Originally, the simultaneous amplification and detection of specific DNA sequences in real-time was achieved by adding ethidium bromide (EtBr) to the PCR reaction so that the accumulation of PCR product could be visualized at each cycle. When EtBr is bound to double-stranded DNA and excited by UV light it fluoresces, therefore an increase in fluorescence in the reaction indicates positive amplification. Soon afterwards real-time PCR product quantitation or "kinetic PCR" was achieved by continuously measuring the increase in EtBr intensity during amplification with a charge-coupled device camera. By creating amplification plots of fluorescence increase versus the cycle number the kinetics of EtBr fluorescence accumulation during thermocycling was directly related to the starting number of DNA copies. When a greater number of target molecules are present fewer cycles are needed to produce a detectable signal.

Kinetic monitoring also provided a means whereby the efficiency of amplification under different conditions could be determined, providing for the first time insight into the fundamental PCR processes. The principle underlying quantitative real-time PCR can be defined as the monitoring of fluorescent signal from one or more PCR reactions, cycle-by-cycle, to completion, where the amount of product produced during the exponential amplification phase can be used to determine the amount of starting material.

The use of EtBr was not ideal since EtBr binds non-specifically to DNA duplexes and non-specific amplification products, such as primer–dimers, can contribute to the fluorescent signal and result in quantification inaccuracies. Subsequent refinements, the most significant of which was the introduction of fluorogenic probes to monitor product accumulation, added a greater element of specificity to real-time PCR and provided greater quantitative precision and dynamic range than previous methods.

These significant advances to the basic PCR technique led to the development of a new generation of PCR platforms and reagents, which allowed simultaneous amplification and quantification of specific nucleic acid sequences cycle-by-cycle. The first commercial platform to become available was the Applied Biosystems ABI Prism 7700 Sequence Detection System, followed by the Idaho Technology LightCycler (later manufactured and sold by Roche Diagnostics). Both of these platforms utilized fluorogenic chemistry and like any real-time PCR platform, they basically consist of a thermal cycler with an integrated optical detection system, which can heat, cool, detect and report. New and improved models have now superseded these two instruments and several other manufacturers have introduced their own real-time PCR platforms.

Real-time PCR offers many advantages over traditional PCR, including the amplification and detection in an integrated system, fluorescent dyes/probes allowing constant reaction monitoring, rapid cycling times (20-40 mins for 35 cycles), high sample throughput (200 to 5000 samples/day), low contamination risk due to sealed reactions, increased sensitivity, detection across a broad dynamic range of 10 - 1010 copies, reproducibility, quantification of results, and software driven operation.

from Logan and Edwards (2009) in Real-Time PCR: Current Technology and Applications

Bibliography:

1. Real-Time PCR: Current Technology and Applications
2. Real-Time PCR in Microbiology: From Diagnosis to Characterization
3. PCR Troubleshooting: The Essential Guide
4. PCR Books

Labels: ethidium bromide, qPCR, quantitation, quantitative real time pcr, real-time pcr, real-time PCR history

Quantitative real-time RT-PCR (qRT-PCR) is widely and increasingly used in any kind of mRNA quantification, because of its high sensitivity, good reproducibility and wide dynamic quantification range. While qRT-PCR has a tremendous potential for analytical and quantitative applications, a comprehensive understanding of its underlying principles is important. Beside the classical RT-PCR parameters, e.g. primer design, RNA quality, RT and polymerase performances, the fidelity of the quantification process is highly dependent on a valid data analysis.

The software provided with real-time PCR instruments allows several types of data analysis:

1. normalisation of the raw data
2. measurement of the cycle number at which any increase in the fluorescence within each reaction vessel reaches significance
3. the data are used in conjunction with the results from internal or external standards to estimate the original number of template copies
4. melting curves are transformed to provide plots of –dF/dT against T (F = fluorescence and T= temperature) in which a peak (melting peak) occurs at the equilibrium temperature for each duplex

In general instrument specific software is easy to use and allows rapid and reproducible data analysis. In addition to the bundled software a range of third party utilities is available to improve the flexibility of real-time PCR data analyses.

from M.W. Pfaffl, J. Vandesompele and M. Kubista in Real-Time PCR: Current Technology and Applications

Further reading: Real-Time PCR

Labels: data analysis, PCR instruments, qPCR, qRT-PCR, quantitation, quantitative real time pcr

A novel circular ferrofluid driven microchip has been developed for rapid polymerase chain reaction (PCR). A closed-loop circular channel was fabricated on one microchip and the PCR mixture together with a small ferrofluid plug was injected into the loop. An external magnet is used to drive the ferrofluid plug, which in turn propels the PCR mixture to move around and flow continuously through three pre-set temperature zones.

Parameters of PCR, such as incubation time, temperatures and number of cycles, can be fully controlled and adjusted. To improve throughput, a multi-loop ferrofluid driven microchip was also developed by designing a series of concentric circular channels on one microchip and the magnet enabled simultaneous actuation of DNA samples in all the channels. High reproducibility was achieved for different channels in the same run and for the same channels in consecutive runs.

