Specificity and Performance of PCR Detection Assays for Microbial Pathogens

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Abstract
PCR has become a widely used tool for detection, identification and differentiation of pathogenic microorganisms in diagnosis of animal and human diseases. However, quite a number of currently used protocols can be further optimized to exclude nonspecific reactions. On the one hand, target sequences as defined by primer binding sites should be checked carefully for the absence of significant homologies to other organisms in order to insure high specificity of detection. A major part of PCR assays is still based on target sequences in the ribosomal RNA operon, but, as the differentiating potential of this region is limited, genes encoding cellular proteins, such as toxins, surface antigens or enzymes, have been shown to be a viable alternative in many instances. On the other hand, various approaches are available to improve the performance of the amplification reaction itself. The kinetics of amplification is known to be heavily dependent on primer-to-template ratio, efficiency of primer annealing and enzyme-to-template ratio. In the present paper, recently published PCR detection assays for microorganisms, particularly bacterial pathogens, are reviewed and optimization strategies are explained. The practical implications and epidemiological consequences of routine use of PCR in the diagnostic laboratory are also discussed.