The European Union (EU) imposes strict regulations on the presence of genetically modified (GM) material in food and feed, requiring thorough testing of samples for various GM lines. Although traditional quantitative real-time PCR (qPCR) methods are sensitive and robust, they are not cost-effective for managing large numbers of GM events due to their limited multiplexing capabilities. Conversely, digital PCR (dPCR) is capable of robust quantitative multiplexing in addition to other benefits such as absolute quantification and better tolerance of PCR inhibitors. In this context, we present a protocol for multiplex quantification of 19 GM soybean lines using dPCR as an improvement over the currently used simplex qPCR approach. This method enables simple and robust quantification of common GM soybean lines with a relatively low number of reactions.

Abstract Digital PCR (dPCR) is a powerful technology for absolute quantification of nucleic acids, valued for its accuracy, sensitivity, and repeatability. Yet, the commercialization of different instruments with proprietary software has introduced challenges to data analysis, interoperability, and comparability. Therefore, we present the Digital PCR Data Essentials Standard (DDES) – a lightweight, human- and machine-readable, and cross-platform data standard developed in collaboration with the dPCR community. The standard consists of three file types designed to enable both manual inspection and automated analysis: (i) a main file summarizing experiment and reaction-level (meta-)data; (ii) an assay file describing targets and detection chemistry, and (iii) intensity files capturing partition-level raw fluorescence data per reaction. DDES supports a wide range of current dPCR applications, including singleplex and multiplex assays, endpoint and real-time readouts, and will be curated to implement future dPCR developments. By harmonizing the data structure, DDES lays out the foundation for FAIR dPCR data practices and supports improved software compatibility, collaborative and reproducible research, and future dPCR data repositories.

Recombinant adeno-associated virus (rAAV) vectors exhibit complex heterogeneity in capsid content. Although a growing array of analytical platforms can interrogate different capsid attributes, no single method provides a comprehensive or unequivocal assessment. Critically, current approaches often fail to evaluate genome integrity, an emerging important quality attribute. Incomplete genomes can contribute to viral genome titers without conferring therapeutic benefit, while simultaneously increasing immunogenic burden and risk of adverse events. Discordant results from orthogonal assays further complicate interpretation and cross-laboratory comparisons. Here, we highlight the limitations of existing characterization strategies and advocate for robust analytical frameworks that directly assess genome completeness. We propose best-practice guidelines, including improved genome integrity assays and harmonized reporting, to better link capsid content analytics with potency and patient safety in gene therapy development.

Plant genetic engineering represents an important aspect of modern agriculture, and new genetically modified (GM) crop varieties are entering the market on a regular basis. This necessitates the development of high throughput multi-target analytical methods to detect and quantify their presence for regulatory compliance. In this study, we present a multiplex dPCR method for discriminative quantification of 19 GM soybean events and the lectin (Le1) endogene on a nanowell plate-based all-in-one dPCR system. The method consists of four 5-plex assays, taking advantage of the platform’s multiple fluorescence detection channels. The assays complied with the minimum performance requirements in terms of specificity, trueness, precision, sensitivity and dynamic range, making them suitable for use in routine detection and quantification of GM crops. This method represents the most comprehensive multi-target GM soybean quantification approach to date without the need for prior screening and features a simplified workflow, making it suitable for widespread adoption. Our study sets a precedent for rapid and straightforward development of multiplex dPCR GM crop quantification assays to address the evolving demands of regulatory monitoring.