The circular ferrofluid-driven PCR microchips combine the cycling flexibility of the chamber PCR and the quick temperature transitions associated with the continuous flow (CF) PCR. Most importantly, the small footprint and simultaneous actuation make it the right candidate for parallel PCR analysis. The simple, reliable and high-throughput PCR microchips will find wide applications in forensic, clinical and biological fields.

from Sun et al in Lab-on-a-Chip Technology (2009) Herold KE and Rasooly A (eds) Published by Caister Academic Press

Further reading:

• Lab-on-a-Chip Technology
• Biomolecular Separation and Analysis

Labels: lab on a chip, microchip, microfluidics, rapid PCR, technology

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

Further reading:

1. Real-Time PCR: Current Technology and Applications
2. Real-Time PCR in Microbiology: From Diagnosis to Characterization
3. PCR Troubleshooting: The Essential Guide
4. Lab-on-a-Chip Technology: Fabrication and Microfluidics
5. Lab-on-a-Chip Technology: Biomolecular Separation and Analysis

Labels: clinical, diagnosis, qPCR, real-time pcr

The Polymerase chain reaction (PCR), which was first described in 1988, is one of the most important methodological developments in molecular biology in the last three decades. In a three-step reaction it is possible to exponentially increase the amount of nucleic acid from complex biological material, which can then be used for further analysis. A further advancement of this method is real-time (RT) PCR, which allows the continuous monitoring of nucleic acid amplification by fluorescent changes measured in real time. There are already numerous books about RT-PCR on the market, all of which stress the importance of this technology nowadays. In fact, this book is considered the second edition of the previously published book Real-Time PCR: An Essential Guide by the same editors in 2004.

The present book comprises 17 chapters, of which the first seven chapters provide an overview of the technology itself, and the following 10 chapters deal with it's applications. The first part of the book contains all information necessary for any scientist working on RT-PCR. The theory behind this technology, different platforms, different chemistries, and data analysis software are well explained. Even the aspect of validation is covered, which is of utmost importance when using this method in, for instance, the pharmaceutical industry.

The second part, which is introduced through Chapter 8 Introduction to the Applications of Real-Time PCR comprises some of the most important applications of the described technology. These applications include mRNA expression analysis, microarray data validation, mutation detection, fungal infections diagnosis, to mention a few. The most comprehensive chapter is Chapter 13 Application in Clinical Microbiology, in which large numbers of microorganisms are mentioned that can be readily identified by RT-PCR. However, the author of this review misses out the difficulties encountered, when a group of microorganisms, such as mycoplasmas, are the target.

Each chapter starts with an abstract to introduce the theme of the chapter. Most reference lists at the end of each chapter are extensive and provide additional sources for the interested reader. Sometimes cross references would be helpful, such as in the case of Locked Nucleic Acid, which is mentioned in Chapter 3, but explained in Chapter 9. Figures are numerous in most chapters, but the accompanying explanations are not always sufficient. Despite these minor criticisms, the editors, who are all affiliated at the Health Protection Agency, Centre of Infections, in London, UK, succeeded in providing a comprehensive overview of the RT-PCR technology, which is as up-to-date as a book can be in such a rapidly advancing field, considering the time span taken to publish such a book. The price for this hardcover book is comparable to that of other scientific books, which is unfortunately on the high side.

from Mareike Viebahn, in Current Issues in Molecular Biology

Further reading: Real-Time PCR: Current Technology and Applications

Other books of interest:

1. Real-Time PCR in Microbiology: From Diagnosis to Characterization
2. PCR Troubleshooting: The Essential Guide
3. Lab-on-a-Chip Technology: Fabrication and Microfluidics
4. Lab-on-a-Chip Technology: Biomolecular Separation and Analysis

Labels: book review, real-time pcr

The methods used to verify the identity of the amplicon(s) produced in real-time PCR are also sufficiently powerful to detect small variations between sequences. Variations in sequence, including single nucleotide polymorphisms (SNPs) have been successfully identified in real-time PCR assays. One common approach to the detection of sequence variation is to compare melting curves. In general, the effect of base substitutions on the melting kinetics of PCR products is too small to be detected reliably (if at all). However, heteroduplexes of relatively long amplicons differing by a SNP can be distinguished from the homoduplexes on the basis of their melting curves.

The melting curves of short fluorescent probes can be used to distinguish between amplicons. This method is sensitive to SNPs, which usually cause a shift in the melting peak of several degrees. A common alternative to the melting curve approach is to use hydrolysis (TaqMan) probes. The efficiency of the 5'-3' endonuclease reaction is greatly impaired when a well-designed probe mismatches its target sequence by even a single base. The detection of mutations by real-time PCR is discussed by Lyon et al Mutation Detection and by Pont-Kingdon Molecular Haplotyping. Although the melting curve and hydrolysis probe methods for mutation analysis are widely used they are only able to detect sequences that represent a large proportion of the population. A quantitative real-time ARMS method can be used (see Lyon et al). ARMS assays are designed to detect the emergence of significant sequence mutants within a background that remains mainly of the parent type.

Bibliography:

1. Real-Time PCR: Current Technology and Applications
2. Real-Time PCR in Microbiology: From Diagnosis to Characterization
3. PCR Troubleshooting: The Essential Guide
4. PCR Books

Labels: molecular haplotyping, mutation detection, real-time pcr, SNP