scholarly journals Application of microfluidic high-throughput quantitative PCR systems for the monitoring and surveillance of aquatic species

2021 ◽  
Vol 4 ◽  
Author(s):  
Luca Mirimin ◽  
Dennis van der Pouw Kraan
Keyword(s):  
Quantitative Pcr ◽  
High Throughput ◽  
Environmental Samples ◽  
Rapid Screening ◽  
Sampling Effort ◽  
Sample Processing ◽  
Important Species ◽  
Short Period ◽  
Wide Range ◽  
Species Specific

Quantitative PCR (qPCR) has been increasingly used for the detection of target organisms in environmental DNA (eDNA) studies, and this is thanks to high sensitivity and ability to quantify DNA targets copy number. However, prior to their implementation, qPCR species-specific assays must be developed and validated and, when implemented, they are limited to relatively low number of targets that can be screened as a multiplex or in parallel. Thanks to recent technological advances, several qPCR-based platforms have become available to increase the throughput capability of qPCR systems as well as lowering time of execution and costs associated to sample processing. The present study describes the use of a microfluidic high-throughput qPCR/dPCR system (Biomark HD, Fluidigm) for the screening of species of ecologic and economic importance in bulk plankton environmental samples from marine coastal areas around the Irish coast. Data was generated using the configuration enabling the highest throughput (in terms of data points) of the system, including Integrated Fluidic Control (IFC) units capable of producing 96 x 96 sample/assay combinations in each run (9,216 individual qPCR tests in a single run). Thanks to such a capability, it was possible to execute the following three main development and implementation phases in a relatively short period of time (weeks as opposed to months/years): (i) development of a panel of species-specific assays targeting a range of crustacean and bivalve species; (ii) assessment of Limits of Detection (LOD), Limits of Quantification (LOQ), and enzymatic inhibition control for selected assays; and (iii) screening of environmental time-series samples (n = 242) obtained from a citizen-like sampling effort that involved a range of stakeholders and locations throughout the Irish marine coastal territory between 2019 and 2020. During the assay-development phase, the IFC system configuration (whereby all assays are tested in parallel against all samples) enabled the rapid screening of species-specific assays against a wide range of (genomic DNA of) non-target organisms, hence enabling for rapid specificity testing. LOD/LOQ experiments showed high levels of sensitivity and thanks to the large number of assays that could be accommodated in a single run, it was possible to include up to four distinct Internal Positive Controls (IPCs) at different concentrations in each run (hence controlling for potential inhibition at different target concentration levels). The inclusion of inhibitor-removal reagents in a pre-amplification step as well as the dilution factor of conducting reactions in small volumes (6.7 nL reaction volumes, hence comparable to a “digital PCR” effect) proved to be an effective strategy to reduce the effect of inhibitors in control experiments (humic acid and EDTA), as well as in actual environmental samples from a range of marine environments. Combining such a high-throughput screening platform with a nation-wide citizen science-like sampling programme enabled the acquisition of large datasets that are being used to monitor occurrence and (spawning) activity of important species that are of conservation concern, commercial value, or non-indigenous and invasive to Irish waters. The Biomark HD system provides a remarkable flexibility to modify existing and/or incorporate new assays because IFCs are customizable just prior to usage (i.e. are not pre-loaded or spotted with primers/probes), thus current work is focussing on increasing the number of species targeted in a single run, and (thanks to the quantitative nature of data) discriminating between different fractions of DNA in heterogeneous bulk samples (e.g. gametes and larvae vs intra- and extracellular eDNA). Thanks to low sample processing cost, assay flexibility and high-throughput capability, microfluidic qPCR platforms behold the potential to significantly advance biomonitoring of aquatic ecosystems.

