In-Depth Comparison of Adeno-Associated Virus Containing Fractions after CsCl Ultracentrifugation Gradient Separation.

Recombinant adeno-associated viruses (rAAVs) play a pivotal role in the treatment of genetic diseases. However, current production and purification processes yield AAV-based preparations that often contain unwanted empty, partially filled or damaged viral particles and impurities, including residual host cell DNA and proteins, plasmid DNA, and viral aggregates. To precisely understand the composition of AAV preparations, we systematically compared four different single-stranded AAV (ssAAV) and self-complementary (scAAV) fractions extracted from the CsCl ultracentrifugation gradient using established methods (transduction efficiency, analytical ultracentrifugation (AUC), quantitative and digital droplet PCR (qPCR and ddPCR), transmission electron microscopy (TEM) and enzyme-linked immunosorbent assay (ELISA)) alongside newer techniques (multiplex ddPCR, multi-angle light-scattering coupled to size-exclusion chromatography (SEC-MALS), multi-angle dynamic light scattering (MADLS), and high-throughput sequencing (HTS)).

Fast and Accurate Multiplex Identification and Quantification of Seven Genetically Modified Soybean Lines Using Six-Color Digital PCR.

The proliferation of genetically modified organisms (GMOs) presents challenges to GMO testing laboratories and policymakers. Traditional methods, like quantitative real-time PCR (qPCR), face limitations in quantifying the increasing number of GMOs in a single sample. Digital PCR (dPCR), specifically multiplexing, offers a solution by enabling simultaneous quantification of multiple GMO targets.

Cold plasma within a stable supercavitation bubble - A breakthrough technology for efficient inactivation of viruses in water.

Water scarcity, one of the most pressing challenges we face today, has developed for many reasons, including the increasing number of waterborne pollutants that affect the safety of the water environment. Waterborne human, animal and plant viruses represent huge health, environmental, and financial burden and thus it is important to efficiently inactivate them. Therefore, the main objective of this study was to construct a unique device combining plasma with supercavitation and to evaluate its efficiency for water decontamination with the emphasis on inactivation of viruses.

Commentary: Multiplex dPCR and SV-AUC are Promising Assays to Robustly Monitor the Critical Quality Attribute of AAV Drug Product Integrity.

Adeno-associated viral (AAV) capsids are an emerging vector technology for a number of novel gene therapy modalities (including transgene delivery and CRISPR gene editing). In this commentary, the proper approach to managing uncertainty (described by Rosenberg et al., 2012) when determining critical quality attributes is stated and applied retrospectively to Peginesatide and prospectively to AAV drug product integrity.

Digital PCR Applications in the SARS-CoV-2/COVID-19 Era: a Roadmap for Future Outbreaks.

The ongoing coronavirus disease 2019 (COVID-19) pandemic, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has led to a global public health disaster. The current gold standard for the diagnosis of infected patients is real-time reverse transcription-quantitative PCR (RT-qPCR). As effective as this method may be, it is subject to false-negative and -positive results, affecting its precision, especially for the detection of low viral loads in samples.

Feasibility of Droplet Digital PCR Analysis of Plasma Cell-Free DNA From Kidney Transplant Patients.

Increasing research demonstrates the potential of donor-derived cell-free DNA (dd-cfDNA) as a biomarker for monitoring the health of various solid organ transplants. Several methods have been proposed for cfDNA analysis, including real-time PCR, digital PCR, and next generation sequencing-based approaches. We sought to revise the droplet digital PCR (ddPCR)-based approach to quantify relative dd-cfDNA in plasma from kidney transplant (KTx) patients using a novel pilot set of assays targeting single nucleotide polymorphisms that have a very high potential to distinguish cfDNA from two individuals.

Inactivation of Pepper Mild Mottle Virus in Water by Cold Atmospheric Plasma.

Water scarcity is one of the greatest threats for human survival and quality of life, and this is increasingly contributing to the risk of human, animal and plant infections due to waterborne viruses. Viruses are transmitted through polluted water, where they can survive and cause infections even at low concentrations. Plant viruses from the genus are highly mechanically transmissible, and cause considerable damage to important crops, such as tomato.

Cold Plasma, a New Hope in the Field of Virus Inactivation.

Viruses can infect all cell-based organisms, from bacteria to humans, animals, and plants. They are responsible for numerous cases of hospitalization, many deaths, and widespread crop destruction, all of which result in an enormous medical, economical, and biological burden. Each of the currently used decontamination methods has important drawbacks.

One-Step Reverse-Transcription Digital PCR for Reliable Quantification of Different Pepino Mosaic Virus Genotypes.

In recent years, pepino mosaic virus (PepMV) has rapidly evolved from an emerging virus to an endemic pathogen, as it causes significant loses to tomato crops worldwide. At present, the main control strategy for prevention of PepMV disease in tomato production remains based on strict hygiene measures. To prevent damage caused by PepMV, cross-protection is used in some countries.

Accurate Quantification and Characterization of Adeno-Associated Viral Vectors.

One of the main challenges in the gene therapy viral vector development is to establish an optimized process for its large scale production. This requires optimization for upstream and downstream processes as well as methods that enable the step-by step analytical characterization of the virus, the results of which inform the iterative refinement of production for yield, purity and potency. The biggest problem here is a plethora of viral vector formulations, many of which interfere with analytical techniques.

