DISEASE TRANSMISSION BY CONTAMINATED WATER - Environmental Engineering: Prevention and Response to Water-,Food-,Soil-,And Air-Borne Disease and Illness - page 87

enzyme-linked immunosorbent assay (ELISA), fluorescent in situ probe (FISH),
flow cytometry, and the polymerase chain reaction (PCR), are available to provide
answers not possible by classical measures. From these has emerged a branch
of epidemiology called molecular epidemiology. Routine use of molecular tools
is nonexistent in many health laboratories, however, owing to the requirement
for relatively expensive equipment, need to employ technicians knowledgeable
about molecular techniques, and the technical issues surrounding detection of
specific genomes present in very low levels in water. Despite these apparent lim-
itations to adopting molecular techniques for routine surveillance of pathogens in
water-quality-control laboratories, molecular protocols have been used to detect
a wide range of pathogenic agents in waters.
A brief introduction to molecular methods for microbiological investigation in
the water environment is given based on descriptions by Rochelle and Schwab. 141
Sample Collection Proper procedures for obtaining water samples are inde-
pendent of the intended use of water. However, taking advantage of the sensitivity
of molecular detection implies that the target organism is probably in very low in
concentration, else it might be prudent to employ a cultural technique (assuming
the target microorganism or virus is in a viable/recoverable state). Therefore,
sample volumes earmarked for molecular applications are usually large and will
require concentration of contents.
Sample Concentration Large water samples are processed by filtration pro-
cedures applicable to bacteria, protozoa, or viruses.
Nucleic Acid Extraction The material of interest to be assayed by molecular
techniques is deoxyribonucleic acid (DNA) or ribonucleic acid (RNA). Extraction
of nucleic acids from filtered/centrifuged biomass containing the target organism
of interest may take place directly or following repeated elution and centrifugation
steps (principally required for virus recovery). Ideally, the extraction step will
be minimally time consuming, produce a high yield of intact nucleic acid, and
preclude carryover of inhibitory substances inimical to the polymerase chain
reaction (PCR) analysis. Special procedures can be introduced prior to nucleic
acid extraction for removal of inhibitors. Published protocols and commercial kits
may be used for postextraction purification of nucleic acids to eliminate inhibitors.
Methods of Detection The basic approach to assaying purified target nucleic
acid is the application of PCR. The purpose of PCR is to amplify the nucleic acid
of the target organism so that workable quantities of product become available for
subsequent sequence analysis. It is important that the PCR procedure be sensitive
and specific. PCR assays are typically operated in three cycles of temperature to
accommodate three steps:
1. Denaturation of the double-stranded, target DNA (92 ◦ -94 ◦ C)
2. Annealing of specific primers to the single-strand form (denatured) of the
target DNA at some prescribed or trial-and-error temperature (45 ◦ -55 ◦ C)