Automated centrifugal microfluidic system for the preparation of adaptor-ligated sequencing libraries.

The intensive workload associated with the preparation of high-quality DNA libraries remains a key obstacle toward widespread deployment of sequencing technologies in remote and resource-limited areas. We describe the development of single-use microfluidic devices driven by an advanced pneumatic centrifugal microfluidic platform, the PowerBlade, to automate the preparation of Illumina-compatible libraries based on adaptor ligation methodology. The developed on-chip workflow includes enzymatic DNA fragmentation coupled to end-repair, adaptor ligation, first DNA cleanup, PCR amplification, and second DNA cleanup. This complex workflow was successfully integrated into simple thermoplastic microfluidic devices that are amenable to mass production with injection molding. The system was validated by preparing, on chip, libraries from a mixture of genomic DNA extracted from three common foodborne pathogens (Listeria monocytogenes, Escherichia coli and Salmonella enterica serovar Typhimurium) and comparing them with libraries made via a manual procedure. The two types of libraries were found to exhibit similar quality control metrics (including genome coverage, assembly, and relative abundances) and led to nearly uniform coverage independent of GC content. This microfluidic technology offers a time-saving and cost-effective alternative to manual procedures and robotic-based automation, making it suitable for deployment in remote environments where technical expertise and resources might be scarce. Specifically, it facilitates field practices that involve mid- to low-throughput sequencing, such as tasks related to foodborne pathogen detection, characterization, and microbial profiling.

Extraction of nucleic acids from blood: unveiling the potential of active pneumatic pumping in centrifugal microfluidics for integration and automation of sample preparation processes.

This paper describes the development of an on-chip nucleic acid (NA) extraction assay from whole blood using a centrifugal microfluidic platform that allows for pneumatic actuation of liquids during rotation. The combination of pneumatic and centrifugal forces makes it possible to perform fluidic operations without the need for integrating active control elements on the microfluidic cartridge. The cartridge is fabricated from thermoplastic polymers (e.g., Zeonor 1060R) and features a simple design that is compatible with injection molding. In addition, the cartridge is interfaced with two external vials for off-chip storage of the blood sample and retrieval of the eluted NA solution, respectively. On-chip capture of NAs is performed using an embedded solid-phase extraction matrix composed of commercial glass microfiber filters (Whatman GF/D and GF/F). The yield of the automated, on-chip extraction protocol, determined by measuring absorbance at 260 nm, is comparable to some of the best manually operated kits (e.g., Qiagen QIAamp DNA Mini Kit) while providing low assay-to-assay variability due to the high level of control provided by the platform for each processing step. The A260/A280 and A260/A230 ratios of the absorbance spectra also reveal that protein contamination of the sample is negligible. The capability of the pneumatic platform to circulate air flux through the microfluidic conduit was used to dry leftover ethanol residues retained in the capture matrix during washing. This method, applied in combination with localized heating, proved effective for reducing ethanol contamination in eluted samples from ∼12% to 1% (v/v).

Preharvest ultraviolet-C irradiation: Influence on physicochemical parameters associated with strawberry fruit quality.

Postharvest ultraviolet-C (UV-C) hormesis has been shown effective for the treatment of the edible part of several horticultural crops such as strawberry fruit; however, there is a lack of information on its potential preharvest impact. In the present study three strawberry cultivars (Fragaria × ananassa Duch. 'Albion', 'Charlotte' and 'Seascape') were exposed to UV-C during two growth seasons for a period of three weeks. Treatment begins when the first flowers were wide open and fruits at commercial maturity were harvested within one week after UV treatment. The physicochemical quality parameters of the fruits harvested from the treated plants were compared to those of the fruits of the untreated control plants. Preharvest UV-C treatment tended to increase fruit firmness in all cultivars with significant differences declared only for 'Albion' and 'Seascape' in season 2. Fruits from treated plants were generally redder but a significant difference was observed only for cultivar 'Charlotte' in the second growing season. Other color attributes were not affected by UV-C, neither were organic acids, simple sugars, soluble solids content (SSC), titratable acidity (TA) and pH, although in most cases slight decreases were noticed. Cultivar and growing season were the factors that mostly influenced on the parameters under study. The present study show that cumulative preharvest UV-C treatment of 3.6 kJ m-2 did not adversely affected important strawberry quality parameters.

