Two-phase wash to solve the ubiquitous contaminant-carryover problem in commercial nucleic-acid extraction kits

The success of fundamental and applied nucleic acid (NA) research depends on NA purity, but obtaining pure NAs from raw, unprocessed samples is challenging. Purification using solid-phase NA extractions utilizes sequential additions of lysis and wash buffers followed by elution. The resulting eluent contains NAs and carryover of extraction buffers. Typically, these inhibitory buffers are heavily diluted by the reaction mix (e.g., 10x dilution is 1 µL eluent in 9 µL reaction mix), but in applications requiring high sensitivity (e.g., single-cell sequencing, pathogen diagnostics) it is desirable to use low dilutions (e.g., 2x) to maximize NA concentration. Here, we demonstrate pervasive carryover of inhibitory buffers into eluent when several commercial sample-preparation kits are used following manufacturer protocols. At low eluent dilution (2–2.5x) we observed significant reaction inhibition of polymerase chain reaction (PCR), loop-mediated isothermal amplification (LAMP), and reverse transcription (RT). We developed a two-phase wash (TPW) method by adding a wash buffer with low water solubility prior to the elution step. The TPW reduces carryover of extraction buffers, phase-separates from the eluent, and does not reduce NA yield (measured by digital PCR). We validated the TPW for silica columns and magnetic beads by demonstrating significant improvements in performance and reproducibility of qPCR, LAMP, and RT reactions

The use of digital microfluidics for a silicon photonic sensors platform

status: publishe

Economic viability of phytoremediation of a cadmium contaminated agricultural area using energy maize: part II: economics of anaerobic digestion of metal contaminated maize in Belgium

This paper deals with remediation of the Campine soil, an agricultural area diffusely contaminated with metals where most farmers raise dairy cattle and grow fodder maize. In a previous study, we calculated the effect of switching from fodder to energy maize on the farmer's income. Selling this energy maize as feedstock for anaerobic digestion to produce renewable energy could lead to a significant increase in his income. This paper explores the economic opportunities for the farmer of digesting the harvested contaminated biomass himself, by performing a Net Present Value (NPV) analysis on the digestion activity and by calculating the probability of a positive NPV of income resulting from the digestion installation. We investigate the trade off between the maximum price for energy maize that can be paid by the digestion activity and the minimum price that the farming activity needs to compensate for covering its production costs. Integrating the previous study in the current analysis results in an increase of total extra income for the farmer (i.e., from both growing energy maize and performing digestion)

Extraction and Identification of Clavine and Lysergic Acid Alkaloids from Morning Glories

Author Institution: Department of Zoology and Microbiology, Ohio UniversityWITTERS, WELDON L. Extraction and identification of clavine and lysergic acid alkaloids from morning glories. Ohio J. Sci. 75(4): 198, 1975

Dilogic-2: A Sparse Data Scan Readout Processor for the HMPID Detector of ALICE

The processing of analog information is always spoiled by additional DC level and noise given by the sensors or their additional readout electronics. The Dilogic-2 ASICcircuit has been developed in a 0.7um n-well CMOS technologyto process the data given by Analog to Digital Converters, in order to eliminate the empty channels, to subtract the base line (pedestal) and to locally store the true analog information.(Abstract only available, full text willfollow

Evaluating 3D printing to solve the sample-to-device interface for LRS and POC diagnostics: example of an interlock meter-mix device for metering and lysing clinical urine samples

This paper evaluates the potential of 3D printing, a semi-automated additive prototyping technology, as a means to design and prototype a sample-to-device interface, amenable to diagnostics in limited-resource settings, where speed, accuracy and user-friendly design are critical components. As a test case, we built and validated an interlock meter-mix device for accurately metering and lysing human urine samples for use in downstream nucleic acid amplification. Two plungers and a multivalve generated and controlled fluid flow through the device and demonstrate the utility of 3D printing to create leak-free seals. Device operation consists of three simple steps that must be performed sequentially, eliminating manual pipetting and vortexing to provide rapid (5 to 10 s) and accurate metering and mixing. Bretherton's prediction was applied, using the bond number to guide a design that prevents potentially biohazardous samples from leaking from the device. We employed multi-material 3D printing technology, which allows composites with rigid and elastomeric properties to be printed as a single part. To validate the meter-mix device with a clinically relevant sample, we used urine spiked with inactivated Chlamydia trachomatis and Neisseria gonorrhoeae. A downstream nucleic acid amplification by quantitative PCR (qPCR) confirmed there was no statistically significant difference between samples metered and mixed using the standard protocol and those prepared with the meter-mix device, showing the 3D-printed device could accurately meter, mix and dispense a human urine sample without loss of nucleic acids. Although there are some limitations to 3D printing capabilities (e.g. dimension limitations related to support material used in the printing process), the advantages of customizability, modularity and rapid prototyping illustrate the utility of 3D printing for developing sample-to-device interfaces for diagnostics