Source obtained from: http://afic.org/Detecting%20Genetically%20Modified%20Foods%20-%20Easier%20said%20than%20done.htm
Rather than measuring the rDNA in a sample, immunoassays measure the levels of proteins expressed by DNA sequences inserted by genetic modification. The test works by using anti-bodies specific for proteins encoded by rDNA sequences. One example of a commonly used immunoassay is the Enzyme-Linked Immunosorbent Assay (or the ELISA).
The ELISA relies on a reaction between antibodies (soluble proteins that are produced by the immune system in response to a foreign substance) and a "foreign substance" (typically the inserted protein), called the "antigen". The reaction is detected by a colour change or a flourometric reaction that can be measured quantitatively.
Immunoassays are less sensitive than PCR methods, which means they are less susceptible to false positives caused by minor levels of contamination. However, careful validation of each food type is needed before the test can be performed because of the large diversity of foods. This means that new assays need to be continually developed as new products of biotechnology are developed. Many hundreds of different assays will eventually be required to enable accurate detection methods.
One of the key advantages of this method of analysis is that the results are generally available within minutes. The costs of immunoassays are also much lower than that for PCR techniques (about US$2-10 per sample) however the costs for the assay development and the generation of antibodies and protein standards mean that the up-front costs can be significant.
One problem with immunoassays is that the technique does not distinguish between different sources of biotechnology-derived rDNA that may express similar protein characteristics. For example, the technique won't detect if a specific protein, such as the Bt protein, is derived from corn or soy. In addition, proteins are denatured by many food processing methods so this technique is more useful for raw foods or food ingredients that have undergone minimal processing.
Sampling methods
The choice of technique largely depends on the type of product being analysed and the availability of equipment and funds for the analysis. Both techniques also share some common problems such as a lack of internationally recognised sampling methods or agreement on the number or size of samples required.
Sunday, July 15, 2007
Enzyme immunoassay
What is Enzyme immunoassay?
Source obtained from: http://en.wikipedia.org/wiki/Enzyme_immunoassay
The Enzyme-Linked ImmunoSorbent Assay, or ELISA, is a biochemical technique used mainly in immunology to detect the presence of an antibody or an antigen in a sample. The ELISA has been used as a diagnostic tool in medicine and plant pathology, as well as a quality control check in various industries.
Performing an ELISA involves at least one antibody with specificity for a particular antigen. The sample with an unknown amount of antigen is immobilized on a solid support (usually a polystyrene microtiter plate) either non-specifically (via adsorption to the surface) or specifically (via capture by another antibody specific to the same antigen, in a "sandwich" ELISA). After the antigen is immobilized the detection antibody is added, forming a complex with the antigen. The detection antibody can be covalently linked to an enzyme, or can itself be detected by a secondary antibody which is linked to an enzyme through bioconjugation. Between each step the plate is typically washed with a mild detergent solution to remove any proteins or antibodies that are not specifically bound. After the final wash step the plate is developed by adding an enzymatic substrate to produce a visible signal, which indicates the quantity of antigen in the sample. Older ELISAs utilize chromogenic substrates, though newer assays employ fluorogenic substrates with much higher sensitivity. In simple terms, an unknown amount of antigen in a sample is immobilized on a surface. One then washes a particular antibody over the surface. This antibody is linked to an enzyme that visibly reacts when activated, say by light hitting it in the case of a fluorescent enzyme; the brightness of the fluorescence would then tell you how much antigen is in your sample.
Source obtained from: http://en.wikipedia.org/wiki/Enzyme_immunoassay
The Enzyme-Linked ImmunoSorbent Assay, or ELISA, is a biochemical technique used mainly in immunology to detect the presence of an antibody or an antigen in a sample. The ELISA has been used as a diagnostic tool in medicine and plant pathology, as well as a quality control check in various industries.
Performing an ELISA involves at least one antibody with specificity for a particular antigen. The sample with an unknown amount of antigen is immobilized on a solid support (usually a polystyrene microtiter plate) either non-specifically (via adsorption to the surface) or specifically (via capture by another antibody specific to the same antigen, in a "sandwich" ELISA). After the antigen is immobilized the detection antibody is added, forming a complex with the antigen. The detection antibody can be covalently linked to an enzyme, or can itself be detected by a secondary antibody which is linked to an enzyme through bioconjugation. Between each step the plate is typically washed with a mild detergent solution to remove any proteins or antibodies that are not specifically bound. After the final wash step the plate is developed by adding an enzymatic substrate to produce a visible signal, which indicates the quantity of antigen in the sample. Older ELISAs utilize chromogenic substrates, though newer assays employ fluorogenic substrates with much higher sensitivity. In simple terms, an unknown amount of antigen in a sample is immobilized on a surface. One then washes a particular antibody over the surface. This antibody is linked to an enzyme that visibly reacts when activated, say by light hitting it in the case of a fluorescent enzyme; the brightness of the fluorescence would then tell you how much antigen is in your sample.
