THE ROLE OF BIOTECHNOLOGY IN BTW AGENT DETECTION. DNA Methods: Polymerase Chain Reaction

摘要

The polymerase chain reaction (PCR) is one of the most powerful biotechnology tools available for the identification of organisms as well as for studying biodiversity and complex ecological systems. The PCR can be used to amplify very small amounts of DNA present in a sample. Theoretically, it is possible to find and identify a single bacterial cell in an environmental sample, even if large numbers of other microorganisms are present, although in practice, the sensitivity of detection of microorganisms using PCR technology varies with the samples under study. By choosing specific primers, microorganisms can be identified at the level of groups, genera, species, or possibly even strains, provided that there are nucleotide base sequences specifying these various categories. There are, however, numerous documentations of problems involving inhibition of the PCR, due primarily to the sensitivity of RNA and DNA poly-merases to inhibitory substances in clinical and environmental samples. In such situations, false negative results may occur, and various methods are being tested that are designed to concentrate and purify samples from inhibitory substances. A very promising technique that is becoming wide-spread in use is the immunocapture-PCR (IC-PCR). Multiplex PCR systems have found many applications in recent years, and have proved to be very successful in distinguishing individual variation at highly polymorphic genetic loci and identifying species- or strain-specific variations in microorganisms. A profile based on a set of seven "housekeeping" genes, that is genes that are relatively conserved and do not change very rapidly with time, is considered useful for identifying and tracking the global spread of antibiotic-resistant bacteria and other virulent pathogens. Rapidly evolving genes on the other hand are useful for short-term, fine-scale epidemiological investigations detecting uncharacterized genomic differences between microbial isolates during an epidemic within a restricted area. Continued developments in PCR equipment towards compactness and automation can be expected in the near future, so that on-site analyses in mobile laboratories will become more feasible.