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.
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
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.
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.
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.
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.