scholarly journals Exploiting the Enzymatic Recognition of an Unnatural Base Pair to Develop a Universal Genetic Analysis System

2003 ◽  
Vol 49 (3) ◽  
pp. 407-414 ◽  
Author(s):  
Michael J Moser ◽  
David J Marshall ◽  
Jennifer K Grenier ◽  
Collin D Kieffer ◽  
Anthony A Killeen ◽  
...  
Keyword(s):  
Energy Transfer ◽  
Real Time ◽  
Real Time Pcr ◽  
Dynamic Range ◽  
Solid Phase ◽  
Rapid Development ◽  
Factor V Leiden ◽  
Dna Chip ◽  
Factor V ◽  
Probe System

Abstract Background: With the invention of the DNA chip, genome-wide analysis is now a reality. Unfortunately, solid-phase detection systems such as the DNA chip suffer from a narrow range in quantification and sensitivity. Today the best methodology for sensitive, wide dynamic range quantification and genotyping of nucleic acids is real-time PCR. However, multiplexed real-time PCR technologies require complicated and costly design and manufacturing of separate detection probes for each new target. Methods: We developed a novel real-time PCR technology that uses universal energy transfer probes constructed from An Expanded Genetic Information System (AEGIS) for both quantification and genotyping analyses. Results: RNA quantification by reverse transcription-PCR was linear over four orders of magnitude for the simultaneous analysis of β-actin messenger RNA and 18S ribosomal RNA. A single trial validation study of 176 previously genotyped clinical specimens was performed by endpoint analysis for factor V Leiden and prothrombin 20210A mutation detection. There was concordance for 173 samples between the genotyping results from Invader® tests and the AEGIS universal energy transfer probe system for both factor V Leiden and prothrombin G20210A. Two prothrombin and one factor V sample gave indeterminate results (no calls). Conclusion: The AEGIS universal probe system allows for rapid development of PCR assays for nucleic acid quantification and genotyping.

ARMS TaqMan real‐time PCR for genotyping factor V Leiden mutation in Han Chinese

2020 ◽  
Vol 41 (23) ◽  
pp. 2015-2020
Author(s):  
Pan Yu ◽  
Yibei Dai ◽  
Jiantao Dong ◽  
Luyan Zhang ◽  
Ying Ping ◽  
...  
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Factor V Leiden ◽  
Han Chinese ◽  
Factor V ◽  
Factor V Leiden Mutation

scholarly journals Rapid Single-Tube Genotyping of the Factor V Leiden and Prothrombin Mutations by Real-Time PCR Using Dual-Color Detection

2000 ◽  
Vol 46 (8) ◽  
pp. 1191-1195 ◽  
Author(s):  
Frank A J T M van den Bergh ◽  
Arletta M van Oeveren-Dybicz ◽  
Michelle A M Bon
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Factor V Leiden ◽  
Factor V ◽  
Single Tube ◽  
Dual Color ◽  
Color Detection

scholarly journals Use of Ethidium Monoazide and PCR in Combination for Quantification of Viable and Dead Cells in Complex Samples

2005 ◽  
Vol 71 (2) ◽  
pp. 1018-1024 ◽  
Author(s):  
Knut Rudi ◽  
Birgitte Moen ◽  
Signe Marie Drømtorp ◽  
Askild L. Holck
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Dynamic Range ◽  
Covalent Binding ◽  
Biological Research ◽  
Complex Samples ◽  
Ethidium Monoazide ◽  
Food Borne ◽  
Pcr Method ◽  
Membrane Cell

ABSTRACT The distinction between viable and dead cells is a major issue in many aspects of biological research. The current technologies for determining viable versus dead cells cannot readily be used for quantitative differentiation of specific cells in mixed populations. This is a serious limitation. We have solved this problem by developing a new concept with the viable/dead stain ethidium monoazide (EMA) in combination with real-time PCR (EMA-PCR). A dynamic range of approximately 4 log10 was obtained for the EMA-PCR viable/dead assay. Viable/dead differentiation is obtained by covalent binding of EMA to DNA in dead cells by photoactivation. EMA penetrates only dead cells with compromised membrane/cell wall systems. DNA covalently bound to EMA cannot be PCR amplified. Thus, only DNA from viable cells can be detected. We evaluated EMA-PCR with the major food-borne bacterium Campylobacter jejuni as an example. Traditional diagnosis of this bacterium is very difficult due to its specific growth requirements and because it may enter a state where it is viable but not cultivable. The conditions analyzed included detection in mixed and natural samples, survival in food, and survival after disinfection or antibiotic treatment. We obtained reliable viable/dead quantifications for all conditions tested. Comparison with standard fluorescence-based viable/dead techniques showed that the EMA-PCR has a broader dynamic range and enables quantification in mixed and complex samples. In conclusion, EMA-PCR offers a novel real-time PCR method for quantitative distinction between viable and dead cells with potentially very wide application.

