New technologies and applications of PCR in clinical diagnosis

Abstract

Polymerase chain reaction (PCR) has been the method of choice for genomic cloning and analysis since its introduction to the scientific community. The production of automated thermal cyclers and thermostable DNA polymerases were the first key factors for the rapid spread of PCR in research and genetic laboratories [1]. PCR, first used to detect mutations associated with genetic diseases or for HLA typing, was then utilized to identify HIV proviral sequences in peripheral blood mononuclear cells and lymphoid tissues [2]. These initial studies clearly showed the potential applications that such a sensitive technique could support. Subsequent progression in the selection, synthesis and purification of primers, in the fidelity and efficiency of DNA polymerases, and in the development of ‘nested’ techniques, greatly increased the sensitivity and specificity of PCR and fostered its use in microbiology It is mainly used for the detection of pathogens that are difficult to identify by conventional techniques. Indeed the lack or scarcity of in vitro culture and antigen detection techniques prompted the development of assays specific for human immunodeficiency virus (HIV) and hepatitis C virus (HCV), this being further facilitated by the introduction of Tth DNA polymerase. This enzyme can in fact carry out reverse transcription and amplification of RNA sequences in a single reaction, with a significant improvement in the overall reaction efficiency and turn-around time [3]. The ever expanding application of PCR to microbiological diagnosis accelerated its introduction to the routine clinical laboratories and in the development of standardized and easy-to-use reagents and protocols. Semi-automated assays that incorporated additional reagents to prevent false positive and negative reactions