Polymerase Chain Reaction (PCR) is a well-known method for amplifying DNA or RNA fragments, and there are several ways to use PCR for identification of bacteria. PCR is extremely powerful because it can 1) detect as few as 10 copies of DNA in a clinical sample 2) be performed in a few hours (Naber).
When employed for bacterial identification, PCR is usually used to amplify a gene of interest, followed by DNA sequencing of the gene. The most widely studied gene for classification of bacteria is the 16S rRNA sequence, because it is small enough in size to be sequenced with a few primer reactions and large enough to provide interesting phylogenetic data. Additionally, conserved regions that have been discovered in the 16S rRNA sequence make it possible to design taxa- specific primers (Fry & Pitcher). Examination of the sequence can be used to identify the bacterial species, and the use of nested primers can reduce the number of false-positives associated with PCR (Garcia-De-Lomas & Navarro).
Another method that is a variation on PCR uses a combination of hybridization and amplification. As with PCR, a forward and a reverse primer are chosen in order to amplify the desired fragment. In addition to the primers, a probe (or multiple probes) is chosen in between the forward primer and the reverse primer. The probe is modified to have a reporter dye on one end and a quencher molecule on the other end. When both reporter and quencher are attached to the probe, the emission intensity of the reporter dye is quenched. Due to the 5' nucleolytic activity of Taq DNA polymerase, the reporter is separated from the quencher, and thus a detectable change in emission intensity proportional to the amount of product is observed. This method has advantages over PCR because the use of a probe allows for real-time detection and the ability to distinguish single base pair differences (Iqbal, et. al., Qi et. al.).
A method related to PCR is the Ligase Chain Reaction (LCR). In LCR, two probes are used that are adjacent to each other on the target strand. The two probes are then ligated and serve as a template for further hybridizations, and the reaction is cycled in a manner similar to PCR. It is possible to distinguish single base pair mismatches with LCR, which exhibits both high sensitivity and high specificity (Garcia-De-Lomas & Navarro). Thus, LCR can be useful for identifying many microorganisms, but in cases where the target sequence is highly variable it is not a useful strategy.
While PCR diagnostics may be successful in some cases, there are also several problems associated with tests that use PCR. First, PCR reactions can be contaminated by extraneous DNA or can result in false positives due to nonspecific amplification. Second, PCR samples can potentially contaminate laboratory equipment and reagents. Sample contaminants can be spread as airborne droplets or by humans (Naber). Finally, PCR requires specialized equipment and personnel, which results in high costs (Garcia-De- Lomas & Navarro).