SPUD qPCR Assay Confirms PREXCEL-Q Software's Ability to Avoid qPCR Inhibition
Real-time quantitative polymerase chain reaction is subject to inhibition by substances that co-purify with nucleic acids during isolation and preparation of samples. Such materials alter the activity of reverse transcriptase (RT) and thermostable DNA polymerase enzymes on which the assay depends. When removal of inhibitory substances by column or reagent-based methods fails or is incomplete, the remaining option of appropriately, precisely and differentially diluting samples and standards to non-inhibitory concentrations is often avoided due to the logistic problem it poses.
To address this, the PREXCEL-Q software program was invented to automate the process of calculating the non-inhibitory dilutions for all samples and standards after a preliminary test plate has been performed on an experimental sample mixture. The SPUD assay was used to check for inhibition in each PREXCEL-Q-designed qPCR reaction. When SPUD amplicons or SPUD amplicon-containing plasmids were spiked equally into each qPCR reaction, all reactions demonstrated complete absence of qPCR inhibition. Reactions spiked with ~15,500 SPUD amplicons yielded a Cq of 27.39 +/- 0.28 (at ~80.8% efficiency), while reactions spiked with ~7,750 SPUD plasmids yielded a Cq of 23.82 +/- 0.15 (at ~97.85% efficiency).
This work demonstrates that PREXCEL-Q sample and standard dilution calculations ensure avoidance of qPCR inhibition.
from Gallup et al in SPUD qPCR Assay Confirms PREXCEL-Q Software's Ability to Avoid qPCR Inhibition
Further reading: PREXCEL-Q Software's Ability to Avoid qPCR Inhibition
To address this, the PREXCEL-Q software program was invented to automate the process of calculating the non-inhibitory dilutions for all samples and standards after a preliminary test plate has been performed on an experimental sample mixture. The SPUD assay was used to check for inhibition in each PREXCEL-Q-designed qPCR reaction. When SPUD amplicons or SPUD amplicon-containing plasmids were spiked equally into each qPCR reaction, all reactions demonstrated complete absence of qPCR inhibition. Reactions spiked with ~15,500 SPUD amplicons yielded a Cq of 27.39 +/- 0.28 (at ~80.8% efficiency), while reactions spiked with ~7,750 SPUD plasmids yielded a Cq of 23.82 +/- 0.15 (at ~97.85% efficiency).
This work demonstrates that PREXCEL-Q sample and standard dilution calculations ensure avoidance of qPCR inhibition.
from Gallup et al in SPUD qPCR Assay Confirms PREXCEL-Q Software's Ability to Avoid qPCR Inhibition
Further reading: PREXCEL-Q Software's Ability to Avoid qPCR Inhibition
Labels: PREXCEL-Q, qPCR, qPCR Inhibition, SPUD
qPCR Web Resources
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
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
qPCR machines
In weighing up the pros and cons of the different real-time PCR platforms or qPCR machines for your laboratory, factors to consider include: supported chemistries; multiplex capability for that chemistry; throughput; flexibility; format; easy-of-use and robust software package; reproducibility; speed; size; technical support; customer support and not least the cost, not only of the initial equipment outlay and servicing but also the associated cost of consumables and reagents.
The following qPCR machines are compared for various features to help you decide which instrument is most suitable for your needs.
Bibliography:
The following qPCR machines are compared for various features to help you decide which instrument is most suitable for your needs.
- Applied Biosystems: ABI 7300, ABI 7500, ABI 7500 Fast, ABI 7900 Fast HT with automation accessory, ABI StepOne
- Roche: LightCycler 480, LightCycler 1.5, LightCycler 2.0
- Stratagene: Mx4000, Mx3000P, Mx3005P
- Cepheid: SmartCycler
- Corbett: Rotor-Gene 6000
- Eppendorf: Mastercycler ep realplex
- BioRad: MiniOpticon, MyiQ, Opticon2, Chromo4, iQ5
Bibliography:
- Real-Time PCR: Current Technology and Applications
- Real-Time PCR in Microbiology: From Diagnosis to Characterization
- PCR Troubleshooting: The Essential Guide
- PCR Books
Labels: qPCR, qPCR instruments, qPCR machines, real time PCR machines
qPCR machines. Part 3
Ideally, the analysis software supplied with the platform should be as user-friendly as possible but it is also important to check that the software can fully analyse results of the chosen probe chemistry. Some platforms and analysis software suites are biased towards certain chemistries. Some real-time instruments also have specific primer and probe design software that is either supplied with the hardware or available at extra cost. Such software can help simplify and speed up the assay design process and is optimised for that system and reagents. The LightCyclers also have specific relative quantification software that is designed to determine the exact relative nucleic acid concentration normalized to a calibrator sample. This software speeds up and greatly simplifies relative quantification.
from Logan and Edwards (2009) in Real-Time PCR: Current Technology and Applications
Bibliography:
from Logan and Edwards (2009) in Real-Time PCR: Current Technology and Applications
Bibliography:
- Real-Time PCR: Current Technology and Applications
- Real-Time PCR in Microbiology: From Diagnosis to Characterization
- PCR Troubleshooting: The Essential Guide
- PCR Books
Labels: qPCR, qPCR software, qPCR thermal cycler, qRT-PCR, RT-PCR software, software
History of 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:
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:
- Real-Time PCR: Current Technology and Applications
- Real-Time PCR in Microbiology: From Diagnosis to Characterization
- PCR Troubleshooting: The Essential Guide
- PCR Books
Labels: ethidium bromide, qPCR, quantitation, quantitative real time pcr, real-time pcr, real-time PCR history
Real-Time PCR Data Analysis
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:
from M.W. Pfaffl, J. Vandesompele and M. Kubista in Real-Time PCR: Current Technology and Applications
Further reading: Real-Time PCR
The software provided with real-time PCR instruments allows several types of data analysis:
- normalisation of the raw data
- measurement of the cycle number at which any increase in the fluorescence within each reaction vessel reaches significance
- the data are used in conjunction with the results from internal or external standards to estimate the original number of template copies
- 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
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
Clinical microbiology and real-time PCR
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:
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:
- 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
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