Principles of DNA probe

A DNA probe can be developed to identify a specific taxonomic group, by directing the probe to a gene which can be found in all genus and species classified under the same taxonomic group or it can be developed to identify a particular strain of the pathogen, in which the targeted gene can only be specific to this strain.

Firstly, the probes are constructed by preparing pure and specific DNA segments. These probes are inserted into plasmids, the plasmids are transformed, thus increasing the amount of DNA probe. The plasmids are purified and can be used for identification of microorganisms.

A DNA probe is made up nucleic acid molecules, or more specifically, double-stranded DNA. The gene it contains has a known function, thus pieces of single-stranded DNA can be synthesized to compliment it. These are commonly referred to as oligonucleotides. Detection of complementary DNA or RNA targets or made possible with both natural and synthetic oligonucleotides.
DNA strands are held together by hydrogen bonds which can be broken by raising pH above 12 or temperature to above 95°C. As such, the DNA is denatured, producing single-stranded molecules. The hydrogen bonds are reformed between the AT and GC pairs, when the pH and temperature are lowered.

Double-stranded DNA probes must be denatured before the hybridization reaction; while single-stranded oligonucleotide and RNA probes do not. The nucleic acids of the tested samples are denatured via high temperature or high pH, before the labeled gene probe is added. Hydrogen bonds will form between the probe and nucleic acid, if the target nucleic acid in the sample has the same nucleotide sequence as the probe. By rinsing, the unreacted, labeled probe is removed, leaving the probe-target complexes, which is signaled by the bound label.


Principles of Enzyme Immunoassay

Detection of pathogens by enzymes immunoassay (EIA) is based on the interaction of a microbial antigen with its specific antibody and can only be used for substances with immunological properties such as proteins (enzymes), glycoprotein, polysaccharides. As hormones, bacterial antigens and antibodies are made up of protein, enzyme immunoassay is able to detect them. EIA can be used on plasma, serum , urine and cell extracts.

The most common type of testing involves an antibody attached to a solid surface. When placed into the sample, the antibody will latch onto substances it has affinity with, such as antigen and thus forms a bond with it. Another substance which consists of an enzyme linked to a antibody is added into the mixture. The enzymes catalyze the reaction of the antigen-antibody complexes, producing a specific colour. The presence and quantity of the tested substance can be determined by measuring the optical density of the colour.

References:
http://www.cfsan.fda.gov/~ebam/bam-24.html
http://fp.okstate.edu/muriana/foodpath/lect7/6LECT.pdf