Technology Used in Green Coral Tech Ltd Kits

For those of you interested in the technologies behind the products found in this web-site, you may find the brief descriptions below useful. Further information is available from Green Coral Tech Ltd. The techniques employed use proven analytical methods formatted in a practical way for our industries. Most involve a change in colour (colorimetric measurement) that allows results to be visually seen, or read and quantified precisely by a relatively inexpensive reading device such as a spectrophotometer or a plate reader.

ELISA immunoassay

Many of our assays (e.g. the toxins, pesticides and drug residues) are classified as ‘competitive ELISAs’. In each kit, there are strips of microwells coated with an antibody that has specific recognition for the analyte of interest. A sample extract is mixed with a conjugate solution (analyte/peroxidase complex) and added to the microwells. The conjugate and target analyte of interest in the sample extract compete to bind to the antibodies coated on the wells. After a short room temperature incubation, the microwells are rinsed out and a substrate is added that changes colour on contact with the enzyme containing conjugate. The colour change is inversely proportional to the amount of target analyte bound to the antibodies. This can be quantified using a series of standards, or simply used as a yes/no screen.

There are other formats of ELISA used. They work on similar principles of specific recognition of the target analyte by an antibody and linking this to a colour change that can be measured.

Enzymatic assays

Addition of an enzyme (or enzymes) specific for converting the target analyte in a sample extract to another metabolite results in the production or reduction in the light absorbing chemicals NADH or NADPH. This can be measured on a spectrophotometer at 340nm. The stoichiometry of the reaction and the extinction coefficients of the substrates are known, so the exact quantity of the original target analyte can be calculated from the change in absorbance at 340nm.

Other enzymatic formats used depend on the inhibitory nature of the target molecule on the activity of the enzyme actually preventing a colour change in the substrate.

Polymerase Chain Reaction (PCR ®)

PCR is a technique for amplifying target DNA, which might be present in a sample in trace amounts, up to a level that can be detected. DNA extracted from the sample is added to a master mix of reagents that includes dNTP’s (building blocks used to make new DNA), MgCl2, buffer, primers (short lengths of DNA that will bind to specific regions of DNA) and Taq polymerase (the enzyme that catalyses the reaction). The mixture is placed into a thermal cycler that by using repeated cycles of three different temperatures allows the manufacture of new strands of DNA.

DNA detection is achieved either by running out the samples on a gel or by using fluorogenic probes and real time PCR technology to quantify the amount of DNA present.

Sandwich Hybridisation

The sandwich hybridisation technique employs probes targeted to rRNA from foodborne bacteria. Samples are firstly enriched for the target pathogen. The enriched sample is then lysed, liberating rRNA from the cells. A cocktail of two synthetic oligonucleotide probes (capture and detector) is then added and the solution is incubated for a set time in a water bath. The capture probe binds to one end of the rRNA and traps it onto a Poly dT-coated surface (microwell or tube) using its poly dA tail. The detector probe which binds to the other end of the target rRNA is conjugated to an enzyme. After washing, substrate is added and a positive colour change (yellow) can be read using a reader at 450nm.

ATP – Luminescence

This technology is used primarily for Hygiene testing, where food or microbial residues may be present. It is based upon the premise that all living organisms or material derived from living organisms contain ATP or related nucleotides.

The swab is taken out of its tube, the target surface is swabbed and the swab pushed right down, back into the tube, breaking the seals on the two chambers found at the bottom of the tube. Any ATP that is present is released from the swab by a solution found in the upper chamber of the tube that then drops into the chamber below containing the reagents luciferase/luciferin. The tube is placed into a reader. On contact with ATP light is emitted, which is measured by the reader. The higher the ATP level (and hence contamination present), the more light is emitted.

Traditional Microbiological Methods

Where traditional methods are required Green Coral Tech Ltd not only has a comprehensive range of microbiological media but also has a number of formats based upon traditional methods that make testing easier, faster or less time consuming.
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