Loop mediated isothermal amplification (LAMP) is a nucleic acid amplification technique performed under isothermal conditions. The output of this amplification technique includes multiple different sizes of deoxyribonucleic acid (DNA) structures which are identified by a banding pattern on gel electrophoresis plots. Although this is a specific amplification technique, the complexity of the primer design and amplification still lead to the issue of obtaining false‐positive results, especially when a positive reading is determined solely by whether there is any banding pattern in the gel electrophoresis plot. Here, we first performed extensive LAMP experiments and evaluated the DNA structures using microchip electrophoresis. We then developed a mathematical model derived from the various components that make up an entire LAMP structure to predict the full LAMP structure size in base pairs. This model can be implemented by users to make predictions for specific, DNA size dependent, banding patterns on their gel electrophoresis plots. Each prediction is specific to the target sequence and primers used and therefore reduces incorrect diagnosis errors through identifying true‐positive and false‐positive results. This model was accurately tested with multiple primer sets in house and was also translatable to different DNA and RNA types in previously published literature. The mathematical model can ultimately be used to reduce false‐positive LAMP diagnosis errors for applications ranging from tuberculosis diagnostics to E. coli to numerous other infectious diseases.
COVID-19 is an infectious disease that caused a global pandemic affecting people worldwide. As disease detection and vaccine rollout continue to progress, there is still a need for efficient diagnostic tools to satisfy continued testing needs. This preliminary study evaluated a novel SARS-CoV-2 diagnostic test called DirectDetect SARS-CoV-2 Direct Real-time reverse transcriptase polymerase chain reaction (RT-PCR) based on a limited sample size of 24 respiratory samples from 14 SARS-CoV-2-positive patients. The test is advantageous compared to others on the market since it does not require viral transport medium or viral RNA extraction prior to nucleic acid amplification and detection. This capability transforms the hours-long sample preparation time into a minutes-long procedure while also eliminating the need for many costly reagents which may be difficult to obtain during the surge in nucleic acid-based testing during the pandemic. The results show a positive agreement of 94.7, 100, and 94.7% between dry sample swabs, treated samples, and untreated samples tested using the DirectDetect SARS-CoV-2 Direct Real-time RT-PCR compared to tests used in a clinical laboratory, respectively. The findings indicate that DirectDetect can be used for multiple different sample types while reducing the number of reagents and time needed for diagnosis. Although this study shows promising results using the DirectDetect results, further validation of this test using a larger sample set is required to assess the true performance of this test.
NA extraction and purification utilitzing a microfluidic chip with applied electric field to induce electroosmotic flow opposite the magnetic NA-bound bead mix.
The advent and dissemination of next-generation sequencing (NGS) technologies such as Illumina's sequencing platforms has brought forth vast reductions in the cost, time, and technical difficulties associated with DNA and RNA sequencing. Despite this trend, the workflow required to generate nucleic acid libraries for sequencing remains time-consuming and laborious. The following research proposes a method for simplifying and streamlining this process by replacing the manual washing steps of the common magnetic bead-based cleanup with a novel microfluidic method by integrating magnetic separation and electrokinetic purification (MSEP). Requiring no pumps, pipette mixing, vortexing, or centrifugation, MSEP relies on selective adsorption of target DNA onto the magnetic beads with subsequent transport of beads through a microchannel undergoing an antiparallel electroosmotic flow. The synergetic flow conditions were optimized using a simple electrohydrodynamic flow model. This work demonstrates that MSEP is as effective in eliminating adapter-dimers from the post-ligation library mix as the manual method while also greatly reducing the hands-on time and amount of pipetting required. Although MSEP has been applied specifically toward NGS library preparation at this time, it has the potential to be adapted and employed for any bead-based separation scheme, namely, solid phase extraction, sequence-specific hybridization, and immunoprecipitation on a microscale.
Plasma has significant utility as an input for diagnostics and screening for conditions such as viral infections, cancer, and more. However, plasma is difficult to obtain at the point-of-care, as separation from whole blood is typically carried out via centrifugation. We have designed and optimized a low-cost, simple-to-operate microfluidic device which carries out the separation of plasma from whole blood. The device utilizes depth filtration as its separation mechanism and collects plasma via capillary action, allowing for operation without components that drive flow externally. We first optimized device dimensions and operating parameters and demonstrated consistent separation efficiencies for the samples with hematocrits ranging from 25–65%. The impact of input sample hematocrit percentage on flow rate through the device was also examined, with samples with hematocrits greater than 45% decreasing plasma flow rate. Lastly, we evaluated the ability of this device to produce plasma with a high protein concentration and found no significant difference between protein levels in samples from the device compared to samples produced via centrifugation. This system produced plasma with a maximum separation efficiency of 88.5% and achieved a maximum plasma volume of ∼14 μl from a 50 μl whole blood input. The low cost, simplicity of operation, and high plasma quality associated with this device give it many advantages in a point-of-care setting. This device could be integrated into plasma-based diagnostic workflows to increase access to various types of disease testing and monitoring.
In many situations, cells migrate through tiny orifices. Examples include the extravasation of immune cells from the bloodstream for fighting infections, the infiltration of cancer cells during metastasis, and the migration of human pathogens. An extremely motile and medically relevant type of human pathogen is Acanthamoeba castellanii. In the study presented here, we investigated how a combination of microparticles and microstructured interfaces controls the migration of A. castellanii trophozoites. The microinterfaces comprised well-defined micropillar arrays, and the trophozoites easily migrated through the given constrictions by adapting the shape and size of their intracellular vacuoles and by adapting intracellular motion. After feeding the trophozoite cells in microinterfaces with synthetic, stiff microparticles of various sizes and shapes, their behavior changed drastically: if the particles were smaller than the micropillar gap, migration was still possible. If the cells incorporated particles larger than the pillar gap, they could become immobilized but could also display remarkable problem-solving capabilities. For example, they turned rod-shaped microparticles such that their short axis fit through the pillar gap or they transported the particles above the structure. As migration is a crucial contribution to A. castellanii pathogenicity and is also relevant to other biological processes in microenvironments, such as cancer metastasis, our results provide an interesting strategy for controlling the migration of cells containing intracellular particles by microstructured interfaces that serve as migration-limiting environments.
Objectives Increase the yield and purity of cell‐free DNA (cfDNA) extracted from plasma for non‐invasive prenatal testing (NIPT) as inefficiencies in this extraction and purification can dramatically affect the sensitivity and specificity of the test. Methods This work integrates cfDNA extraction from plasma with a microfluidic chip platform by combining magnetic bead‐based extraction and electroosmotic flow on the microfluidic chip. Various wash buffers and voltage conditions were simulated using COMSOL Multiphysics Modeling and tested experimentally. Results When performing the first wash step of this assay on the microfluidic chip with 300 V applied across the channel there was a six‐fold increase in the A260/A230 ratio showing a significant improvement (p value 0.0005) in the purity of the extracted sample all while maintaining a yield of 68.19%. These values are critical as a high yield results in more sample to analyze and an increase in A260/A230 ratio corresponds to a decrease in salt contaminants such as guanidinium thiocyanate which can interfere with downstream processes during DNA library preparation and potentially hinder the NIPT screening results. Conclusions This technique has the potential to improve NIPT outcomes and other clinically relevant workflows that use cfDNA as an analyte such as cancer detection.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.