PCR and Related Methods

DNA and RNA hybridization tests have been developed to detect organisms whose specific gene sequence of interest is known. At first, radioactive compounds were used to re port the hybridization, but recently, the reaction has been reported by enzyme and color reactions. The truly revolutionary development in genetic type of identification of microorganisms is in the field of PCR technology. In this technology, the gene sequence of the organism of interest (an anaerobe, for example) is known at the DNA level. A piece of the DNA can be unfolded by heat, and after cooling of the unfolded DNA, two primers will interact with specific regions of the single DNA strands. A heat-stable polymer-ase will complete the complementary strand in the presence of nucleotides from the 3' end and the 5' end. Thus, in one cycle, one DNA molecule will become two, and after another cycle, two will become four, etc. In about 2 hours, one molecule of DNA can be amplified to 106. To use this technology, the genetic sequence of the target organism must be known, and the appropriate primers must be developed for successful use of this powerful tool. There are many new variations of this technology, such as the search for a cold amplification method to bypass the heating cycle and the development of an ELISA-type detection scheme to bypass the need to do electrophoresis of PCR products.

Another technology is called ribotyping. In this technology, the DNA of a target organism is extracted from the cells, an appropriate enzyme or a collection of enzymes is then used to cut the DNA. The DNA fragments are then separated by electrophoresis, and the pattern of the bands are photographed and quantified, resulting in special fingerprints.

The fingerprints can be matched with fingerprints of known cultures for identification of unknowns. One added feature is that for certain organisms (e.g., E. coll) there are different fingerprints, even for the same organism. This becomes very important when one wants to trace the occurrence of an organism in the case of an outbreak of food-borne disease. For example, finding E. coliO157:H7 insev-eral foods and the environment in an outbreak is not good enough to trace the etiology of the outbreak. With ribotyp-ing, the culprit E. coli O157:H7 can be traced to a particular food by matching the ribotyping pattern of the strain that caused the outbreak with the origin of the strain in certain foods or in the environment. This will help pinpoint the source of the infection. Dupont's Qualicon Division is leading the way for the development and commercialization of this technology.

There are, of course, many other methods for identification of unknowns, such as impedance, conductance, capacitance, pyrolysis pattern, microcalorimetry, flow cytometry, etc. Automated Microbial Identification and Quantitation: Technologies for the 2000s, an excellent recent reference book edited by Olson (55), highlights many of these developments. There will be an explosion of technologies in applied microbiology in the near future that will directly and indirectly influence the enumeration, characterization, isolation, and identification ofanaerobes.

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