Getting rid of rain and stars: mitigating inhibition effects in ddPCR assays, the case of the invasive crayfish Pacifastacus leniusculus in the streams of Luxembourg

ddPCR is getting more and more popular in the field of eDNA-based aquatic monitoring. Even if emulsion PCR used in ddPCR confers a partial resistance to inhibition due to the high number of reactions for the same sample (between 10K and 20K), it is not impervious to it. Inhibition impacts the fluorescence amplitude of positive droplets, affecting both their dispersion and their position relatively to the negative droplets cloud. This fluctuation could jeopardize the use of a shared threshold among several samples and thus the objective assignation of the positive droplets. This is even more critical for low concentration samples such as eDNA samples: the positive droplets are scarce and it is thus crucial to objectively discriminate if they can be counted as positive by establishing an appropriate threshold. Another issue is the artifactual generation of high fluorescence droplets that could be counted as positive with a single threshold solution. Here we propose a double threshold method to take both high fluorescence droplets and PCR inhibition impact into account allowing for an objective sorting of the positive and negative droplets in ddPCR assays.

Selection of DNA Aptamers for Subcellular Localization of RBSDV P10 Protein in the Midgut of Small Brown Planthoppers by Emulsion PCR-Based SELEX

Rice black-streaked dwarf virus (RBSDV), classified under the Reoviridae, Fijivirus genus, caused an epidemic in the eastern provinces of China and other East Asian countries and resulted in severe yield loss in rice and wheat production. RBSDV is transmitted by the small brown planthopper (SBPH, Laodelphax striatellus Fallén) in a persistent manner. In order to provide a stable and cost-effective detection probe, in this study we selected three DNA aptamers (R3, R5 and R11) by an optimized, standardized and time saving emulsion PCR-based SELEX, for the detection of RBSDV outer-shell P10 protein for in situ localization studies in the midgut of SBPH. The specificity of these three DNA aptamers was tested through detection of the P10 protein using an enzyme-linked oligonucleotide assay (ELONA) and aptamer-based dot-blot ELISA. All three DNA aptamers can be used to detect RBSDV P10 protein by immunofluorescent labeling in the midgut of RBSDV-infected SBPH. These data show that the selected aptamers can be used for the detection of RBSDV P10 protein in vitro and in vivo. This is the first report of aptamers being selected for detection of a rice virus capsid protein.

Liquid drop of DNA libraries reveals total genome information

Conventional “bulk” PCR often yields inefficient and nonuniform amplification of complex templates in DNA libraries, introducing unwanted biases. Amplification of single DNA molecules encapsulated in a myriad of emulsion droplets (emulsion PCR, ePCR) allows the mitigation of this problem. Different ePCR regimes were experimentally analyzed to identify the most robust techniques for enhanced amplification of DNA libraries. A phenomenological mathematical model that forms an essential basis for optimal use of ePCR for library amplification was developed. A detailed description by high-throughput sequencing of amplified DNA-encoded libraries highlights the principal advantages of ePCR over bulk PCR. ePCR outperforms PCR, reduces gross DNA errors, and provides a more uniform distribution of the amplified sequences. The quasi single-molecule amplification achieved via ePCR represents the fundamental requirement in case of complex DNA templates being prone to diversity degeneration and provides a way to preserve the quality of DNA libraries.

Emulsion PCR (ePCR) as a Tool to Improve the Power of DGGE Analysis for Microbial Population Studies

To the authors’ knowledge, this is the first report of the use of emulsion-Polymerase chain reaction (e-PCR) coupled with denaturing gradient gel electrophoresis (DGGE) analysis. In the present work the effectiveness of ePCR in improving the power of the DGGE technique for microbial population studies was tested. Our results indicated that ePCR results in uniform amplification of several DNA molecules, overcoming the major limitations of conventional PCR, such as preferential amplification and DNA concentration dependence. Moreover, ePCR-DGGE resulted in higher sensitivity when compared to conventional PCR-DGGE methods used for studying microbial populations in a complex matrix. In fact, compared to conventional PCR, the DGGE profiles of ePCR products permitted the detection of a higher number of the species that were present in the tested sample.

Emulsion PCR made easy

Emulsion PCR (ePCR) is an important technique that permits amplification of DNA molecules in physically separated picoliter-volume water-in-oil droplets, and thus avoids formation of unproductive chimeras and other artifacts between similar DNA sequences. However, the recovery of ePCR products involves repeated extraction with hazardous organic solvents followed by purification using silica-based columns, making the overall process cumbersome. In this benchmark, we have described a quick ePCR extraction protocol for the purification of ePCR products, which directly employs silica-based DNA purification columns; products purified using this method have been found to be compatible with gene cloning and next-generation sequencing applications. The method described here makes ePCR easy, safe and within the reach of every laboratory.

Surfactant and oil formulations for monodisperse droplet emulsion PCR

A systematic survey of the oil and surfactant components of stable monodisperse w/o droplets suitable for various methods.