scholarly journals qPCR detection versus microscopy observations for assessing presence–absence of Didymosphenia geminata in Quebec rivers (Canada)

2015 ◽  
Vol 52 (2) ◽  
pp. 109-120
Author(s):  
Sandra Kim Tiam ◽  
Vincent Laderriere ◽  
Carole-Anne Gillis ◽  
Claude Fortin ◽  
Isabelle Lavoie
Keyword(s):  
Polymerase Chain Reaction ◽  
Dna Extraction ◽  
Large Scale ◽  
Dna Isolation ◽  
Quantitative Polymerase Chain Reaction ◽  
Cost Effective ◽  
Chain Reaction ◽  
Didymosphenia Geminata ◽  
Large Scale Assessment ◽  
Polymerase Chain

Microscopic versus qPCR (quantitative polymerase chain reaction) analyses were compared for the detection of Didymosphenia geminata in biofilms and water filtrates from seven Gaspésie rivers (Canada). For the qPCR approach, two DNA extraction kits (QIAamp DNA Micro Kit, Qiagen and PowerSoil DNA Isolation Kit, Mo Bio Laboratories) and two pairs of primers were considered. The pair of primers D602F/D753Rext did not amplify D. geminata DNA whereas the pair of primers D602F/D753R was specific for D. geminata. Presence-absence diagnosis based on qPCR and microscopic analyses were consistent: D. geminata was detected in six of the seven rivers, both in the biofilm and filtrate samples. However, technical replications were needed at certain sites to observe the presence of D. geminata cells by microscopy. This underscores the necessity of replicate analyses, which is cost-effective to achieve when using qPCR due to the capacity to process tens of samples in a single PCR run in the context of a large scale assessment.

scholarly journals Method Comparison of DNA Isolation and Quantification for Fish and Seafood Authenticity Determination

2018 ◽  
Vol 13 (3) ◽  
pp. 115
Author(s):  
Dwiyitno Dwiyitno ◽  
Stefan Hoffman ◽  
Koen Parmentier ◽  
Chris Van Keer
Keyword(s):  
Polymerase Chain Reaction ◽  
Dna Extraction ◽  
Dna Isolation ◽  
Blue Mussel ◽  
Pcr Amplification ◽  
Amplification Efficiency ◽  
Chain Reaction ◽  
Seafood Products ◽  
Polymerase Chain ◽  
Commercial Kit

Fish and seafood products has been commonly targeted for fraudulent activities. For that reason, authentication of fish and seafood products is important to protect consumers from fraudulent and adulteration practices, as well as to implement traceability regulation. From the viewpoint of food safety, authenticity is beneficial to protect public from serious food poisoning incidents, such as due to ingestion of toxic species. Since DNA based identification depends on the nucleic acid polymerase chain reaction (PCR), the quantity and quality/purity of DNA will contribute significantly to the species authentication. In the present study, different DNA extraction and purification methods (3 classical methods and one commercial kit) were compared to produce the better isolated DNA for PCR amplification. Additionally, different methods for the estimation of DNA concentration and purity which is essential for PCR amplification efficiency were also evaluated. The result showed that classical DNA extraction methods (based on TNES-Urea) yielded a higher amount of DNA (11.30-323.60 ng/g tissue) in comparison to commercial kit/Wizard Promega (5.70-83.45 ng/g tissue). Based on the purity of DNA extract (A260/280), classical DNA extraction method produced relatively similar on DNA quality to the commercial kit (1.79-2.12). Interestingly, all classical methods produced DNA with A260/280 ratio of more than 2.00 on the blue mussel, in contrast with commercial kit. The commercial kit also produced better quality of DNA compared to the classical methods, showing the higher efficiency in PCR amplification. NanoDrop is promising as cheap, robust and safe UV-spectrophotometer method for DNA quantification, as well as the purity evaluation.Keywords: seafood authenticity, DNA isolation, polymerase chain reaction, NanoDrop, Picogreen

scholarly journals Novel Dual Labeled Fluorescence Probe Based Assay to Measure the Telomere Length

2018 ◽  
Author(s):  
Itty Sethi ◽  
Gh. Rasool Bhat ◽  
Rakesh Kumar ◽  
Ekta Rai ◽  
Swarkar Sharma
Keyword(s):  
Polymerase Chain Reaction ◽  
Telomere Length ◽  
Fluorescence Probe ◽  
Quantitative Polymerase Chain Reaction ◽  
Cost Effective ◽  
Sybr Green ◽  
Telomeric Dna ◽  
Chain Reaction ◽  
Specific Pcr ◽  
Polymerase Chain

ABSTRACTTelomeres are highly repetitive regions capping the chromosomes and composed of multiple units of hexa-nucleotides, TTAGGG, making their quantification difficult. Most of the methods developed to estimate telomeres are extensively cumbersome and expensive. The quantitative polymerase chain reaction (qPCR) based assay is relatively easy and cheaper method that applies SyBr Green dye chemistry to measure telomere length. As SyBr Green dye fluoresces after intercalation into the dsDNA, lack of differentiation between specific PCR target products and unspecific products is a limitation and it affects accuracy in quantitation of telomeres. To overcome the limitations of SyBr Green, we developed a dual labeled fluorescence probe based quantitative polymerase chain reaction (qPCR) to measure the telomere length. This robust, accurate and highly reproducible (R2=0.96) proprietary method (patent pending), yet cost effective and easy, utilizes a probe that targets specifically the telomeric DNA.

