This comprehensive review provides a systematic classification and a comparative evaluation of current sequence-specific detection methods for LAMP.

Apomixis, the asexual formation of seed, has been known in angiosperms for more than a century yet the genetic mechanisms that control this trait remain poorly understood. Most members of the genus Hieracium are apomicts, forming predominantly asexual seed. Some purely sexual forms, however, also exist. In this paper we present a study of the inheritance of apomixis using two apomictic species of Hieracium which utilize very different forms of megagametogenesis. In both cases the progeny inherited apomixis as a monogenic, dominant trait that could be transferred by both haploid and diploid male gametes. In common with observations from other systems, no diploid apomictic progeny were recovered from these crosses. This appears to have been caused by selection against the survival of diploid zygotes, rather than against the mediation of haploid gametes as has been noted in other systems. Crosses between the two apomicts showed that the dominant determinants in the two forms examined were closely linked, possibly allelic. The significance of these data is discussed with respect to current theories on the associative link between gametophytic apomixis and polyploidy.

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A variety of real-time detection techniques for loop-mediated isothermal amplification (LAMP) based on the change in fluorescence intensity during DNA amplification enable simultaneous detection of multiple targets. However, these techniques depend on fluorogenic probes containing target-specific sequences. That complicates the adaption to different targets leading to time-consuming assay optimization. Here, we present the first universal real-time detection technique for multiplex LAMP. The novel approach allows simple assay design and is easy to implement for various targets. The innovation features a mediator displacement probe and a universal reporter. During amplification of target DNA the mediator is displaced from the mediator displacement probe. Then it hybridizes to the reporter generating a fluorescence signal. The novel mediator displacement (MD) detection was validated against state-of-the-art molecular beacon (MB) detection by means of a HIV-1 RT-LAMP: MD surpassed MB detection by accelerated probe design (MD: 10 min, MB: 3-4 h), shorter times to positive (MD 4.1 ± 0.1 min shorter than MB, n = 36), improved signal-to-noise fluorescence ratio (MD: 5.9 ± 0.4, MB: 2.7 ± 0.4; n = 15), and showed equally good or better analytical performance parameters. The usability of one universal mediator-reporter set in different multiplex assays was successfully demonstrated for a biplex RT-LAMP of HIV-1 and HTLV-1 and a biplex LAMP of Haemophilus ducreyi and Treponema pallidum, both showing good correlation between target concentration and time to positive. Due to its simple implementation it is suggested to extend the use of the universal mediator-reporter sets to the detection of various other diagnostic panels.

Automated point-of-care testing system for rapid single cell detection of MRSA directly from nasal swab samples.

In this study we optimised the fluorescence signal generation of contact quenched universal reporter oligonucleotides.

Inkjet technology as a maskless, direct-writing technology offers the potential for structured deposition of functional materials for the realization of electrodes for, e.g., sensing applications. In this work, electrodes were realized by inkjet-printing of commercial nanoparticle gold ink on planar substrates and, for the first time, onto the 2.5D surfaces of a 0.5 mm-deep microfluidic chamber produced in cyclic olefin copolymer (COC). The challenges of a poor wetting behavior and a low process temperature of the COC used were solved by a pretreatment with oxygen plasma and the combination of thermal (130 • C for 1 h) and photonic (955 mJ/cm 2 ) steps for sintering. By performing the photonic curing, the resistance could be reduced by about 50% to 22.7 µΩ cm. The printed gold structures were mechanically stable (optimal cross-cut value) and porous (roughness factors between 8.6 and 24.4 for 3 and 9 inkjet-printed layers, respectively). Thiolated DNA probes were immobilized throughout the porous structure without the necessity of a surface activation step. Hybridization of labeled DNA probes resulted in specific signals comparable to signals on commercial screen-printed electrodes and could be reproduced after regeneration. The process described may facilitate the integration of electrodes in 2.5D lab-on-a-chip systems.

We present a versatile tool for the generation of monodisperse water-in-fluorinated-oil droplets in standard reaction tubes by centrifugal step emulsification. The microfluidic cartridge is designed as an insert into a standard 2 mL reaction tube and can be processed in standard laboratory centrifuges. It allows for droplet generation and subsequent transfer for any downstream analysis or further use, does not need any specialized device, and manufacturing is simple because it consists of two parts only: A structured substrate and a sealing foil. The design of the structured substrate is compatible to injection molding to allow manufacturing at large scale. Droplets are generated in fluorinated oil and collected in the reaction tube for subsequent analysis. For sample sizes up to 100 µL with a viscosity range of 1 mPa·s-4 mPa·s, we demonstrate stable droplet generation and transfer of more than 6 × 10 5 monodisperse droplets (droplet diameter 66 µm ± 3 µm, CV ≤ 4%) in less than 10 min. With two application examples, a digital droplet polymerase chain reaction (ddPCR) and digital droplet loop mediated isothermal amplification (ddLAMP), we demonstrate the compatibility of the droplet production for two main amplification techniques. Both applications show a high degree of linearity (ddPCR: R 2 ≥ 0.994; ddLAMP: R 2 ≥ 0.998), which demonstrates that the cartridge and the droplet generation method do not compromise assay performance. resistance than the sample supply channel (ii), as long as the viscosity of the sample remains ≥ 4 mPa·s. Details of the fluidic design calculations and channel and chamber dimensions are listed in ESI 2, S1.Molecules 2020, 25, x 3 of 11