Rapid Seafood Species Identification Using Chip-Based Capillary Electrophoresis and Protein Pattern Matching

2017 ◽  
Vol 100 (5) ◽  
pp. 1500-1510 ◽  
Author(s):  
Calvin C Walker ◽  
Cheryl L Lassitter ◽  
Shannara N Lynn ◽  
Courtney B Ford ◽  
Kevin R Rademacher ◽  
...  
Keyword(s):  
Protein Extraction ◽  
Protein Pattern ◽  
Screening Method ◽  
Fish Muscle ◽  
Protein Profiling ◽  
Rapid Screening ◽  
Important Species ◽  
Commercially Important ◽  
Species Specific ◽  
High Level

Abstract Authenticity is crucial to the seafood industry, as substitution and mislabeling have important economic, environmental, and food safety consequences. Toaddress this problem, protein profiling and softwarealgorithm techniques were developed to classify fishmuscle samples by species. The method uses water-based protein extraction, chip-based microfluidic electrophoresis (Agilent 2100 Bioanalyzer) for the analysis of high abundance fish muscle proteins, and a novel data analysis method for species-specific proteinpattern recognition. The method's performance in distinguishing commercially important fish from commonly reported substitutions was evaluated using sensitivity, specificity, and accuracy determinations with all three performance measures at >98% for commonsubstitutions. This study demonstrates that uncookedseafood products of commercially important species of catfish, snapper, and grouper can be rapidly distinguished from commonly substituted species with a high level of confidence. A tiered testing approach toseafood species verification by sequentially applying a rapid screening method and DNA testing is proposed to more effectively ensure accurate product labeling.

scholarly journals Accurate Quantification of Microorganisms in PCR-Inhibiting Environmental DNA Extracts by a Novel Internal Amplification Control Approach Using Biotrove OpenArrays

2009 ◽  
Vol 75 (22) ◽  
pp. 7253-7260 ◽  
Author(s):  
R. van Doorn ◽  
M. M. Klerks ◽  
M. P. E. van Gent-Pelzer ◽  
A. G. C. L. Speksnijder ◽  
G. A. Kowalchuk ◽  
...  
Keyword(s):  
High Throughput ◽  
Environmental Samples ◽  
False Negative ◽  
Environmental Dna ◽  
Target Range ◽  
Gene Target ◽  
Soil Dna ◽  
Control Approach ◽  
Wide Range ◽  
Accurate Quantification

ABSTRACT PCR-based detection assays are prone to inhibition by substances present in environmental samples, thereby potentially leading to inaccurate target quantification or false-negative results. Internal amplification controls (IACs) have been developed to help alleviate this problem but are generally applied in a single concentration, thereby yielding less-than-optimal results across the wide range of microbial gene target concentrations possible in environmental samples (J. Hoorfar, B. Malorny, A. Abdulmawjood, N. Cook, M. Wagner, and P. Fach, J. Clin. Microbiol. 42:1863-1868, 2004). Increasing the number of IACs for each quantitative PCR (qPCR) sample individually, however, typically reduces sensitivity and, more importantly, the reliability of quantification. Fortunately, current advances in high-throughput qPCR platforms offer the possibility of multiple reactions for a single sample simultaneously, thereby allowing the implementation of more than one IAC concentration per sample. Here, we describe the development of a novel IAC approach that is specifically designed for the state-of-the-art Biotrove OpenArray platform. Different IAC targets were applied at a range of concentrations, yielding a calibration IAC curve for each individual DNA sample. The developed IACs were optimized, tested, and validated by using more than 5,000 unique qPCR amplifications, allowing accurate quantification of microorganisms when applied to soil DNA extracts containing various levels of PCR-inhibiting compounds. To our knowledge, this is the first study using a suite of IACs at different target concentrations to monitor PCR inhibition across a wide target range, thereby allowing reliable and accurate quantification of microorganisms in PCR-inhibiting DNA extracts. The developed IAC is ideally suited for high-throughput screenings of, for example, ecological and agricultural samples on next-generation qPCR platforms.

Application of Self-Control in Droplet-Based Microfluidics

2008 ◽  
Author(s):  
Mark Kielpinski ◽  
Danie´ll Malsch ◽  
Nils Gleichmann ◽  
Gu¨nter Mayer ◽  
Thomas Henkel
Keyword(s):  
Time Delay ◽  
High Throughput ◽  
Self Control ◽  
External Control ◽  
Fusion Partner ◽  
Pressure Gradients ◽  
Sample Processing ◽  
Flow Rates ◽  
Wide Range

