A comprehensive list of qPCR Web Resources including qPCR machine manufacturers' websites, PCR web resources, and PCR news groups.

from Julie Logan, Kirstin Edwards and Nick Saunders in Real-Time PCR: Current Technology and Applications

Further reading: qPCR Web Resources

Labels: qPCR, real-time pcr

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

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

Unlike standard PCR, real-time PCR instruments measure the kinetics of product accumulation in each PCR reaction tube. Generally, no product is detected during the first few PCR cycles as the fluorescent signal is below the detection threshold of the instrument.

Most combinations of machine and fluorescence reporter are capable of detecting the accumulation of amplicons before the end of the exponential amplification phase. During this time the efficiency of PCR is often close to 100% giving a doubling of the quantity of product at each cycle. As product concentrations approach the nanogram per microlitre level the efficiency of amplification falls primarily because the amplicons re-associate during the annealing step. This leads to a phase during which the accumulation of product is approximately linear with a constant level of net synthesis at each cycle. Finally, a plateau is reached when net synthesis approximates zero.

Quantification in real-time PCR is done by measuring the number of cycles required for the fluorescent signal to reach a threshold level or the second derivative maximum of the fluorescence versus cycle curve. This cycle number is proportional to the number of copies of template in the sample. Real-time PCR quantification applications are discussed in detail in Bustin and Nolan (2009) Analysis of mRNA Expression by Real-Time PCR In: Real-Time PCR Logan, Edwards and Saunders, eds.; Wurmbach (2009) Validation of Array DataIn: Real-Time PCR Logan, Edwards and Saunders, eds.; and Wiseman (2009) Real-Time PCR: Application to Food Authenticity and Legislation In: Real-Time PCR Logan, Edwards and Saunders, eds.

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: fluorescence, PCR instruments, PCR machines, quantitation, real-time pcr

PCR machines with integrated fluorimeters and a mechanism for transferring excitation light from a source into the reaction vessel and then from the sample to a detector are required for real-time PCR. The heating blocks that are the mainstay of the standard PCR machine present several technical challenges in conversion to application in real-time PCR machines. The main problem being that the light must be channelled through the lid of the block and the cap of the reaction vessel across an air gap and then into the sample. Emitted light must then take the return path. Although blocks are used by several real-time PCR machines including the first commercial real-time PCR machine (Applied Biosystems 7700), the difficulties associated with them have led to the development of alternative designs for real-time pcr machines.

The LightCycler (LC24) was the forerunner of PCR machines that use air as the heating/cooling medium. Thermal transfer via air has the advantage of greater uniformity and rapidity than can be achieved on block-based PCR machines, besides allowing shortening of the light path. As well as differing in the choice of heating medium real-time PCR machines also provide a range of options for the light source and detection of fluorescence. Current, real-time PCR machines tend to allow the excitation and detection of multiple dyes so that internal standards and multiplex reactions are possible. There is also a tendency to build in a bias toward the use of either universal donor or universal recipient chemistry.

The cost of real-time PCR machines has fallen in tandem with continual improvement in their capability and accuracy. This has been the result of competition, the volume of sales and the introduction into the marketplace of improved designs dependent on new technology. These trends are unlikely to be reversed and will contribute to the growth in the popularity of real-time PCR. Real-time PCR machines are described and discussed in more detail in Real-Time PCR Machines (Logan and Edwards 2009. Chapter 2. Real-Time PCR: Current Technology and Applications. Caister Academic Press, Norfolk, UK.

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: fluorimeters, PCR instruments, PCR machines, real-time pcr, thermal cyclers

Real-Time PCR: Current Technology and Applications
Publisher: Caister Academic Press
Editor: Julie Logan, Kirstin Edwards and Nick Saunders
Publication date: January 2009
ISBN: 978-1-904455-39-4
This essential manual presents a comprehensive guide to the most up-to-date technologies and applications as well as providing an overview of the theory of this increasingly important technique. This timely and authoritative volume describes the latest PCR platforms, fluorescent chemistries, validation software, data analysis, and internal and external controls and discusses a wide range of RT-PCR applications including: clinical diagnostics, biodefense, RNA expression studies, validation of array data, mutation detection, food authenticity and legislation, NASBA, molecular halotyping, and much more.
further information

