Package 2- Main difference between PCR & ELISA

Package 2- Polymerase Chain Reaction (PCR)

Polymerase Chain Reaction (PCR) is the quick and easy method for generating unlimited copies of any fragment of DNA. This technique is used mainly in two areas: detection of infectious disease organisms & detection of variations and mutations in genes.

Why is PCR so useful?
The genetic material of each living organism (plant, animal, bacterium or virus) possesses sequences of its nucleotide building blocks (usually DNA, sometimes RNA) are uniquely and specifically present only in its own species. Complex organisms such as human beings possess DNA sequences that are uniquely and specifically present only in particular individuals. These unique variations make it possible to trace genetic material back to its origin, identifying with precision at least what species of organism it came from, and often which particular member of that species.

What PCR requires?
This technique requires a template molecule (the DNA or RNA you want to copy) and two primer molecules to get the copying process started. The primers are short chains of the four different chemical components that make up any strand of genetic material. These four components are like bricks or building blocks that are used to construct genetic molecules; also known as nucleotides or bases in laboratory. For PCR, primers must be duplicates of nucleotide sequences on either side of the piece of DNA of interest, which means that the exact same order of the primers’ nucleotides must be known already.

Procedures of PCR:
There are 3 basic steps in PCR.

1st step --> The target genetic material must be denatured, where the strands of its helix must be wound and separated by heating to 90-96°C.

2nd step --> Hybridization/annealing, where the primers bind to their complementary bases on the now single-stranded DNA.

3rd step --> DNA synthesis by a polymerase. Starting from the primer; the polymerase can read a template strand and match it with a complementary nucleotides very quickly.

The result is two new helixes in place of the first, each composed of one of the original strands plus its newly assembled complementary strand.



Quoted from: http://www.faseb.org/opa/bloodsupply/pcr.html

Package 2- Immunoassays

Besides the conventional agar plate testing of foodborne pathogens, there are other methods such as Immunoassays, PCR, DNA hybridization etc.

Immunoassay refers to the qualitative or quantitative determination of antigen or antibody in a specimen by an immunological reaction. In addition, immunoassays are among the most commonly used techniques in clinical, food and environmental microbiology for rapidly detecting pathogenic bacteria.

There are 5 types of immunoassays techniques:

1) Immunofluorescence
This technique is the first type of immunoassay used to detect bacteria in biological specimens. Immunofluorescence kits for detecting Salmonella in food have been commercially available. In this method, bacteria from an enrichment culture are fixed to a microscope slide and the fixed cells are treated with fluoresin-conjugated somatic and flagellar antibodies specific for Salmonella. After excess reagent is removed, the slide is observed under fluorescence microscope for cells with fluorescent cell wall and/or flagella. Although immunofluorescence is a useful tool in the research laboratory, it is not used in most food microbiology laboratories because final evaluation of the reaction is performed by microscopic examination, which is tedious. Furthermore, trained and experienced personnel are needed to obtain reliable results.

2) Coagglutination (agglutination-enhancement)
This technique is popular in clinical microbiology because it is simple, rapid, moderately sensitive and does not require instrumentation. The most common type of antigen can be immobilized on inert latex particles by either passive adsorption or covalent bonding. Latex reagents prepared in this manner are stable for as long as 1 year when stored at 4°C. The test is performed as follows:

· After centrifugation of an enrichment culture, the pellet containing bacteria of interest is re-suspended in an appropriate buffer.
· A drop of the bacterial suspension is placed on a slide.
· A drop of the sensitized latex reagent is added and mixed thoroughly with the specimen.
· The slide is rocked for 1-5minutes and then visually examined under a high-intensity lamp for agglutination. The sensitivity of the latex agglutination test for bacteria is generally in the range 10^7-10^8 cells per ml.

It is important to perform appropriate controls each time to ensure that the senitized latex has retained reactivity and to detect false-positives caused by spontaneous agglutination of the senitized latex.

3) Immunoaffinity Chromatography
A monoclonal or polyclonal antibody to the target molecule- for example: aflatoxin B1, is immobilized on a solid inert support contained in a mini-column. A sample is suspected to contain the target antigen is allowed to flow through the column. The target molecule is retained while other materials are removed by extensive washing. The retained target molecule is eluted by changing the composition of the mobile phrase and quantitated by an appropriate method (for example: fluorometry).

4) Immunoimmobilization
Immunoimmobilization is a procedure that takes advantage of the immobilization of motile bacteria cells in a semisolid medium by a specific antibody directed against the flagella of the bacteria. Motile bacteria traverse the semi-solid medium until they meet the antiflagellar antibody diffusing from the opposite direction. A visible arc of immobilization forms at the antibody/bacterial interface; this arc indicated that target bacteria are present.

5) Enzyme Immunoassays
Enzyme immunoassays (EIA) also know as Enzyme-Linked-Immunosorbent Assay (ELISA), is the most commonly used format for the immunological detection of microorganisms and their metabolites (toxins). EIA depends on 3 principles:

Ø The exquisite specifity of antigen-antibody reactions
Ø Biological amplification of the antigen-antibody reaction by an enzyme
Ø The antibody’s ability to retain its immunoreactivity after conjugation with an enzyme.

Traditional immunoassays that used polyclonal antibodies often suffered from shortcoming caused by the presence of antibodies cross-reactive to bacteria other than the target organism and from batch-to-batch variations in antibody specificity.

Package 2- GM Labeling

GM Labeling

According to an article in the Food Standards Agency:

In the EU, if a food contains or consists of genetically modified organisms (GMOs), or contains ingredients produced from GMOs, this must be indicated on the label. For GM products sold 'loose', information must be displayed immediately next to the food to indicate that it is GM.

On 18 April 2004, new rules for GM labelling came into force in all EU Member States.

The GM Food and Feed Regulation (EC) No. 1829/2003 lays down rules to cover all GM food and animal feed, regardless of the presence of any GM material in the final product.

This means products such as flour, oils and glucose syrups will have to be labelled as GM if they are from a GM source.

Products produced with GM technology (cheese produced with GM enzymes, for example) will not have to be labelled.

Products such as meat, milk and eggs from animals fed on GM animal feed will also not need to be labelled. Details on the labelling rules can be found on the table below.

Any intentional use of GM ingredients at any level must be labelled. But there is no need for small amounts of GM ingredients (below 0.9% for approved GM varieties and 0.5% for unapproved GM varieties that have received a favourable assessment from an EC scientific committee) that are accidentally present in a food to be labelled.

The above article is quoted from: http://www.food.gov.uk/gmfoods/gm_labelling

Package 2- Genetically Modified (GM) Food

What is GM food?

GM food stands for Genetically Modified Food. It is the altering of the genetic make-up of a living organisms/food by using a particular method to transfer one or more genes from one organism to another. Usually, through this process, the nutrient value, yield or resistant against pest of the crops will increase. Thereby, increasing the food supply.

Definition of genes:
Genes are the instruction code for all characteristics that are inherited from one generation to the next and are found in nucleus in every cell in every living organism.

Difference between GM and biotechnology:
Genetically Modified Process is different from biotechnology. Genetically Modified Process involves the altering of genes whereas biotechnology makes use of the natural process/products of living things for medical, industrial and environmental.

Example of GM food:
· Tomatoes --> modified to ripen slowly to improve flavour and shelf life.
· Soybeans --> modified to resist herbicide to improve crop yields.
· Canola Oil --> modified to have a higher content of monounsaturated fatty acids, which is healthier.
· Rice --> modified to produce beta-carotene to prevent vitamin A deficiency in developing countries.