qPCR 2010 Event
April 7 - 9, 2010
Vienna, Austria Further information
Forum to exchange ideas, share experiences, and discuss the future of the perhaps most powerful analytical technology ever developed in the life sciences area, the quantitative real-time polymerase chain reaction (qPCR). Forty scientific talks from invited international scientists and diagnostic companies in the qPCR field who will show their latest research findings and newest technologies. Focus of the event will be the ongoing evolution of qPCR.
Suggested reading: PCR Books
Vienna, Austria Further information
Forum to exchange ideas, share experiences, and discuss the future of the perhaps most powerful analytical technology ever developed in the life sciences area, the quantitative real-time polymerase chain reaction (qPCR). Forty scientific talks from invited international scientists and diagnostic companies in the qPCR field who will show their latest research findings and newest technologies. Focus of the event will be the ongoing evolution of qPCR.
Suggested reading: PCR Books
Labels: Conference
PCR methods for the detection of Borrelia burgdorferi
PCR is now a standard approach for direct detection of B. burgdorferi infection in tick vectors, host reservoirs, experimentally infected laboratory animals, and clinical specimens. PCR methods that are available for detection of B. burgdorferi DNA in various specimens include standard PCR, nested PCR, competitive PCR and real-time PCR (Wang et al., 2010).
The efficiency of a PCR assay is determined by several factors. Among these, the selection of an appropriate gene target and primer set for PCR amplification are the most important. In general, a PCR primer set yielding an amplicon of 100-300 bp is recommended, as it has high amplification efficiency under standard PCR conditions and can attentuate the effects of DNA fragmentation during sample processing.
Although PCR assays targeting numerous B. burgdorferi genes have been employed extensively in research settings, only a few of these genes have been widely utilized for PCR-based detection of B. burgdorferi s. l. These include the chromosomally encoded genes rrs, flaB, recA and p66, and the plasmid-encoded gene ospA (Wang et al., 2010).
Given that the number of spirochetes in infected tissues or body fluids of patients is generally very low, appropriate procedures for sample collection, transport and DNA preparation from clinical samples are critical for yielding reliable and consistent PCR results. Sensitivity of PCR assays may be reduced by degradation of the B. burgdorferi DNA during sample transport, storage, and processing. If the tissue is kept in BSK medium for over 24 h, some spirochetes may have migrated from the tissue to the culture medium. In this case, DNA prepared from both the tissue and the medium will increase the positivity rate of the PCR assay (Wang et al., 2010).
Clinical specimens collected from patients should be subjected to DNA extraction and PCR analysis shortly after collection; if this is not possible, specimens should be kept frozen until analysis. Studies on infected animal tissues suggest that PCR with DNA prepared from fresh frozen tissues has higher yields than that from paraffin-embedded, formalin-fixed tissues. As host DNA can interfere with PCR detection of B. burgdorferi in clinical and tick samples, an optimized DNA extraction procedure is essential to yield reliable PCR results for certain samples. Also, PCR inhibitors may be present in various biological materials (blood, urine, synovial fluid and cerebrospinal fluid) obtained from patients.
Real-time PCR has been used for detection, quantification and genotyping of pathogenic B. burgdorferi species in ticks and patients (Wang et al., 2010). The sensitivity of DNA extraction using manual or automated nucleic acid preparation systems is comparable for a variety of specimen types. Only a few genes have been used as targets in real-time PCR (e.g., ospA, rrs, hbb, recA) and these assays usually have used smaller fragments (100-200 bp) than those targeted in standard PCR tests. The sensitivity, specificity and reproducibility of real-time PCR and real-time quantitative PCR (qPCR) can be affected by a number of factors, including the properties of selected primers or probe, the quality of template DNA, the detection format (SYBR Green I-based or hybridization probe-based), and the amplification conditions (Wang et al., 2010).
References:
Wang, G., Aguero-Rosenfeld, M.E., Wormser, G.P. and Schwartz, I. (2010) Detection of Borrelia burgdorferi. In: Borrelia: Molecular Biology, Host Interaction and Pathogenesis (D. Scott Samuels and Justin D. Radolf, eds.). Caister Academic Press, Norfolk, UK.
Mackay, I.M. (2007) Real-Time PCR in Microbiology: From Diagnosis to Characterization. Caister Academic Press, Norfolk, UK.
The efficiency of a PCR assay is determined by several factors. Among these, the selection of an appropriate gene target and primer set for PCR amplification are the most important. In general, a PCR primer set yielding an amplicon of 100-300 bp is recommended, as it has high amplification efficiency under standard PCR conditions and can attentuate the effects of DNA fragmentation during sample processing.
Although PCR assays targeting numerous B. burgdorferi genes have been employed extensively in research settings, only a few of these genes have been widely utilized for PCR-based detection of B. burgdorferi s. l. These include the chromosomally encoded genes rrs, flaB, recA and p66, and the plasmid-encoded gene ospA (Wang et al., 2010).
Given that the number of spirochetes in infected tissues or body fluids of patients is generally very low, appropriate procedures for sample collection, transport and DNA preparation from clinical samples are critical for yielding reliable and consistent PCR results. Sensitivity of PCR assays may be reduced by degradation of the B. burgdorferi DNA during sample transport, storage, and processing. If the tissue is kept in BSK medium for over 24 h, some spirochetes may have migrated from the tissue to the culture medium. In this case, DNA prepared from both the tissue and the medium will increase the positivity rate of the PCR assay (Wang et al., 2010).
