The one-phrase answer is "the polymerase chain reaction (PCR)"
The Tetrad "DNA engine" thermal cycler (MJ Research) allows seven DNA targets to be amplified simultaeneously.
PCR allows us to target specific regions of DNA which have been proven to be unique to the organism we are interested in and create millions of copies of these gene fragments. This allows us to produce enough DNA to see on an agarose gel. The beauty of the technique is that total DNA (the huge majority of which will be host-derived) does not have to be treated in any way after extraction. PCR will seek out those few bacterial chromosomes (if any) present and generate enough new copies for us to see and thus make a positive diagnosis.
Class II microbiological safety cabinet used for the initial stages of DNA isolation from blood and tissue samples.
The exquisite sensitivity of PCR is its own downfall in many environments. If one drop of a successful PCR reaction was mixed in an Olympic-sized swimming pool then one drop of this mixture would contain enough DNA to give a positive result if water from the pool was tested!
To avoid the problems of contamination - leading to false positive results - we have designed the laboratory so that DNA extraction, PCR preparation and analysis of the products are done in separate, environmentally controlled rooms. Extreme care is taken to avoid cross -contamination of samples. In addition the laboratory has been designed bearing in mind that these micro-organisms can constitute a hazard to human health.
Using the scientific literature we have identified DNA sequences unique to each of the organisms being studied and have established methods for amplifying these regions of DNA selectively and reproducibly. The identity of the PCR products will be confirmed using restriction enzyme analysis, DNA sequencing and denaturing gradient gel electrophoresis (DGGE).
Agarose gel electrophoresis and imaging room where the analysis of the PCR reactions takes place.
The final result looks like this:
Agarose gel showing results of PCR for Bartonella sp. A 400 bp segment of the citrate synthase gene is amplified from the sample (Cat) and culture-derived Bartonella DNA (+ve). DNA from a control cat produces no band (-ve). Size markers (M) are shown on the left (bp=base pair)
Using the Polymerase Chain Reaction (PCR) to detect microbial infections - A Summary
