PCR Methodology

PCR methodologies have become firmly entrenched in many clinical laboratories for the detection of a wide range of organisms, because they offer major advantages of improved sensitivity and rapidity over traditional diagnostic methods. However, many variables need to be considered in performing a reliable PCR assay, ranging from nucleic acid extraction, storage, composition of the PCR reaction mix used, to the dynamics of the amplification reaction. To control for these variables, there is an obvious need for standardised reagents and quality assurance programmes to obtain reproducible and clinically significant results. The diagnostic potential of the PCR technology has been greatly enhanced with the development of multiplex, real-time, and quantitative PCR methods, and these are now routinely performed in many diagnostic laboratories. More recently, PCR has been applied to bacterial typing, and a reasonable prediction is that in the near future, bacterial typing will be performed by either some variant of next-generation sequencing, or by HRM analysis of selected markers, depending on the amount of information required.