Comparative assessment of DNA extraction procedures for Ascaris spp. eggs

2019 ◽  
Vol 94 ◽  
Author(s):  
I.D. Amoah ◽  
G. Singh ◽  
K. Troell ◽  
P. Reddy ◽  
T.A. Stenström ◽  
...  
Keyword(s):  
Dna Extraction ◽  
Molecular Detection ◽  
Dna Isolation ◽  
Quantitative Polymerase Chain Reaction ◽  
High Yield ◽  
Chain Reaction ◽  
Removal Technology ◽  
Polymerase Chain ◽  
Microbial Dna ◽  
Accurate Quantification

Abstract A central and critical step in the molecular detection of soil-transmitted helminths from environmental sources is the extraction of DNA from the eggs. In this study, we investigated the yield of DNA extracted from known quantities (500, 100, 50, 20, 10 and 5) of Ascaris suum eggs, as well as directly from wastewater and sludge samples containing Ascaris spp. eggs, using six commercial DNA extraction kits. The amount of DNA extracted was quantified with NanoDrop, Qubit and Ct values from quantitative polymerase chain reaction (qPCR) assay using CFX96 Touch™ real-time PCR equipment. The PowerLyzer Ultraclean Microbial DNA isolation kit and PowerSoil DNA isolation kit gave the highest yield of DNA based on the NanoDrop, Qubit and Ct values. However, the qPCR results indicate that in some of the kits, PCR inhibitors may have been carried over to the PCR reaction. DNA extraction kits that incorporate a bead-beating step as well as other mechanical eggshell disruption steps were superior in extracting DNA from Ascaris spp. eggs. Additionally, for the accurate quantification of extracted DNA, the use of Ct values from qPCR and Qubit readings gives better results compared to the NanoDrop readings. For efficient downstream applications, the use of DNA extraction kits with superior inhibitor removal technology is essential, in addition to a high yield of DNA.

scholarly journals Species identification of felis and vulpes by a novel loop-mediated isothermal amplification assay in fur products

2019 ◽  
Vol 14 ◽  
pp. 155892501882072
Author(s):  
Shunji Yu ◽  
Wenjia Gu ◽  
Yi Yu ◽  
Qinfeng Qu ◽  
Yi’nan Zhang
Keyword(s):  
Polymerase Chain Reaction ◽  
Species Identification ◽  
Quantitative Polymerase Chain Reaction ◽  
Cost Effective ◽  
Isothermal Amplification ◽  
Chain Reaction ◽  
Species Identity ◽  
Loop Mediated Isothermal Amplification ◽  
Amplification Method ◽  
Polymerase Chain

In the chaotic market of fur goods, genetic distinction is increasingly important for identifying species. A vast diversity of species identification methods has been proposed, while little is developed, particularly those easy, fast, and cost-effective ones. In this study, a simple and reliable novel loop-mediated isothermal amplification method for identifying cytochrome c oxidase I of felis and vulpes was established. It saves laborious post–polymerase chain reaction procedures and shortens the time for high-fidelity gene amplification. The sensitivity of this method for felis and vulpes identification, which is well matched to quantitative polymerase chain reaction, could be 10 or 1.0 pg, respectively. Predominantly, the sensitivity of loop-mediated isothermal amplification is more tolerant to those polymerase chain reaction inhibitors such as pigments, dyes, or other fur ingredients, compared to quantitative polymerase chain reaction. Even without costly specialized equipment, a water bath is sufficient for genetic distinction. Our approach is a new technique with broad application perspective, such as on-site species identity tests, commercial fraud, and wildlife crimes.

AN IMPROVED METHOD FOR INHIBITOR FREE NUCLEIC ACIDS FROM POLYPHENOL RICH LEAVES OF MUSA SPP

2017 ◽  
Vol 23 (1) ◽  
Author(s):  
N.NANDHA KUMAR ◽  
K. SOURIANATHA SUNDARAM ◽  
D. SUDHAKAR ◽  
K.K. KUMAR
Keyword(s):  
Polymerase Chain Reaction ◽  
Quantitative Polymerase Chain Reaction ◽  
Proteinase K ◽  
Chain Reaction ◽  
Banana Plant ◽  
High Molecular Weight Dna ◽  
Musa Spp ◽  
Leaf Tissues ◽  
Polymerase Chain

Excessive presence of polysaccharides, polyphenol and secondary metabolites in banana plant affects the quality of DNA and it leads to difficult in isolating good quality of DNA. An optimized modified CTAB protocol for the isolation of high quality and quantity of DNA obtained from banana leaf tissues has been developed. In this protocol a slight increased salt (NaCl) concentration (2.0M) was used in the extraction buffer. Polyvinylpyrrolidone (PVP) and Octanol were used for the removal of polyphenols and polymerase chain reaction (PCR) inhibitors. Proteins like various enzymes were degraded by Proteinase K and removed by centrifugation from plant extract during the isolation process resulting in pure genomic DNA, ready to use in downstream applications including PCR, quantitative polymerase chain reaction (qPCR), ligation, restriction and sequencing. This protocol yielded a high molecular weight DNA isolated from polyphenols rich leaves of Musa spp which was free from contamination and colour. The average yields of total DNA from leaf ranged from 917.4 to 1860.9 ng/ìL. This modified CTAB protocol reported here is less time consuming 4-5h, reproducible and can be used for a broad spectrum of plant species which have polyphenol and polysaccharide compounds.

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