Identification of Foodborne Bacterial Pathogens
Identification of Foodborne Bacterial Pathogens by Gene Probes
Source obtained from: http://www.cfsan.fda.gov/~ebam/bam-24.html
The above website contains information on:
Source obtained from: http://www.cfsan.fda.gov/~ebam/bam-24.html
The above website contains information on:
- Some gene probes used to detect pathogenic bacteria in foods
- Description of individual probes to identify targets.
Example of Probes and their targets:
Campylobacter jejuni: Ribosomal RNA
A probe that is specific for C. jejuni ribosomal RNA genes has been developed (86,87) and is available commercially. A pool of randomly selected and tested chromosomal fragments is also specific for C. jejuni, but the target has not been reported (83).
Escherichia coli: Heat-labile enterotoxin genes
The heat-labile enterotoxins (LT) of E. coli are a closely related group of proteins; they are distinguished from heat-stable enterotoxins (ST) by being immunogenic and are inactivated by heating at 60°C for 10 Min (31). The toxins stimulate adenylate cyclase (30) and can be detected by tissue culture assays of Chinese hamster ovary cells (30) or mouse Y-l adrenal cells (13). Using these tests, So et al. (102) localized and cloned the structural gene for LT; Dallas et al. (8) recloned a smaller fragment into plasmid pEWD299. Although there are several different genes for LT, as evidenced by their nucleotide sequences (56,73,103,110,111), they all share a significant amount of genetic similarity. The region of the LT genes chosen as a gene probe target is identical in each of these genes, so that all strains with the genetic potential to produce LTs should be detected.
The LT probe, eltA11, is a 20 base synthetic oligonucleotide that encodes amino acids 45-51 of the A subunit of the E. coli LT (111).
Methods
Four different techniques can be used for colony hybridization tests:
- Direct plating of samples for enumeration.
- Direct plating of cultures after enrichment to determine presence/absence.
- Spotting of individual colonies or pure cultures for an additional hybridization assay to confirm a positive result from colony hybridization with a mixed culture.
- Returning to a "master" replica plate to make pure cultures of positive colonies for further study and analysis.
The first three techniques differ as to when solid media are inoculated. In the first, aliquots of the homogenized sample are plated immediately after blending. In the second, plates are inoculated after aliquots of the homogenized sample have been incubated under selective conditions. Samples from the first and second techniques are plated onto selective agar media whenever such appropriate media are available. For the third technique, individual positive colonies are re-streaked and an additional colony hybridization test is conducted to ensure that the initial positive or negative results can be repeated. With the last technique, a pure culture can be obtained without selective enrichment, and additional microbiological tests requiring a pure culture can then be performed.
Control cultures
Strains that are positive or negative for the various probe tests must be properly stored and periodically tested for the appropriate phenotypic characteristics. A test methodology other than a gene probe must be used to independently verify the genotype of the control microorganisms. Control cultures must also be stored appropriately to minimize the possibility of genetic change. Usually, freezing liquid cultures at -70°C in 10-25% glycerol will suffice, except for Vibrio species, which are particularly sensitive to cold. Appropriate control strains have been listed, but other strains can be used if they have been properly characterized.
DNA PROBES
What are DNA Probes?
Source obtained from: http://en.wikipedia.org/wiki/DNA_probe
In molecular biology, a hybridization probe is a fragment of DNA of variable length (usually 100-1000 bases long), which is used to detect in DNA or RNA samples the presence of nucleotide sequences that are complementary to the sequence in the probe. The hybridization probe is labeled radioactively (commonly with 32P) or with immunological markers, such digoxigenin. The labeled probe is then denatured (by heating) into single DNA strands and hybridized to target DNA (Southern blotting) or RNA (northern blotting) immobilized on a membrane or in situ. DNA sequences or RNA transcripts that have moderate to high (depending on the stringency in the hybridization) sequence similarity to the probe, are detected by visualizing the hybridized probe via autoradiography or other imaging techniques. Hybridization probes used in DNA microarrays refer to DNA covalently attached to an inert surface, such as coated glass slides or gene chips, and to which a mobile DNA target is hybridized.