scholarly journals Development of Quantitative Real-Time PCR Assays for Detection and Quantification of Surrogate Biological Warfare Agents in Building Debris and Leachate

2007 ◽  
Vol 73 (20) ◽  
pp. 6557-6565 ◽  
Author(s):  
Pascal E. Saikaly ◽  
Morton A. Barlaz ◽  
Francis L. de los Reyes
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Genomic Dna ◽  
Dynamic Range ◽  
Limit Of Detection ◽  
Fate And Transport ◽  
Biological Warfare ◽  
Quantitative Real Time Pcr ◽  
Pcr Assays ◽  
Detection And Quantification

ABSTRACT Evaluation of the fate and transport of biological warfare (BW) agents in landfills requires the development of specific and sensitive detection assays. The objective of the current study was to develop and validate SYBR green quantitative real-time PCR (Q-PCR) assays for the specific detection and quantification of surrogate BW agents in synthetic building debris (SBD) and leachate. Bacillus atrophaeus (vegetative cells and spores) and Serratia marcescens were used as surrogates for Bacillus anthracis (anthrax) and Yersinia pestis (plague), respectively. The targets for SYBR green Q-PCR assays were the 16S-23S rRNA intergenic transcribed spacer (ITS) region and recA gene for B. atrophaeus and the gyrB, wzm, and recA genes for S. marcescens. All assays showed high specificity when tested against 5 ng of closely related Bacillus and Serratia nontarget DNA from 21 organisms. Several spore lysis methods that include a combination of one or more of freeze-thaw cycles, chemical lysis, hot detergent treatment, bead beat homogenization, and sonication were evaluated. All methods tested showed similar threshold cycle values. The limit of detection of the developed Q-PCR assays was determined using DNA extracted from a pure bacterial culture and DNA extracted from sterile water, leachate, and SBD samples spiked with increasing quantities of surrogates. The limit of detection for B. atrophaeus genomic DNA using the ITS and B. atrophaeus recA Q-PCR assays was 7.5 fg per PCR. The limits of detection of S. marcescens genomic DNA using the gyrB, wzm, and S. marcescens recA Q-PCR assays were 7.5 fg, 75 fg, and 7.5 fg per PCR, respectively. Quantification of B. atrophaeus vegetative cells and spores was linear (R 2 > 0.98) over a 7-log-unit dynamic range down to 101 B. atrophaeus cells or spores. Quantification of S. marcescens (R 2 > 0.98) was linear over a 6-log-unit dynamic range down to 102 S. marcescens cells. The developed Q-PCR assays are highly specific and sensitive and can be used for monitoring the fate and transport of the BW surrogates B. atrophaeus and S. marcescens in building debris and leachate.

scholarly journals Interlaboratory Comparison of Six Real-Time PCR Assays for Detection of Bovine Leukemia Virus Proviral DNA

2018 ◽  
Vol 56 (7) ◽  
pp. e00304-18 ◽  
Author(s):  
J. P. Jaworski ◽  
A. Pluta ◽  
M. Rola-Łuszczak ◽  
S. L. McGowan ◽  
C. Finnegan ◽  
...  
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Bovine Leukemia Virus ◽  
Dynamic Range ◽  
Leukemia Virus ◽  
Large Variability ◽  
Proviral Dna ◽  
International Panel ◽  
Pcr Assays ◽  
Dna Copy Numbers

ABSTRACTQuantitative real-time PCR (qPCR) is increasingly being used for the detection of bovine leukemia virus (BLV) proviral DNA. Nevertheless, quality control for the validation and standardization of such tests is currently lacking. Therefore, the present study was initiated by three Office International des Epizooties (OIE) reference laboratories and three collaborating laboratories to measure the interlaboratory variability of six already developed and available BLV qPCR assays. For that purpose, an international panel of 58 DNA samples reflecting the dynamic range of the majority of the assays was distributed to six testing centers. Based on qualitative results, the overall agreement among all six laboratories was moderate. However, significant variability in the measurement of the BLV proviral DNA copy number was observed among different laboratories. Quantitative PCR assays, even when performed by experienced staff, can yield large variability in BLV proviral DNA copy numbers without harmonization. Further standardization of different factors (i.e., utilization of unified protocols and unique calibrators) should increase interlaboratory agreement.

scholarly journals Erratum: Corrigendum: Solid-phase proximity ligation assays for individual or parallel protein analyses with readout via real-time PCR or sequencing

2015 ◽  
Vol 10 (4) ◽  
pp. 644-644 ◽  
Author(s):  
Rachel Yuan Nong ◽  
Di Wu ◽  
Junhong Yan ◽  
Maria Hammond ◽  
Gucci Jijuan Gu ◽  
...  
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Solid Phase ◽  
Proximity Ligation
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