confirmed immunologically and, if desired, characterized biochemically, in about 24 hours.

http://www.ncbi.nlm.nih.gov/sites/entrez?cmd=Retrieve&db=PubMed&list_uids=3283171&dopt=Abstract
http://seafood.ucdavis.edu/HACCP/Compendium/Chapt17.htm#Top

REVEAL for Salmonella

It is a test system that provides for the rapid recovery of Salmonella in food and allowing detection and identification of Salmonella within 21 hours.

Following selective enrichment in Rappaport-Vasiliadis broth, a portion of the sample enrichment is placed into the sample port of the Reveal Device initiating flow. The Reveal Device contains antibodies with high specificity to Salmonella antigens. These antibodies are bound to colloidal gold and, separately, to a solid support matrix. Any Salmonella antigen present will bind to the gold conjugated antibodies forming an antigen – antibody – chromogen complex. This complex flows across a lateral flow membrane and is subsequently bound by antibody immobilized on the membrane. This causes the gold conjugate to precipitate, forming a visible line and indicating a positive reaction. Proper test completion and flow is indicated by a control line which forms further up in the test window and verifies a valid test run. Absence of a control line invalidates the test. Record results at 10 minutes incubation time.

Immunodiffusion

The principle behind the technique relies on the fact that antibodies produced by the immune system are incredibly sensitive to differences in the structure of molecules they are exposed to. The antibodies of one species will react when exposed to blood serum, containing the proteins, of another. The antibodies that respond against a specific blood protein are manufactured and collected from an outgroup species. These antibodies, in the form of an antiserum are then exposed to the blood proteins of the species under investigation.

If an organism is only very distantly related to the outgroup species a strong reaction will take place between the blood serum (containing the protein) and the antibodies, and there is very tight binding between the two. The degree of relatedness between the species being tested is reflected by the strength of reaction when exposed to the antiserum. Species more closely related to the outgroup will exhibit a weaker reaction. The experiment is usually conducted on an agar gel with the test blood protein serums arranged in wells around the test antiserum, or some arrangement that will enable comparisons of the test species.

http://www.nhc.ed.ac.uk/index.php?page=236.438.445

Transia Card

For E. coli 0157, salmonella, Listeria, Staphylococcal enterotoxins.
The Transia Plate method uses only one broth, in a two-step protocol, followed by a rapid immunoassay. It uses only one broth, in a two-step protocol, followed by a rapid immunoassay. The Transia ELISA technology, with ready-to-use reagents and a microtiterplate with divisible strips, makes the kit ideal for both single tests and high-throughput automation.

http://www.diffchamb.com/website/Archive/Templates/Item/product.asp?iSecId=36

Production of Monoclonal Antibodies

Antibodies which neutralize botulinum neurotoxin serotype F are produced using biologically active botulinum neurotoxin instead of toxoid for immunization and exploiting the importance of cross reaction between various serotypes to obtain immune responses, or monoclonal antibodies, to additional serotypes of interest. Methods of preparation and uses of the neutralizing botulinum neurotoxin antibodies are described.

PCR (polymerase chain reaction)

In this technique, double-stranded target DNA is denatured to provide single-stranded templates to which specific oligonucleotide primers are hybridized, followed by primer extension with a thermostable DNA polymerase. Primer pairs complementary to opposite strands of a DNA region are chosen. Repetitive denaturation, annealing, and primer extension cycles exponentially amplify a unique DNA fragment bordered by the primers. PCR-based methods have been developed to detect foodborne pathogens, including Listeria monocytogenes, enterotoxigenic Escherichia coli, V. vulnificus, V. cholerae, Shigella flexneri, Yersinia enterocolitica, various Salmonella and Campylobacter species.

http://www.cfsan.fda.gov/~ebam/bam-28.html

Analytical Method for testing GMOs

PCR (polymerase chain reaction)

1. Identifies genetically-modified organisms in a wide variety of materials and differentiates between different GMO components and quantifies them.
2. Determines if there are GMOs in the materials examined.
3. Determines the relative percentage of genetically-modified food.
4. Identifies animal derived ingredients by differentiating of species on the DNA level and analysis of allergens.
5. Detects proteins and even the smallest traces of allergen.
However, it is time consuming and relatively expensive ( up to $300 per analysis)

Gel electrophoresis
Clumsy and less than optimal for screening multiplex PCR products.

