The first PCR methods to be described for clinical microbiology utilized gel electrophoresis for the detection of PCR amplification products (Real-Time PCR in Microbiology: From Diagnosis to Characterization
). Although these assays proved useful, their specificity and sensitivity was compromised by this rather cumbersome end-point detection method. Specificity of detection could be improved by incorporating a solid phase hybridization such as Southern blotting; however, this was labour intensive and time consuming requiring further manipulation of the PCR product.
Detection of PCR products by solid phase hybridization also limited the numbers of samples that could be processed, and the methods used were difficult to standardize between laboratories. The overall time taken to produce a result from a PCR assay could be two or three days and the test required a significant level of technical skill limiting the use of PCR to specialized laboratories. The introduction of enzyme-linked hybridization probe formats (PCR-ELISA) for the detection of amplification products did improve the detection process; however, they still required manipulation of the amplification products following PCR. Manipulation of the amplified product increases the likelihood of contaminating subsequent PCR reactions leading to false positives a phenomenon known as amplicon carryover.
PCR-ELISA facilitated the introduction of quantitative PCR (QPCR) assays; however, the range and accuracy of quantitation was limited. The more recent introduction of real-time platforms for PCR
has revolutionized molecular diagnostic detection methods in clinical microbiology. These closed tube systems virtually eliminate the risk of amplicon carryover because the samples are not opened following thermal cycling. Many of these new platforms process samples more rapidly than conventional block-based thermal cyclers making pathogen testing much more rapid. In addition, the ability to monitor the reaction in real-time provides results immediately after cycling and facilitates quantitation of the original target sequence over many orders of magnitude. Realtime platforms can differentiate between several closely related sequences within the same reaction therefore assays can be multiplexed to detect a range of pathogens within the same tube. Many of the assays described to date have utilized the Idaho LightCycler or the Roche LightCycler instrument. Some of the other commonly used platforms for real-time PCR
are the Applied Biosystems ABI Prism 7000, 7500, and 7900 Sequence Detection Systems, and the Cepheid Smart Cycler.
The real-time PCR
method has been applied in virtually all areas of clinical microbiology and has proven useful in a wide range of applications.
Quality control has an important role in the implementation of molecular diagnostic testing
for the diagnosis of infectious disease. Quality control encompasses measures such as the inclusion of appropriate positive, negative, and inhibition controls in assay runs. The results of positive controls should be monitored over time to ensure the assay is performing consistently and that inter-assay reproducibility remains high. External quality control schemes will play a very crucial role to ensure high standards in molecular diagnostic in the future.
The first external quality control scheme to be developed was the European Union Quality Control Concerted Action for Nucleic Acid Amplification in Diagnostic Virology. This temporary entity has been superseded by Quality Control for Molecular Diagnostics, a non-profit organization for the standardization and quality control of molecular diagnostics and genomic technologies. This organization sends out proficiency panels of simulated clinical samples containing a wide range of viral and bacterial pathogens for molecular diagnostic assays. Over 100 laboratories from more than 60 countries regularly participate in the program which is endorsed by the European Society for Clinical Virology and the European Society for Microbiology and Infectious Disease. Laboratories providing molecular diagnostic testing should participate in this scheme to ensure quality of testing.
The introduction of real-time PCR methods in clinical microbiology has improved the detection of infectious disease agents and led to improvements in patient management and care. In the future new developments in real-time molecular diagnostics will lead to further benefits to the patient consolidating the role of real-time PCR as an essential tool in the clinical microbiology laboratory.from
Andrew David Sails in Real-Time PCR: Current Technology and Applications
- Real-Time PCR: Current Technology and Applications
- Real-Time PCR in Microbiology: From Diagnosis to Characterization
- PCR Troubleshooting: The Essential Guide
- Lab-on-a-Chip Technology: Fabrication and Microfluidics
- Lab-on-a-Chip Technology: Biomolecular Separation and Analysis
Labels: clinical, diagnosis, qPCR, real-time pcr