Assessment of Primer/Template Mismatch Effects on Real-Time PCR Amplification of Target Taxa for GMO Quantification

2009 ◽  
Vol 57 (20) ◽  
pp. 9370-9377 ◽  
Author(s):  
Rim Ghedira ◽  
Nina Papazova ◽  
Marnik Vuylsteke ◽  
Tom Ruttink ◽  
Isabel Taverniers ◽  
...  
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Pcr Amplification ◽  
Gmo Quantification

Real-Time PCR Quantification of Vibrio parahaemolyticus in Oysters Using an Alternative Matrix

2004 ◽  
Vol 67 (11) ◽  
pp. 2424-2429 ◽  
Author(s):  
G. E. KAUFMAN ◽  
G. M. BLACKSTONE ◽  
M. C. L. VICKERY ◽  
A. K. BEJ ◽  
J. BOWERS ◽  
...  
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Vibrio Parahaemolyticus ◽  
Pcr Amplification ◽  
Oyster Tissue ◽  
Pcr Assay ◽  
Colony Hybridization ◽  
The Real ◽  
Mantle Fluid ◽  
June July

This study examined the relationship between levels of total Vibrio parahaemolyticus found in oyster tissues and mantle fluid with the goal of using mantle fluid as a template matrix in a new quantitative real-time PCR assay targeting the thermolabile hemolysin (tlh) gene for the enumeration of total V. parahaemolyticus in oysters. Oysters were collected near Mobile Bay, Ala., in June, July, and September and tested immediately after collection and storage at 26°C for 24 h. Initial experiments using DNA colony hybridization targeting tlh demonstrated that natural V. parahaemolyticus levels in the mantle fluid of individual oysters were strongly correlated (r = 0.85, P < 0.05) with the levels found in their tissues. When known quantities of cultured V. parahaemolyticus cells were added to real-time PCR reactions that contained mantle fluid and oyster tissue matrices separately pooled from multiple oysters, a strong linear correlation was observed between the real-time PCR cycle threshold and the log concentration of cells inoculated into each PCR reaction (mantle fluid: r = 0.98, P < 0.05; and oyster: r = 0.99, P < 0.05). However, the mantle fluid exhibited less inhibition of the PCR amplification than the homogenized oyster tissue. Analysis of natural V. parahaemolyticus populations in mantle fluids using both colony hybridization and real-time PCR demonstrated a significant (P < 0.05) but reduced correlation (r =−0.48) between the two methods. Reductions in the efficiency of the real-time PCR that resulted from low population densities of V. parahaemolyticus and PCR inhibitors present in the mantle fluid of some oysters (with significant oyster-to-oyster variation) contributed to the reduction in correlation between the methods that was observed when testing natural V. parahaemolyticus populations. The V. parahaemolyticus–specific real-time PCR assay used for this study could estimate elevated V. parahaemolyticus levels in oyster mantle fluid within 1 h from sampling time.

scholarly journals Detection and Enumeration of Aromatic Oxygenase Genes by Multiplex and Real-Time PCR

2003 ◽  
Vol 69 (6) ◽  
pp. 3350-3358 ◽  
Author(s):  
Brett R. Baldwin ◽  
Cindy H. Nakatsu ◽  
Loring Nies
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Simultaneous Detection ◽  
Gene Copy Number ◽  
Pcr Amplification ◽  
Gene Copy ◽  
Hydrocarbon Biodegradation ◽  
Polymerization Temperature ◽  
Primer Sets ◽  
The Impact

ABSTRACT Our abilities to detect and enumerate pollutant-biodegrading microorganisms in the environment are rapidly advancing with the development of molecular genetic techniques. Techniques based on multiplex and real-time PCR amplification of aromatic oxygenase genes were developed to detect and quantify aromatic catabolic pathways, respectively. PCR primer sets were identified for the large subunits of aromatic oxygenases from alignments of known gene sequences and tested with genetically well-characterized strains. In all, primer sets which allowed amplification of naphthalene dioxygenase, biphenyl dioxygenase, toluene dioxygenase, xylene monooxygenase, phenol monooxygenase, and ring-hydroxylating toluene monooxygenase genes were identified. For each primer set, the length of the observed amplification product matched the length predicted from published sequences, and specificity was confirmed by hybridization. Primer sets were grouped according to the annealing temperature for multiplex PCR permitting simultaneous detection of various genotypes responsible for aromatic hydrocarbon biodegradation. Real-time PCR using SYBR green I was employed with the individual primer sets to determine the gene copy number. Optimum polymerization temperatures for real-time PCR were determined on the basis of the observed melting temperatures of the desired products. When a polymerization temperature of 4 to 5�C below the melting temperature was used, background fluorescence signals were greatly reduced, allowing detection limits of 2 � 102 copies per reaction mixture. Improved in situ microbial characterization will provide more accurate assessment of pollutant biodegradation, enhance studies of the ecology of contaminated sites, and facilitate assessment of the impact of remediation technologies on indigenous microbial populations.

scholarly journals Whole blood Nested PCR and Real-time PCR amplification ofTalaromyces marneffeispecific DNA for diagnosis

2015 ◽  
Vol 54 (2) ◽  
pp. 162-168 ◽  
Author(s):  
Sha Lu ◽  
Xiqing Li ◽  
Richard Calderone ◽  
Jing Zhang ◽  
Jianchi Ma ◽  
...  
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Whole Blood ◽  
Nested Pcr ◽  
Pcr Amplification

scholarly journals The sensitivity of real-time PCR amplification targeting invasive Salmonellaserovars in biological specimens