Clinical specimens collected from patients should be subjected to DNA extraction and PCR analysis shortly after collection; if this is not possible, specimens should be kept frozen until analysis. Studies on infected animal tissues suggest that PCR with DNA prepared from fresh frozen tissues has higher yields than that from paraffin-embedded, formalin-fixed tissues. As host DNA can interfere with PCR detection of B. burgdorferi in clinical and tick samples, an optimized DNA extraction procedure is essential to yield reliable PCR results for certain samples. Also, PCR inhibitors may be present in various biological materials (blood, urine, synovial fluid and cerebrospinal fluid) obtained from patients.
Real-time PCR has been used for detection, quantification and genotyping of pathogenic B. burgdorferi species in ticks and patients (Wang et al., 2010). The sensitivity of DNA extraction using manual or automated nucleic acid preparation systems is comparable for a variety of specimen types. Only a few genes have been used as targets in real-time PCR (e.g., ospA, rrs, hbb, recA) and these assays usually have used smaller fragments (100-200 bp) than those targeted in standard PCR tests. The sensitivity, specificity and reproducibility of real-time PCR and real-time quantitative PCR (qPCR) can be affected by a number of factors, including the properties of selected primers or probe, the quality of template DNA, the detection format (SYBR Green I-based or hybridization probe-based), and the amplification conditions (Wang et al., 2010).
References:
Wang, G., Aguero-Rosenfeld, M.E., Wormser, G.P. and Schwartz, I. (2010) Detection of Borrelia burgdorferi. In: Borrelia: Molecular Biology, Host Interaction and Pathogenesis (D. Scott Samuels and Justin D. Radolf, eds.). Caister Academic Press, Norfolk, UK.
Mackay, I.M. (2007) Real-Time PCR in Microbiology: From Diagnosis to Characterization. Caister Academic Press, Norfolk, UK.
Labels: B. burgdorferi, Borrelia, Lyme disease
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
Real-Time PCR book review
I am pleased to provide the following excerpt from a recently published book review:
Real-Time PCR: Current Technology and Applications
"... written by international authors expert in specific technical principles and applications ... a useful compendium of basic and advanced applications for laboratory scientists. It is an ideal introductory textbook and will serve as a practical handbook in laboratories where the technology is employed." from Christopher J. McIver, Microbiology Department, Prince of Wales Hospital, New South Wales, Australia writing in Australian J. Med. Sci. 2009. 30(2): 59-60
Real-Time PCR: Current Technology and Applications
"... written by international authors expert in specific technical principles and applications ... a useful compendium of basic and advanced applications for laboratory scientists. It is an ideal introductory textbook and will serve as a practical handbook in laboratories where the technology is employed." from Christopher J. McIver, Microbiology Department, Prince of Wales Hospital, New South Wales, Australia writing in Australian J. Med. Sci. 2009. 30(2): 59-60
 | Edited by: Julie Logan, Kirstin Edwards and Nick Saunders Published: 2009 ISBN: 978-1-904455-39-4 Price: GB £150 or US $310 A comprehensive guide to the most up-to-date real-time PCR technology and applications. The latest PCR platforms, fluorescent chemistries, validation software, data analysis, internal and external controls,clinical diagnostics, biodefense, RNA expression studies, validation of array data, mutation detection, food authenticity and legislation, NASBA, molecular halotyping. read more ... |
Labels: book review
PCR Troubleshooting
An expert guide to PCR troubleshooting. The online guide covers problems with the template DNA, inadequate dNTPs, primer concentration, Taq concentration, Mg concentration, and KCl concentration. Most PCR problems are caused by one or other of these variables.
Find out the theory and possible solutions to these problems at PCR troubleshooting.
Find out the theory and possible solutions to these problems at PCR troubleshooting.
Labels: trouble shooting, Troubleshooting
Cloning PCR Fragments
Zuo and Rabie describe a new one-step "Quick Assemble" method of precisely and simultaneously joining multiple DNA fragments followed by intramolecular ligation. This technique of plasmid assembly and circularization can be used to construct new plasmids with any promoter, resistance gene marker, restriction site, or tag.
The new 2-day PCR-based method can be used to generate both circularized and linear final assembled products. The method makes the synthesis and assembly of large molecules in a quick and efficient way, while circumventing the use of DNA ligases. The ligase-free method also offers an additional improvement over the combination of long PCR and overlap extension PCR, and is expected to facilitate various kinds of complex genetic engineering projects that require precise in-frame assembly of multiple fragments, such as multiple site-direct mutagenesis and whole-DNA library gene shuffling.
from Zuo and Rabie in Curr. Issues Mol. Biol. 12: 11-16
Further reading: Assembly of DNA fragments into circular constructs
The new 2-day PCR-based method can be used to generate both circularized and linear final assembled products. The method makes the synthesis and assembly of large molecules in a quick and efficient way, while circumventing the use of DNA ligases. The ligase-free method also offers an additional improvement over the combination of long PCR and overlap extension PCR, and is expected to facilitate various kinds of complex genetic engineering projects that require precise in-frame assembly of multiple fragments, such as multiple site-direct mutagenesis and whole-DNA library gene shuffling.
from Zuo and Rabie in Curr. Issues Mol. Biol. 12: 11-16
Further reading: Assembly of DNA fragments into circular constructs
Labels: cloning, PCR cloning
PCR Chip
The polymerase chain reaction (PCR) has been widely used for amplification of DNA sequences. The PCR technique can be coupled with a variety of detection assays for the identification of a wide range of DNA targets. Reducing PCR to the microchip level is of interest for portable detection technologies and high-throughput, massively parallel analytical systems. The basic process of creating a miniaturized, microfluidic PCR chip and a simple method for thermally cycling this microchip during PCR amplification have been described (Herold and Rasooly, 2009. Lab-on-a-Chip Technology. ISBN: 978-1-904455-47-9) These methods can be broadly applied to a variety of microchip architectures, materials, and downstream analytical methods.
Labels: PCR chips, PCR on a chip
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