Innovative technological solutions are needed for water decontamination to combat the diverse pollutants present in water systems, as no single optimal decontamination technique is appropriate for all circumstances. Vacuum-ultraviolet (V-UV) radiation is a source of energetic photons that break molecular bonds, producing a plethora of chemically reactive agents, most notably OH center dot radicals, which can cause the degradation of harmful pollutants. Low-pressure gaseous plasma is a good source of V-UV radiation; however, its application to liquid water poses challenges. We constructed an inductively coupled radiofrequency plasma to produce high-intensity V-UV radiation, which was applied to contaminated water via a V-UV-transparent window. Plasma was sustained in hydrogen, as it produces the highest V-UV intensity among all gases at selected discharge parameters. Bacteriophage MS2 was used as an indicator of microbial decontamination efficiency. Reactive oxygen and nitrogen species were measured at various treatment setups to quantify their effect on MS2 inactivation and elucidate the primary inactivation factors. At optimal conditions, the concentration of active virus dropped by 9 log10 PFU/mL in 60 s. The optimal experimental setup was then used to treat bacteria E. coli, S. aureus, antibiotic tetracycline, and synthetic dye methylene blue as representatives of other types of pollutants, all of which were effectively removed/degraded within 10 min of treatment. A comparison of energy efficiency (EEO) to other disinfection setups was made for bacteriophage inactivation. With a low EEO value, we showcase the potential of this technique for further work in this field.

Cell therapy product (CTP) developers face the significant challenge of developing appropriate potency tests for their CTPs. A review of the known potency tests used for the 31 United States Food and Drug Administration-approved CTPs (US FDA) can guide developers in designing effective potency tests for future CTPs. Data on these tests were primarily collected from publicly available regulatory documentation on the US FDA website (90%) as well as other sources (literature, company communications, etc.). Based on these data, an estimated 104 total potency tests have been used for the 31 CTPs. Of these, 33 are redacted (32%), leaving 71 non-redacted potency tests. On average, each CTP has 3.4 potency tests (standard deviation 2.0). The 71 non-redacted potency tests were categorized into 5 bins: "Viability and count" (37 tests, 52%), "Expression" (19 tests, 27%), "Bioassays" (7 tests, 7%), "Genetic modification" (6 tests, 9%) and "Histology" (2 tests, 3%). Measurements of gene or protein expression were used by 20 of the 31 CTPs (65%), and 19 CTPs (61%) used measurements of cell viability or cell count as a potency test. "Viability and count" and "Expression" are the two tests that have most often been used together for the same product, occurring for 16 CTPs (52%). It is unclear if bioassays are commonly used as potency tests since only 7 of 31 CTPs (23%) reported bioassays as potency tests. However, due to redactions, as many 24 (77%) CTPs could potentially have a bioassay as a potency test. Additionally, 26 of the 31 CTPs (84%) cite physicochemical assays (non-bioassays) as a potency test. This analysis of potency tests for approved CTPs provides valuable insights for developing potency tests for new CTPs.

Water disinfection is a critical treatment step for removing harmful organisms from contaminated water sources. Vacuum ultra-violet (V-UV) radiation, generated by a low-pressure gaseous plasma discharge, consists of photons with high enough energy to break molecular bonds. In this work, we constructed and characterized a low-pressure capacitively coupled gaseous plasma and used it as a V-UV radiation source to inactivate MS2 bacteriophage, a surrogate for human enteric viruses. The treatment system allows for variation of gas composition inside the sample chamber and the modulation of V-UV radiation intensity. Both were used to separate the actual germicidal contribution of V-UV radiation from producing germicidal species by using virus inactivation to determine efficiency. OH* radical production was determined through the terephthalic acid chemical probe, which showed that when air was present in the sample chamber, it resulted in the highest OH* production and the best inactivation of MS2. Furthermore, we showcase that the OH* production rate was wavelength dependent and that ozone, generated by plasma treatment in the gas phase, leads to hydroxy terephthalic acid degradation, allowing us to determine better the actual OH* production rate with different treatment regimes. Lastly, by adding an OH* scavenger to the liquid, we were able to elucidate it as the primary inactivation agent in this setup while also providing evidence of its production in the bulk liquid by the transport and subsequent decomposition of the longer-lasting ozone molecule. This study demonstrates, for the first time, the applicability of low-pressure plasma radiation as a water treatment method.

Pepino mosaic virus (PepMV) is a plant pathogen causing significant economic losses in tomato production. Sensitive, reliable, and robust detection methods are crucial for containing the spread of PepMV and reducing its damaging effects. Digital PCR (dPCR) presents several advantages to conventional real-time quantitative PCR (qPCR), including absolute quantification ability, robust quantitative multiplexing capabilities, and straightforward result analysis. Furthermore, dPCR is especially suitable for analysis of complex samples due to its remarkable tolerance to PCR inhibitors, which makes it a promising method for plant virus genotyping. In this chapter, we present two protocols for PepMV genotyping and quantification using one-step reverse transcription digital PCR (RT-dPCR). The first protocol outlines four simplex assays, while the second describes two duplex assays for precise and comprehensive genotyping of PepMV variants.