Droplet-based microfluidics provide a powerful platform for high-throughput operations applied in micro analytics, micro reaction technology and live sciences. Todays research interests focus on the development of highly integrated fluidic networks for sample processing according to a microchemical or microanalytical protocol. Normally, fluidic networks with integrated fluidic loops and bypasses are very complicated systems that require a huge effort for external control and integration of actor components. In contrast, in droplet-based microfluidics interface generated forces can be used to temporarily seal bypasses or to generate well defined pressure gradients at strictures. This potential can be used to implement self-control and self-synchronization at functional nodes in order to minimize the effort for external control and actors integration. Here we report on progress in development of functional nodes for self-synchronized 1:1 coalescence of two independently generated droplet sequences at a Y-shaped junction and on approaches for droplet aliquotation at a Y-shaped bifurcation. The droplet connector automatically balances the time delay between two droplets arriving at the junction. On this account, strictures are integrated into the Y-junction and an additional bypass connects the arriving channels. The first arriving droplet stops at the stricture until its fusion partner arrives. The droplet splitter performs an 1:1 aliqoutation of all elements of a droplet sequence. The main challenges are the balancing of pressure differences at the outlets and the correct aliquotation of droplets independent of their volume at a wide range of flow rates. The splitter design is based on the rule that forces required for splitting are always lower than the forces required for complete droplet inflow into only one of the outlet channels without splitting.

scholarly journals Parallel, targeted analysis of environmental samples via high‐throughput quantitative PCR

2020 ◽  
Vol 2 (4) ◽  
pp. 544-553 ◽  
Author(s):  
Taylor M. Wilcox ◽  
Kevin S. McKelvey ◽  
Michael K. Young ◽  
Cory Engkjer ◽  
Richard F. Lance ◽  
...  
Keyword(s):  
Quantitative Pcr ◽  
High Throughput ◽  
Environmental Samples ◽  
Targeted Analysis

scholarly journals The Band-Gap Studies of Short-Period CdO/MgO Superlattices

2021 ◽  
Vol 16 (1) ◽  
Author(s):  
Ewa Przeździecka ◽  
P. Strąk ◽  
A. Wierzbicka ◽  
A. Adhikari ◽  
A. Lysak ◽  
...  
Keyword(s):  
Band Gap ◽  
Layer Thickness ◽  
Energy Gap ◽  
Band Gaps ◽  
Short Period ◽  
Wide Range ◽  
Salt Structure ◽  
Short Period Superlattices ◽  
Direct Band ◽  
Sublayer Thickness

AbstractTrends in the behavior of band gaps in short-period superlattices (SLs) composed of CdO and MgO layers were analyzed experimentally and theoretically for several thicknesses of CdO sublayers. The optical properties of the SLs were investigated by means of transmittance measurements at room temperature in the wavelength range 200–700 nm. The direct band gap of {CdO/MgO} SLs were tuned from 2.6 to 6 eV by varying the thickness of CdO from 1 to 12 monolayers while maintaining the same MgO layer thickness of 4 monolayers. Obtained values of direct and indirect band gaps are higher than those theoretically calculated by an ab initio method, but follow the same trend. X-ray measurements confirmed the presence of a rock salt structure in the SLs. Two oriented structures (111 and 100) grown on c- and r-oriented sapphire substrates were obtained. The measured lattice parameters increase with CdO layer thickness, and the experimental data are in agreement with the calculated results. This new kind of SL structure may be suitable for use in visible, UV and deep UV optoelectronics, especially because the energy gap can be precisely controlled over a wide range by modulating the sublayer thickness in the superlattices.

Species-specific duplex PCR for the detection of Pratylenchus penetrans

Nematology ◽  
2009 ◽  
Vol 11 (6) ◽  
pp. 847-857 ◽  
Author(s):  
Lieven Waeyenberge ◽  
Nicole Viaene ◽  
Maurice Moens
Keyword(s):  
Internal Control ◽  
Dna Sequences ◽  
Rrna Gene ◽  
Duplex Pcr ◽  
Specific Primers ◽  
5.8S Rrna Gene ◽  
5.8S Rrna ◽  
Wide Range ◽  
Pcr Products ◽  
Species Specific