Labels: books, publications, real-time pcr

There are two general approaches used to obtain a fluorescent signal from the synthesis of product in Real-time PCR. The first depends upon the property of fluorescent dyes such as SYBR Green I to bind to double stranded DNA and undergo a conformational change that results in an increase in their fluorescence. The second approach is to use fluorescent resonance energy transfer (FRET). These methods use a variety of ways to alter the relative spatial arrangement of photon donor and acceptor molecules. These molecules are attached to probes, primers or the PCR product and are usually selected so that amplification of a specific DNA sequence brings about an increase in fluorescence at a particular wavelength.

A major advantage of the real-time PCR instruments and signal transduction systems currently available is that it is possible to characterise the PCR amplicon in situ on the machine. This is done by analysis of the melting temperature and/or probe hybridisation characteristics of the amplicon within the PCR reaction mixture. In the intercalating dye system, the melting temperature of the amplicon can be estimated by measuring the level of fluorescence emitted by the dye as the temperature is increased from below to above the expected melting temperature. The methods that rely upon probe hybridisation to produce a fluorescent signal are generally less liable to produce false positive results than alternative methods such as the use of intercalating dyes to detect net synthesis of double stranded DNA (dsDNA) followed by melting analysis of the product.

Hybridisation, ResonSense and hydrolysis probe systems give fluorescent signals that are only produced when the target sequence is amplified and are unlikely to give false positive results. An additional feature of the hybridisation, ResonSense and related methods is the possibility to measure the temperature at which the probes disassociate from their complementary sequences. This measurement gives a further verification of the specificity of the amplification reaction. An important feature of many of the probe systems is their compatibility with multiplexing due to the availability of fluorophores with resolvable emission spectra.

from N.A. Saunders 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: fluorescence, fluorescent dyes, hybridisation, multiplexing, PCR instruments, real-time pcr, ResonSense, signal transduction

Standard PCR requires the identification of the amplified fragment(s) by post-PCR analysis, usually by gel electrophoresis. These methods rely on either the size or sequence of the amplicon. Gel electrophoresis, often used to measure the amplicon size, is both inexpensive and simple to implement. However, size analysis has limited specificity since different molecules of approximately the same molecular weight cannot be distinguished. Gel electrophoresis alone is not a sufficient PCR end-point in many instances, including most clinical applications. Characterisation of the product by its sequence is far more reliable and informative and probe hybridisation assays can be used for this purpose. Such methods are time-consuming and care must be taken to ensure that amplicons accidentally released into the laboratory environment do not contaminate the DNA preparation and clean rooms.

Real-time PCR greatly simplifys amplicon recognition by providing the means to monitor the accumulation of specific products continuously during cycling. All current instruments designed for real-time PCR, measure the progress of amplification by monitoring changes in fluorescence within the PCR reaction vessel. Changes in fluorescence can be linked to product accumulation by a variety of methods.

A further advantage of the real-time format is that the analysis can be performed without opening the tube which can then be disposed of without the risk of dissemination of PCR amplicons or other target molecules into the laboratory environment. Although alternative methods for avoiding PCR contamination are available, containment within the PCR vessel is likely to be the most efficient and cost-effective.

A major drawback of standard PCR formats that rely on end-point analysis is that they are not quantitative because the final yield of product is not primarily dependent upon the concentration of the target sequence in the sample. Real-time PCR overcomes this limitation.

from N.A. Saunders 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: contamination, quantitation, real-time pcr

The development of instruments that allow real-time monitoring of fluorescence within a PCR reaction vessel led to a significant advance in PCR technology and applications. Many different instruments and fluorescent probe systems have been developed and are currently available.

Real-time PCR assays can be completed rapidly since no manipulations are required post-amplification. Identification of the amplification products by probe detection in real-time is highly accurate compared with 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 real-time PCR products within the original reaction mixture using probes and melting analysis can detect sequence variants including single base mutations.

Real-time PCR has 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, real-time machines are also compatible with alternative amplification methods such as NASBA, provided a fluorescence end-point is available.

Bibliography:

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

from N.A. Saunders in Real-Time PCR: Current Technology and Applications

Labels: applications, assays, NASBA, probes, real-time pcr