Source obtained from: http://en.wikipedia.org/wiki/DNA_probe
In molecular biology, a hybridization probe is a fragment of DNA of variable length (usually 100-1000 bases long), which is used to detect in DNA or RNA samples the presence of nucleotide sequences that are complementary to the sequence in the probe. The hybridization probe is labeled radioactively (commonly with 32P) or with immunological markers, such digoxigenin. The labeled probe is then denatured (by heating) into single DNA strands and hybridized to target DNA (Southern blotting) or RNA (northern blotting) immobilized on a membrane or in situ. DNA sequences or RNA transcripts that have moderate to high (depending on the stringency in the hybridization) sequence similarity to the probe, are detected by visualizing the hybridized probe via autoradiography or other imaging techniques. Hybridization probes used in DNA microarrays refer to DNA covalently attached to an inert surface, such as coated glass slides or gene chips, and to which a mobile DNA target is hybridized.
Bacillus thuringiensis (Bt)
What is Bt?
Bt is the abbreviated name given to the common bacterium Bacillus thuringiensis. Different strains of this bacterium produce a range of different crystal proteins that are toxic to different types of insects. Most are only toxic to Lepidopterans (butterflies and moths), Coleopterans (beetles) or Dipterans (flies) but some are toxic to more than one of these insect groups.
These crystal proteins are not toxic to any other insects or living organisms, including humans.
Bt has a long history of safe use as a natural insecticide that is used widely by the organic
food industry and in many developing countries to protect agricultural and forestry crops from insect pests. It has probably always been present (in trace amounts) in the human diet due to its common presence in soil and water and on the surfaces of plants.
Genes coding for Bt crystal proteins have recently been introduced into GM plants to protect them from insect attack.
The toxic effect of the Bt crystal proteins requires a particular gut environment and special gut receptors that are only present in some insects.The crystal proteins must be eaten by the insect larvae and converted into a toxic form by enzymes in the insect gut.The toxic protein then binds to specific receptors on the insect gut wall, leading to death of the cells lining the gut wall and paralysis of the gut. The insect stops feeding and dies.
A large number of scientific studies have shown that Bt is not toxic to humans or other
vertebrate animals. Rats, mice, rhesus monkeys and humans do not contain receptors for the toxin proteins.
There are several environmental concerns associated with the use of Bt in agriculture—
such as the possibility of toxic effects on beneficial insect populations and the possibility
that insects will become resistant to the action of Bt.These concerns are addressed by other government agencies before GM crops are allowed to be grown.
Bt is the abbreviated name given to the common bacterium Bacillus thuringiensis. Different strains of this bacterium produce a range of different crystal proteins that are toxic to different types of insects. Most are only toxic to Lepidopterans (butterflies and moths), Coleopterans (beetles) or Dipterans (flies) but some are toxic to more than one of these insect groups.
These crystal proteins are not toxic to any other insects or living organisms, including humans.
Bt has a long history of safe use as a natural insecticide that is used widely by the organic
food industry and in many developing countries to protect agricultural and forestry crops from insect pests. It has probably always been present (in trace amounts) in the human diet due to its common presence in soil and water and on the surfaces of plants.
Genes coding for Bt crystal proteins have recently been introduced into GM plants to protect them from insect attack.
The toxic effect of the Bt crystal proteins requires a particular gut environment and special gut receptors that are only present in some insects.The crystal proteins must be eaten by the insect larvae and converted into a toxic form by enzymes in the insect gut.The toxic protein then binds to specific receptors on the insect gut wall, leading to death of the cells lining the gut wall and paralysis of the gut. The insect stops feeding and dies.
A large number of scientific studies have shown that Bt is not toxic to humans or other
vertebrate animals. Rats, mice, rhesus monkeys and humans do not contain receptors for the toxin proteins.
There are several environmental concerns associated with the use of Bt in agriculture—
such as the possibility of toxic effects on beneficial insect populations and the possibility
that insects will become resistant to the action of Bt.These concerns are addressed by other government agencies before GM crops are allowed to be grown.
Toxins in GM Foods
Source obtained from: http://www.foodstandards.gov.au/_srcfiles/GM%20Foods_text_pp_final.pdf
What about toxins in GM foods?
All substances — both natural and human-made — are toxic at some dose. However, substances classed as toxins are those that can be harmful to health at typical levels of exposure. A number of different toxins are found naturally in various foods, but the vast majority of these are present at concentrations well below the level that would harm the consumer.
Examples of toxic substances found in foods include:
• glycoalkaloids found in green potatoes
• fungal toxins that sometimes contaminate food
• glucosinolates in cabbage, cauliflower, broccoli, brussels sprouts and canola
• erucic acid in canola
• psoralens in celery
• cyanogenic glycosides in bitter almonds
• substances in poisonous species of fish and mushrooms.
Thus, toxic substances are naturally present in many conventional foods that are subsequently genetically modified. For example, both GM and conventional varieties of canola naturally contain erucic acid, and there is a limit on the amount of erucic acid that is allowed in foods derived from canola, such as canola oil. These limits apply whether the food is derived from conventional or GM
canola.