Cold Atmospheric Plasma as a Novel Method for Inactivation of Potato Virus Y in Water Samples.

While one of the biggest problems we are facing today is water scarcity, enormous quantities of water are still being used in irrigation. If contaminated, this water can act as an effective pathway for the spread of disease-causing agents, like viruses. Here, we present a novel, environmentally friendly method known as cold atmospheric plasma for inactivation of viruses in water used in closed irrigation systems.

Validated reverse transcription droplet digital PCR serves as a higher order method for absolute quantification of Potato virus Y strains.

RNA viruses have a great potential for high genetic variability and rapid evolution that is generated by mutation and recombination under selection pressure. This is also the case of Potato virus Y (PVY), which comprises a high diversity of different recombinant and non-recombinant strains. Consequently, it is hard to develop reverse transcription real-time quantitative PCR (RT-qPCR) with the same amplification efficiencies for all PVY strains which would enable their equilibrate quantification; this is specially needed in mixed infections and other studies of pathogenesis.

Multiplex Droplet Digital PCR Protocols for Quantification of GM Maize Events.

The standard-curve based simplex quantitative polymerase chain reaction (qPCR) has been the gold standard for DNA target quantification for more than a decade. The large and growing number of individual analyses needed to test for genetically modified organisms (GMOs) is reducing the cost-effectiveness of qPCR. Droplet digital PCR (ddPCR) enables absolute quantification without standard curves, avoids the amplification efficiency bias observed with qPCR, allows more accurate estimations at low target copy numbers and, in combination with multiplexing, significantly improves cost efficiency.

Critical assessment of digital PCR for the detection and quantification of genetically modified organisms.

The number of genetically modified organisms (GMOs) on the market is steadily increasing. Because of regulation of cultivation and trade of GMOs in several countries, there is pressure for their accurate detection and quantification. Today, DNA-based approaches are more popular for this purpose than protein-based methods, and real-time quantitative PCR (qPCR) is still the gold standard in GMO analytics.

Salicylic Acid Perturbs sRNA-Gibberellin Regulatory Network in Immune Response of Potato to Infection.

is the most economically important potato viral pathogen. We aimed at unraveling the roles of small RNAs (sRNAs) in the complex immune signaling network controlling the establishment of tolerant response of potato cv. Désirée to the virus.

Inter-laboratory analysis of selected genetically modified plant reference materials with digital PCR.

Digital PCR (dPCR), as a new technology in the field of genetically modified (GM) organism (GMO) testing, enables determination of absolute target copy numbers. The purpose of our study was to test the transferability of methods designed for quantitative PCR (qPCR) to dPCR and to carry out an inter-laboratory comparison of the performance of two different dPCR platforms when determining the absolute GM copy numbers and GM copy number ratio in reference materials certified for GM content in mass fraction. Overall results in terms of measured GM% were within acceptable variation limits for both tested dPCR systems.

Droplet volume variability as a critical factor for accuracy of absolute quantification using droplet digital PCR.

Accurate and precise nucleic-acid quantification is crucial for clinical and diagnostic decisions, as overestimation or underestimation can lead to misguided treatment of a disease or incorrect labelling of the products. Digital PCR is one of the best tools for absolute nucleic-acid copy-number determination. However, digital PCR needs to be well characterised in terms of accuracy and sources of uncertainty.

Development and inter-laboratory assessment of droplet digital PCR assays for multiplex quantification of 15 genetically modified soybean lines.

Quantification of genetically modified organisms (GMOs) in food and feed products is often required for their labelling or for tolerance thresholds. Standard-curve-based simplex quantitative polymerase chain reaction (qPCR) is the prevailing technology, which is often combined with screening analysis. With the rapidly growing number of GMOs on the world market, qPCR analysis becomes laborious and expensive.

Multiplex quantification of four DNA targets in one reaction with Bio-Rad droplet digital PCR system for GMO detection.

The advantages of the digital PCR technology are already well documented until now. One way to achieve better cost efficiency of the technique is to use it in a multiplexing strategy. Droplet digital PCR platforms, which include two fluorescence filters, support at least duplex reactions and with some developments and optimization higher multiplexing is possible.

Solanum venturii, a suitable model system for virus-induced gene silencing studies in potato reveals StMKK6 as an important player in plant immunity.

Background: Virus-induced gene silencing (VIGS) is an optimal tool for functional analysis of genes in plants, as the viral vector spreads throughout the plant and causes reduced expression of selected gene over the whole plant. Potato (Solanum tuberosum) is one of the most important food crops, therefore studies performing functional analysis of its genes are very important. However, the majority of potato cultivars used in laboratory experimental setups are not well amenable to available VIGS systems, thus other model plants from Solanaceae family are used (usually Nicotiana benthamiana).

Multiplex quantification of 12 European Union authorized genetically modified maize lines with droplet digital polymerase chain reaction.

Presence of genetically modified organisms (GMO) in food and feed products is regulated in many countries. The European Union (EU) has implemented a threshold for labeling of products containing more than 0.9% of authorized GMOs per ingredient.