Real-time PCR has removed many of the limitations of standard end-point PCR and since its introduction in the mid-1990s there has been an explosion both in the number of publications and available instrumentation describing real-time PCR applications across many disciplines. Real-time PCR (RT-PCR) technology is highly flexible and many alternative instruments and fluorescent probe systems have been developed recently. The decreased hands-on time, increased reliability and improved quantitative accuracy of RT-PCR methods have contributed to the adoption of RT-PCR for a wide range of new applications.

The development of instruments that allowed real-time monitoring of fluorescence within PCR reaction vessels was a significant advance. The technology is flexible and many alternative instruments and fluorescent probe systems are available. RT-PCR assays can be completed rapidly since no manipulations are required after the amplification. Identification of the amplification products by probe detection in real-time is highly accurate compared with the traditional PCR method of size analysis on gels. Analysis of the progress of the reaction allows accurate quantification of the target sequence over a very wide dynamic range, provided suitable standards are available. Further investigation of the RT-PCR products within the original reaction mixture using probes and melting analysis can detect sequence variants including single base mutations. RT-PCR has found applications in many branches of biological science. Applications include gene expression analysis, the diagnosis of infectious disease and human genetic testing. Due to their fluorimetry capabilities, these real-time machines are also compatible with alternative amplification methods such as NASBA, provided a fluorescence end-point is available.

The introduction of RT-PCR assays to the clinical microbiology laboratory has led to significant improvements in the diagnosis of infectious disease. The technology has applications in clinical bacteriology, parasitology and virology. There are few areas of clinical microbiology which remain unaffected by this new method. It has been particularly useful to detect slow growing or difficult to grow infectious agents.Its greatest impact is probably its use for the quantitation of target organisms in samples. The ability to monitor the PCR reaction in real-time allows accurate quantitation of target sequence over at least six orders of magnitude. The closed-tube format which removes the need for post-amplification manipulation of the PCR products also reduces the likelihood of amplicon carryover to subsequent reactions reducing the risk of false-positives. Standardisation of assay protocols for use in diagnostic clinical microbiology and external quality control schemes is required to ensure quality of testing.

Further reading:
Real-Time PCR: Current Technology and Applications
Real-Time PCR in Microbiology: From Diagnosis to Characterization

Labels: diagnostics, PCR, qPCR, real-time PCR, RT-PCR