Unless any toxins present in a conventional food are specifically removed, they will remain in the GM version of the food. FSANZ compares the levels of naturally occurring toxins in the conventional food with those in the GM food. If a safety assessment found that levels of naturally occurring toxins were
higher in a GM food, FSANZ would need to assess how much of the food is normally eaten (that is, the dietary intake), to ensure that the levels of toxins consumed would not be harmful to health.
What about toxins in GM foods?
All substances — both natural and human-made — are toxic at some dose. However, substances classed as toxins are those that can be harmful to health at typical levels of exposure. A number of different toxins are found naturally in various foods, but the vast majority of these are present at concentrations well below the level that would harm the consumer.
Examples of toxic substances found in foods include:
• glycoalkaloids found in green potatoes
• fungal toxins that sometimes contaminate food
• glucosinolates in cabbage, cauliflower, broccoli, brussels sprouts and canola
• erucic acid in canola
• psoralens in celery
• cyanogenic glycosides in bitter almonds
• substances in poisonous species of fish and mushrooms.
Thus, toxic substances are naturally present in many conventional foods that are subsequently genetically modified. For example, both GM and conventional varieties of canola naturally contain erucic acid, and there is a limit on the amount of erucic acid that is allowed in foods derived from canola, such as canola oil. These limits apply whether the food is derived from conventional or GM
canola.
Unless any toxins present in a conventional food are specifically removed, they will remain in the GM version of the food. FSANZ compares the levels of naturally occurring toxins in the conventional food with those in the GM food. If a safety assessment found that levels of naturally occurring toxins were
higher in a GM food, FSANZ would need to assess how much of the food is normally eaten (that is, the dietary intake), to ensure that the levels of toxins consumed would not be harmful to health.
Saturday, July 14, 2007
Genetic Modification
Source obtained from: http://www.gurupedia.com/g/gm/gmo.htm#Genetic_modification_of_bacteria
Genetic Modification of Bacteria
Three processes are known by which the genetic composition of bacteria can be altered: transformation, conjugation and transduction.
Genetic Modification of Bacteria
Three processes are known by which the genetic composition of bacteria can be altered: transformation, conjugation and transduction.
- Transformation is a process by which some bacteria are naturally capable of taking up DNA to acquire new genetic traits. This phenomenon was discovered by Fred Griffith in 1928, although the fact that it was specifically DNA molecules that carried the genetic information was not proven until 1944. Bacteria that are competent to undergo transformation are frequently used in molecular biology.
- In conjugation, DNA is transferred from one bacteria to another via a temporary connecting strand of DNA called a pilus (a process analogous to but biologically distinct from mating). Conjugation is not widely used for the artificial genetic modification of bacteria.
- Transduction refers to the introduction of new DNA into a bacterial cell by a bacteriophage (a virus that infects bacteria).
Genetic Modification of plants
The principle technique for the genetic modification of plants is based on a natural ability of the bacteria Agrobacterium tumefaciens. This bacteria infects plants and causes a tumor-like growth termed a crown gall. Agrobacterium causing crown galls contains a plasmid (a circular piece of DNA) that transfers from the bacteria into the infected plant and integrates into the plant's genome. The transferred genes cause the plant to form the gall, which houses the bacteria and produces nutrients that support the bacteria's growth. A number of scientists contributed to this discovery throughout the late 1960s and the 1970s, with key discoveries by Jeff Schell, Marc Van Montagu, Georges Morel and Jacques Tempé. By 1983 biotechnology had reached the point where it was possible to insert additional genes of interest into Agrobacterium and thus transfer those genes into plants.
Genetic Modification of animals
Like bacteria and plants, animals can be genetically modified by viral infection. However, the genetic modification occurs only in those cells that become infected, and in most cases these cells are eventually eliminated by the immune system. In some cases it is possible to use the gene-transferring ability of viruses for gene therapy, i.e. to correct diseases caused by defective genes by supplying a normal copy of the genes. Permanent genetic modification of whole animals can be accomplished in mice. The process begins by first genetically modifiying a mouse embryonic stem cell. This is normally done by physically introducing into the cell a plasmid that can integrate into the genome by homologous recombination. This altered cell is implanted into a blastocyst (an early embryo), which is then implanted into the uterus of a female mouse. A pup born from this blastocyst will be a chimera containing some cells derived from the unmodified cells of the blastocyst and some derived from the modified stem cell. By selecting mice whose germ cells (sperm or egg producing cells) developed from the modified cell and interbreeding them, pups that contain the genetic modification in all of their cells will be born.
Subscribe to:
Posts (Atom)