We investigated the spatial dynamics of potato (Solanum tuberosum) responses to herbivory and mechanical wounding. We first followed the spatiotemporal response of jasmonic acid (JA) signaling, known to be involved in the response. We generated two potato sensor lines: a JAZ degradation sensor and a downstream multicystatin (MC) transcriptional reporter. Both sensors revealed concentric, locally restricted responses on wounded leaves. Notably, JA-dependent gene expression was absent in cells immediately adjacent to the wound, whereas JAZ degradation spread continuously outward from the wound site. This pattern occurred after both herbivore attack and mechanical injury by the needle. To probe the mechanism, a salicylic acid (SA) reporter showed SA accumulation near the wound. Introducing the MC reporter into SA-depleted NahG plants produced a uniform spread of MC expression, confirming that SA attenuates the JA response in proximal cells. Together, these results show that a locally distinct, spatiotemporal SA-JA crosstalk shapes wound responses in potato, extending principles known from pathogen-plant interactions to herbivory and mechanical damage.

During the COVID-19 pandemic, face masks were the first line of defense against the spread of infection. However, infectious viruses may remain on medical textiles, potentially serving as an additional source of infection. Due to their chemical inertness, many textiles cannot be enhanced with antiviral functionalities. Through treatment with low-pressure gaseous plasma, we have activated the surface of a medical-grade meltblown, non-woven polypropylene textile so that it can absorb sodium dodecyl sulfate, an antimicrobial surfactant. Within two hours of contact time, the functionalized textile has been able to inactivate over 7 log(10) PFU mL(-1) of bacteriophage phi6, a surrogate of enveloped viruses such as SARS-CoV-2, and it has retained its antiviral properties for over 100 days. The functionalized material has not disrupted facial mask filtration efficiency or breathability. In addition, the in vitro biocompatibility testing in accordance with ISO 10993-5 for testing of medical devices has demonstrated that the selected formulation causes no adverse effects on the mouse fibroblast cell line L-929. With the treatment processes that have been completed within seconds, the method seems to have great potential to produce antiviral textiles against future outbreaks.

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)). Suboptimal particle separation within the fractions resulted in unexpectedly similar infectivity levels. No single technique could simultaneously provide comprehensive insights in the presence of both bioactive particles and contaminants. Notably, multiplex ddPCR revealed distinct vector genome fragmentation patterns, differing between ssAAV and scAAV. This highlights the urgent need for innovative analytical and production approaches to optimize AAV vector production and enhance therapeutic outcomes.

One of the most challenging aspects of developing advanced cell therapy products (CTPs) is defining the mechanism of action (MOA), potency and efficacy of the product. This perspective examines these concepts and presents helpful ways to think about them through the lens of metrology. A logical framework for thinking about MOA, potency and efficacy is presented that is consistent with the existing regulatory guidelines, but also accommodates what has been learned from the 27 US FDA-approved CTPs. Available information regarding MOA, potency and efficacy for the 27 FDA-approved CTPs is reviewed to provide background and perspective. Potency process and efficacy process charts are introduced to clarify and illustrate the relationships between six key concepts: MOA, potency, potency test, efficacy, efficacy endpoint and efficacy endpoint test. Careful consideration of the meaning of these terms makes it easier to discuss the challenges of correlating potency test results with clinical outcomes and to understand how the relationships between the concepts can be misunderstood during development and clinical trials. Examples of how a product can be “potent but not efficacious” or “not potent but efficacious” are presented. Two example applications of the framework compare how MOA is assessed in cell cultures, animal models and human clinical trials and reveals the challenge of establishing MOA in humans. Lastly, important considerations for the development of potency tests for a CTP are discussed. These perspectives can help product developers set appropriate expectations for understanding a product’s MOA and potency, avoid unrealistic assumptions and improve communication among team members during the development of CTPs.