Quantitative Polymerase Chain Reaction Based on a Dual-Analyte Chemiluminescence Hybridization Assay for Target DNA and Internal Standard

1998 ◽  
Vol 70 (19) ◽  
pp. 4120-4125 ◽  
Author(s):  
Monique Verhaegen ◽  
Theodore K. Christopoulos
Keyword(s):  
Polymerase Chain Reaction ◽  
Quantitative Polymerase Chain Reaction ◽  
Internal Standard ◽  
Hybridization Assay ◽  
Chain Reaction ◽  
Target Dna ◽  
Polymerase Chain

Development of AFLP-derived, functionally specific markers for environmental persistence studies of fungal strains

2006 ◽  
Vol 52 (5) ◽  
pp. 451-461 ◽  
Author(s):  
S S Hynes ◽  
O Chaudhry ◽  
M A Providenti ◽  
M L Smith
Keyword(s):  
Polymerase Chain Reaction ◽  
Real Time ◽  
Genetically Modified Organisms ◽  
Quantitative Polymerase Chain Reaction ◽  
Pcr Primers ◽  
Chain Reaction ◽  
Environmental Persistence ◽  
Fungal Strains ◽  
Target Dna ◽  
Polymerase Chain

The ability to rapidly identify and quantify a microbial strain in a complex environmental sample has widespread applications in ecology, epidemiology, and industry. In this study, we describe a rapid method to obtain functionally specific genetic markers that can be used in conjunction with standard or real-time polymerase chain reaction (PCR) to determine the abundance of target fungal strains in selected environmental samples. The method involves sequencing of randomly cloned AFLP (amplified fragment length polymorphism) products from the target organism and the design of PCR primers internal to the AFLP fragments. The strain-specific markers were used to determine the fate of three industrially relevant fungi, Aspergillus niger, Aspergillus oryzae, and Chaetomium globosum, during a 4 month soil microcosm experiment. The persistence of each of the three fungal strains inoculated separately into intact soil microcosms was determined by PCR analyses of DNA directly extracted from soil. Presence and absence data based on standard PCR and quantification of the target DNA by real-time PCR showed that all three strains declined after inoculation (~14-, 32-, and 4-fold for A. niger, A. oryzae, and C. globosum, respectively) but remained detectable at the end of the experiment, suggesting that these strains would survive for extended periods if released into nature.Key words: Canada domestic substances list (DSL), Canadian Environmental Protection Act (CEPA), genetically modified organisms (GMO), quantitative polymerase chain reaction (qPCR).

scholarly journals Evaluation of Resistance to Rhabdocline Needlecast in Douglas Fir Variety Shuswap, with Quantitative Polymerase Chain Reaction

2010 ◽  
Vol 100 (4) ◽  
pp. 337-344 ◽  
Author(s):  
M. Catal ◽  
G. C. Adams ◽  
D. W. Fulbright
Keyword(s):  
Polymerase Chain Reaction ◽  
Quantitative Polymerase Chain Reaction ◽  
Douglas Fir ◽  
Chain Reaction ◽  
Disease Symptoms ◽  
Seed Sources ◽  
Release Times ◽  
Spore Release ◽  
Target Dna ◽  
Polymerase Chain

A quantitative polymerase chain reaction assay was developed that could detect DNA of Rhabdocline pseudotsugae and R. oblonga among DNA of Douglas fir needles to a limit as low as three copies of target DNA. Differential infection rates of two varieties (seed sources) of Douglas fir interplanted in a field were studied in relation to staggered bud breaks. Infection of Douglas fir var. San Isabel corresponded to ascospore release times for Rhabdocline spp., whereas infection of var. Shuswap Lake did not occur throughout the spore release period during 2 years of study, despite abundant inoculum and adequate moisture during bud break. Rhabdocline spp. DNA was never detected in Shuswap Lake and disease symptoms were not observed in any year. We provide evidence that Shuswap Lake is resistant and probably immune to Rhabdocline spp. infection and Rhabdocline needlecast under Michigan conditions.

scholarly journals Enumeration of viable and non-viable larvated Ascaris eggs with quantitative PCR

2012 ◽  
Vol 10 (4) ◽  
pp. 594-604 ◽  
Author(s):  
Maria Raynal ◽  
Eric N. Villegas ◽  
Kara L. Nelson
Keyword(s):  
Polymerase Chain Reaction ◽  
Detection Limit ◽  
Quantitative Polymerase Chain Reaction ◽  
High Heat ◽  
Chain Reaction ◽  
Direct Microscopy ◽  
Development Stages ◽  
Target Dna ◽  
Qpcr Method ◽  
Polymerase Chain

The goal of this study was to further develop an incubation-quantitative polymerase chain reaction (qPCR) method for quantifying viable Ascaris eggs by characterizing the detection limit and number of template copies per egg, determining the specificity of the method, and testing the method with viable and inactivated larvated eggs. The number of template copies per cell was determined by amplifying DNA from known numbers of eggs at different development stages; the value was estimated to be 32 copies. The specificity of the method was tested against a panel of bacteria, fungi, protozoa and helminths, and no amplification was found with non-target DNA. Finally, fully larvated eggs were inactivated by four different treatments: 254 nm ultraviolet light, 2,000 ppm NH3-N at pH 9, moderate heat (48 °C) and high heat (70 °C). Concentrations of treated eggs were measured by direct microscopy and incubation-qPCR. The qPCR signal decreased following all four treatments, and was in general agreement with the decrease in viable eggs determined by microscopy. The incubation-qPCR method for enumerating viable Ascaris eggs is a promising approach that can produce results faster than direct microscopy, and may have benefits for applications such as assessing biosolids.

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