Abstract ITS1, the 5.8S rRNA gene and ITS2 of the rDNA region were sequenced from 20 different Pratylenchus species. Additionally, the same region was sequenced from seven populations of P. penetrans. After purifying, cloning and sequencing the PCR products, all sequences were aligned in order to find unique sites suitable for the design of species-specific primers for P. penetrans. Since ITS regions showed variability between and even within populations of P. penetrans, only three small DNA sequences were suitable for the construction of three potentially useful species-specific primers. New species-specific primers were paired with existing universal ITS primers and tested in all possible primer combinations. The best performing primer set, supplemented with a universal 28S rDNA primer set that served as an internal control, was tested in duplex PCR. The ideal annealing temperature, Mg2+ concentration and primer ratios were then determined for the most promising primer set. The optimised duplex PCR was subsequently tested on a wide range of different Pratylenchus spp. and 25 P. penetrans populations originating from all over the world. To test the sensitivity, the duplex PCR was conducted on DNA extracted from a single P. penetrans nematode mixed with varying amounts of nematodes belonging to another Pratylenchus species. Results showed that a reliable and sensitive P. penetrans species-specific duplex PCR was constructed.

scholarly journals Optimization of Plant Extract Purification Procedure for Rapid Screening Analysis of Sixteen Phenolics by Liquid Chromatography

Separations ◽  
2021 ◽  
Vol 8 (2) ◽  
pp. 13
Author(s):  
Petra Ranušová ◽  
Ildikó Matušíková ◽  
Peter Nemeček
Keyword(s):  
Liquid Chromatography ◽  
High Performance ◽  
Solid Phase ◽  
Low Cost ◽  
Sinapic Acid ◽  
Rapid Screening ◽  
Coumaric Acid ◽  
Laboratory Equipment ◽  
Wide Range ◽  
Methoxycinnamic Acid

A solid-phase extraction (SPE) procedure was developed for simultaneous monitoring of sixteen different phenolics of various polarity, quantified by high-performance liquid chromatography (HPLC). The procedure allowed screening the accumulation of intermediates in different metabolic pathways that play a crucial role in plant physiology and/or are beneficial for human health. Metabolites mostly involved in phenylpropanoid, shikimate, and polyketide pathways comprise chlorogenic acid, gentisic acid, vanillic acid, caffeic acid, protocatechuic acid, ferulic acid, rutin, quercetin, epicatechin, gallic acid, sinapic acid, p-coumaric acid, o-coumaric acid, vanillin; two rarely quantified metabolites, 2,5-dimethoxybenzoic acid and 4-methoxycinnamic acid, were included as well. The procedure offered low cost, good overall efficiency, and applicability in laboratories with standard laboratory equipment. SPE recoveries were up to 99.8% at various concentration levels. The method allowed for routine analysis of compounds with a wide range of polarity within a single run, while its applicability was demonstrated for various model plant species (tobacco, wheat, and soybean), as well as different tissue types (shoots and roots).

Carpatho-Ukraine’s defeat as a precursor of WWII

Rusin ◽  
2021 ◽  
pp. 201-222
Author(s):  
A.I. Kudriachenko ◽  
Keyword(s):  
International Relations ◽  
Third Reich ◽  
Historical Period ◽  
International Context ◽  
Short Period ◽  
Wide Range ◽  
Depth Study ◽  
Territorial Claims ◽  
The Third Reich

The paper analyzes the course of events and the international context of Сarpatho- Ukrainian state’s rise and defeat in terms of role and impact of the leading European actors at the end of the 1930’s. Based on an in-depth study of the wide range of literature, documents and relevant archives, the author highlights the role of Carpatho-Ukraine, which for a short period happened to be at the epicenter of the geopolitical interests of the states whose actions or inaction fueled the warmongers. The revival and strengthening of a number of European countries at that historical period deteriorized international relations. The erosion of the Versailles-Washington bases intensified the contradictions in the foreign policy between the victorious and vanquished states. The defeated countries reinforced their positions, since the victors, who were expanding their military might at the expense of Czechoslovak Republic, including Transcarpathia, were becoming more submissive in accommodating territorial claims. This situation largely updated the approaches to the Ukrainian question. The variability of the ways to solve it largely depended on the situational decisions of the Third Reich political leadership.

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