Next job for me

Microbiological techniques isolation and identification of foodborn pathogens

-What are the principals? (examples of techniques)
e.g. immunoassay and DNA probes.

Molecular markers and MAS used

Molecular markers are identifiable DNA sequences, found at specific locations of the genome and associated with the inheritance of a trait or linked gene.

Molecular markers can be used for
(a) marker-assisted breeding,
(b) understanding and conserving genetic resources and
(c) genotype verification.

Genetic linkage maps can be used to locate and select for genes affecting traits of economic importance in plants or animals. The potential benefits of marker-assisted selection (MAS) are greatest for traits that are controlled by many genes.

Markers can also be used to increase the speed or efficiency of introducing new genes from one population to another, for example when wishing to introduce genes from wild relatives into modern plant varieties. When the desired trait is found within the same species, it may be transferred with traditional breeding methods, with molecular markers being used to track the desired gene.

Genetic engineering can be used when insufficient natural variation in the desired nutrient exists within a species. Biofortification (the development of nutritionally enhanced foods) can be advanced through the application of several biotechnologies in combination. Genomic analysis and genetic linkage mapping are needed to identify the genes responsible for natural variation in nutrient levels of common foods. These genes can then be transferred into familiar cultivars through conventional breeding and MAS or, if sufficient natural variation does not occur within a single species, through genetic engineering. Non-transgenic approaches are being used, for example, to enhance the protein content in maize, iron in rice, and carotene in sweet potato.

References: http://www.greenfacts.org/en/gmo/index.htm#4
The State of Food and Agriculture 2003-2004

Genetic engineering using bacterium species

Genetic engineering differs from conventional plant breeding. In conventional plant breeding half of the genes of an individual come from each parent, whereas in genetic engineering one or a few specially selected genes are added to the plant genome.

Moreover, conventional plant breeding can only combine closely related plants. Genetic engineering permits the transfer of genes between organisms that are not normally able to cross breed because they are not genetically compatible. The transferred genes are called transgenes. They can come from another plant species, or even from a completely different organism (e.g., bacterial genes). These transgenes are then replicated and inherited in the same way as natural plant genes.

When the desired trait is found in an organism that is not sexually compatible with the host, it may be transferred using genetic engineering.

There are 2 ways of genetic engineering, i.e. biologically, and physically.

Biologically, in plants, the most common method for genetic engineering uses the soil bacterium Agrobacterium tumefasciens as a vector. Researchers insert the desired gene or genes into the bacterium and then infect the host plant. The desired genes are transmitted to the host along with the infection. This method is used mainly with species such as tomato and potato.

In the most common transformation technique for these crops, physical means are used. The desired gene is coated on gold or tungsten particles and a “gene gun” is used literally to shoot the gene into the host at high velocity. Once the DNA reaches the cell nucleus, it inserts itself at random into one of the host chromosomes and can express the desired character. The genetically modified plant is then grown from the transformed cell.


Overview of how transgenic crops are created:
When the bacterium infects the plant, it penetrates the plants cells and transfers its modified DNA to the plant.

Three distinctive types of genetically modified crops exist:
(a) “distant transfer”, in which genes are transferred between organisms of different kingdoms (e.g. bacteria into plants);
(b) “close transfer”, in which genes are transferred from one species to another of the same kingdom (e.g. from one plant to another); and
(c) “tweaking”, in which genes already present in the organism's genome are manipulated to change the level or pattern of expression.

Once the gene has been transferred, the crop must be tested to ensure that the gene is expressed properly and is stable over several generations of breeding.

A number of economically valuable characteristics have been introduced into plants by genetic engineering. Most of the genetically modified crop plants used so far have transgenes that provide resistance to herbicides or insects. To improve crop production and soil management, research is now exploring how to increase the variety of transgenic characteristics to include resistance to drought, heat, cold, acid soils, and heavy metals.

Transgenic plants can provide food with enhanced nutritional content. For example, genetically modified “Golden Rice” contains two daffodil genes and one bacterial gene that together result in elevated levels of provitamin A.

These techniques could be applied to improve many characteristics in other crop species.