Effects of preharvest ultraviolet-C irradiation on fruit phytochemical profiles and antioxidant capacity in three strawberry (Fragaria × ananassa Duch.) cultivars.

BACKGROUND: Ultraviolet-C (UV-C) has proven effective in extending shelf-life, reducing disease incidence and increasing the levels of health-promoting compounds in several crops. While most studies were conducted at the postharvest stage, our study examined the effect of preharvest UV-C application in three strawberry cultivars (Fragaria × ananassa Duch. 'Albion', 'Charlotte' and 'Seascape'). UV-C treatment was applied from the onset of flowering until the fruits reached commercial maturity on plants grown for two consecutive seasons under greenhouse conditions. The phytochemical profiles and antioxidant capacity of the fruits were assessed at harvest. RESULTS: The ellagic acid and kaempferol-3-glucuronide contents were significantly increased only in fruits of the cultivar 'Albion' collected from UV-C-treated plants in season 1. UV-C did not consistently affect the other phenolic compounds that were measured. Based on the results of the ferric-reducing antioxidant power, oxygen radical absorbance capacity and total phenolic content assays, the antioxidant capacity of the three strawberry cultivars was not affected by UV-C. Season and cultivar had a decisive impact on these parameters. CONCLUSION: The effect of preharvest UV-C on the levels of bioactive compounds in strawberry fruits appears to be cultivar- dependent, with season or growing conditions having a significant impact.

First Report of Ceratocystis fimbriata Infecting Balsam Poplar (Populus balsamifera).

This is the first report of Ceratocystis fimbriata Ellis & Halst. (Ophiostomatales) (anamorph: Chalara sp. (Corda) Rabenh.) associated with symptoms of perennial canker on small branches and wilt of leaves and twigs on balsam poplar (Populus balsamifera L.). The fungus is known to occur on trembling aspen (P. tremuloides Michx.) (1) throughout the mountainous West and extends eastward to Quebec and Pennsylvania (2).These tree species are common in the boreal and Rocky Mountain areas of North America. In April 1999, the disease was observed on cuttings (15 × 1 cm) collected from the terminal branches of young balsam poplar trees (h = 4.5 m) near Baie Comeau located 350 km northeast of Quebec City (48°61', 68°21'), Quebec, Canada. Apparently healthy cuttings were potted in a soil mix (peat moss and vermiculite, 3:1 vol:vol), grown in a greenhouse at 22 ± 1°C and watered twice a week. About 2 weeks after bud break, a number of cuttings were symptomatic, showing necrotic bark lesions and cankers, discoloration and wilting of leaves, and reddish brown exudate surrounding leaf petioles. During the following two weeks, 22% of the cuttings died (212 out of 958). The fungus was isolated from diseased cuttings on 2% MEA (malt extract agar), from the necrotic bark around active cankers (2 × 1 cm), from brown to black streaks found in vascular tissue, and from the ascomata found on the canker margin and inconspicuous necrotic roots. From necrotic petioles, one other opportunistic fungus, Aureobasidium pullulans (de Bary) G. Arnaud, was occasionally isolated. The inoculum for infection of the balsam poplar may have originated from neighboring trembling aspen trees. We believe that C. fimbriata could have a negative impact on reforestation efforts unless phytosanitary inspections are made of the planting stock. References: (1) D. F. Farr et al. 1989. Fungi on Plants and Plant Products in the United States. The American Phytopathological Society, St. Paul, MN. (2) W. A. Sinclair et al. 1993. Diseases of Trees and Shrubs. 3rd ed. Cornell University Press, Ithaca, NY.