2010 ◽  
Vol 10 (1) ◽  
Author(s):  
Tran Vu Thieu Nga ◽  
Abhilasha Karkey ◽  
Sabina Dongol ◽  
Hang Nguyen Thuy ◽  
Sarah Dunstan ◽  
...  
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Pcr Amplification

scholarly journals In-House Validation and Comparison of Two Wheat (Triticum aestivum) Taxon-Specific Real-Time PCR Methods for GMO Quantification Supported by Droplet Digital PCR

2017 ◽  
Vol 11 (5) ◽  
pp. 1281-1290 ◽  
Author(s):  
Annalisa Paternò ◽  
Daniela Verginelli ◽  
Pamela Bonini ◽  
Marisa Misto ◽  
Cinzia Quarchioni ◽  
...  
Keyword(s):  
Triticum Aestivum ◽  
Real Time ◽  
Real Time Pcr ◽  
Digital Pcr ◽  
Droplet Digital Pcr ◽  
Gmo Quantification

scholarly journals Endpoint PCR Detection of Sars-CoV-2 RNA

2020 ◽  
Author(s):  
Samuel Elliot Moses ◽  
Claire Warren ◽  
Phil Robinson ◽  
Jon Curtis ◽  
Steve Asquith ◽  
...  
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Large Scale ◽  
Pcr Amplification ◽  
Positive Sample ◽  
Pcr Detection ◽  
Rate Limiting Step ◽  
Copy Numbers ◽  
Qualitative Detection ◽  
Large Scale Testing

Quantitative real-time PCR methods have been used to perform approximately 278 million tests for COVID-19 up to mid-July 2020. Real-time PCR involves a rate limiting step where the samples are measured in situ during each PCR amplification cycle. This creates a bottleneck limiting scalability and as a consequence reducing access to inexpensive reliable testing at national and international scales. We investigated endpoint PCR for the qualitative detection of SARS-CoV-2 sequences on synthetic RNA standards and hospital patient samples. The endpoint PCR detection limit is constrained only by the stochastics of low copy numbers and reliably detected single copies of synthetic RNA standards. On a set of 30 patient samples, endpoint PCR found one additional positive sample and was able to confirm an indeterminate sample as negative. These results were found using 4 μl reagent and 1 μl of sample representing an 80% reduction relative to the NHS protocol (20 μl reagent and 5 μl sample). These results indicate that endpoint PCR should be the method of choice for large scale testing programmes. Based on the experience from ultra-high throughput genotyping efforts a single workflow using 384-well plates has similar PCR capacity (250 Million) to that required for all testing done worldwide during the first 7 month of the pandemic.

scholarly journals Real-Time Polymerase Chain Reaction-Based Approach for Quantification of the pat Gene in the T25 Zea mays Event

2004 ◽  
Vol 87 (6) ◽  
pp. 1342-1355 ◽  
Author(s):  
Florian Weighardt ◽  
Cristina Barbati ◽  
Claudia Paoletti ◽  
Maddalena Querci ◽  
Simon Kay ◽  
...  
Keyword(s):  
Polymerase Chain Reaction ◽  
Zea Mays ◽  
Real Time ◽  
Real Time Pcr ◽  
Reference Genes ◽  
Genetically Modified ◽  
Mean Value ◽  
Chain Reaction ◽  
Polymerase Chain ◽  
Gmo Quantification

Abstract In Europe, a growing interest for reliable techniques for the quantification of genetically modified component(s) of food matrixes is arising from the need to comply with the European legislative framework on novel food products. Real-time polymerase chain reaction (PCR) is currently the most powerful technique for the quantification of specific nucleic acid sequences. Several real-time PCR methodologies based on different molecular principles have been developed for this purpose. The most frequently used approach in the field of genetically modified organism (GMO) quantification in food or feed samples is based on the 5′-3′-exonuclease activity of Taq DNA polymerase on specific degradation probes (TaqMan® principle). A novel approach was developed for the establishment of a TaqMan quantification system assessing GMO contents around the 1% threshold stipulated under European Union (EU) legislation for the labeling of food products. The Zea mays T25eliteeventwas chosen as a model for the development of the novel GMO quantification approach. The most innovative aspect of the system is represented by the use of sequences cloned in plasmids as reference standards. In the field of GMO quantification, plasmids are an easy to use, cheap, and reliable alternative to Certified Reference Materials (CRMs), which are only available for a few of the GMOs authorized in Europe, have a relatively high production cost, and require further processing to be suitable for analysis. Strengths and weaknesses of the use of novel plasmid-based standards are addressed in detail. In addition, the quantification system was designed to avoid the use of a reference gene (e.g., a single copy, species-specific gene) as normalizer, i.e., to perform a GMO quantification based on an absolute instead of a relative measurement. In fact, experimental evidences show that the use of reference genes adds variability to the measurement system because a second independent real-time PCR-based measurement must be performed. Moreover, for some reference genes no sufficient information on copy number in and among genomes of different lines is available, making adequate quantification difficult. Once developed, the method was subsequently validated according to IUPAC and ISO 5725 guidelines. Thirteen laboratories from 8 EU countries participated in the trial. Eleven laboratories provided results complying with the predefined study requirements. Repeatability (RSDr)values ranged from 8.7 to 15.9%, with a mean value of 12%. Reproducibility (RSDR) values ranged from 16.3 to 25.5%, with a mean value of 21%. Following Codex Alimentarius Committee guidelines, both the limits of detection and quantitation were determined to be <0.1%.

scholarly journals Detection and quantification of minority HIV isolates harbouring the D30N mutation by real-time PCR amplification

2007 ◽  
Vol 60 (4) ◽  
pp. 881-884 ◽  
Author(s):  
Maria G. Detsika ◽  
Becky Chandler ◽  
Saye H. Khoo ◽  
Craig Winstanley ◽  
Pat Cane ◽  
...  
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Pcr Amplification ◽  
Detection And Quantification
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