5: RAPID MOLECULAR METHODS FOR IDENTIFICATION OF PATHOGENIC MICROORGANISMS AND TOXINS

5 RAPID MOLECULAR METHODS FOR IDENTIFICATION OF PATHOGENIC MICROORGANISMS AND TOXINS

Conventional detection methods fail to provide a timely and adequate assessment of the risk from waterborne disease or to accurately reflect water quality and treatment requirements. ^[9] Recent advances have been made in a range of new molecular detection technologies based on immunological or gene probe methods which are applicable to water testing. ^[23] In the Polymerase Chain Reaction (PCR), sample DNA is combined with DNA polymerase, nucleotides, and DNA primers that are specific for a given nucleotide sequence. By applying a rapid cycle of heating and cooling it is possible to replicate the DNA sequence and so amplify the signal, creating millions of copies. The amplified DNA can then be detected by a variety of methods. As little as a single copy of a particular sequence can be specifically amplified and detected. The development of real-time quantitative PCR has eliminated the variability traditionally associated with quantitative PCR, allowing the routine and reliable quantitation of PCR products. Fast PCR involves rapid cycling and replication of DNA from specific microorganisms and can also be used for quantitative assay.

Enzyme-Linked Immunosorbent Assay (ELISA) can be used for detecting particulate material but is particularly well suited to detecting toxins in water [24]. In this technique, specific binding of the labeled antibody (or antigen) is detected by reacting an enzyme label with a substrate that generates a visible colour in the reaction mixture and the results read by photometry. ELISA can...