Commercial stocks of SARS-CoV-2 RNA may report low concentration values, leading to artificially increased apparent sensitivity of diagnostic assays

In response to the rapidly evolving COVID-19 pandemic, the U.S. Food and Drug Administration (FDA) has rapidly issued 49 emergency use authorizations (EUAs) for SARS-CoV-2 in vitro diagnostic test-kits. A critical metric in the performance evaluation for a diagnostic test kit is the analytical sensitivity, which is measured by the limit of detection (LOD). Commercial RNA stocks with known titers are used to determine LOD. We identified a problem with the titer reported for the commercial stocks when examining the analytical sensitivity of the reverse transcription quantitative PCR (RT-qPCR) protocol that is recommended by the Centers for Disease Control and Prevention (CDC) using plasmid DNA from Integrated DNA Technologies (IDT), synthetic RNA from BEI Resources (BEI), and extracted genomic RNA from BEI. We detected 3/3 positives for reactions containing synthetic RNA at a concentration of 0.1 copies/reaction (based on the supplier's label concentration). The apparent better-than-single-molecule performance is a statistically highly unlikely event, indicating a potential inaccuracy in the supplier's quantification of the stock material. Using an ultrasensitive and precise assay, reverse transcription digital PCR (RT-dPCR), we independently quantified concentrations of commercial SARS-CoV-2 plasmid DNA and SARS-CoV-2 RNA stocks. For plasmid DNA, the actual concentration measured by RT-dPCR was 11% of the nominal label concentration. For synthetic RNA, the actual concentration measured by RT-dPCR for one lot was 770% of the label concentration and for a different lot was 57% of the label concentration. For genomic RNA, the concentration measured by RT-dPCR for one lot was 240% of the label concentration and for a different lot it was 300% of the label concentration. This SARS-CoV-2 genomic RNA from BEI Resources has been used in at least 11 approved FDA Emergency Use Authorizations as of April 27, 2020. Such deviations of reported RNA or DNA stock concentrations from true concentrations can result in inaccurate quantification and calculation of LOD. Precise and accurate reporting of DNA and RNA stock concentrations by commercial suppliers will enable accurate quantification of assay performance, which is urgently needed to improve evaluation of different assays by diagnostic developers and regulatory bodies

Millisecond kinetics on a microfluidic chip using nanoliters of reagents

This paper describes a microfluidic chip for performing kinetic measurements with better than millisecond resolution. Rapid kinetic measurements in microfluidic systems are complicated by two problems: mixing is slow and dispersion is large. These problems also complicate biochemical assays performed in microfluidic chips. We have recently shown (Song, H.; Tice, J. D.; Ismagilov, R. F. Angew. Chem., Int. Ed. 2003, 42, 768-772) how multiphase fluid flow in microchannels can be used to address both problems by transporting the reagents inside aqueous droplets (plugs) surrounded by an immiscible fluid. Here, this droplet-based microfluidic system was used to extract kinetic parameters of an enzymatic reaction. Rapid single-turnover kinetics of ribonuclease A (RNase A) was measured with better than millisecond resolution using sub-microliter volumes of solutions. To obtain the single-turnover rate constant (k = 1100 +/- 250 s(-1)), four new features for this microfluidics platform were demonstrated: (i) rapid on-chip dilution, (ii) multiple time range access, (iii) biocompatibility with RNase A, and (iv) explicit treatment of mixing for improving time resolution of the system. These features are discussed using kinetics of RNase A. From fluorescent images integrated for 2-4 s, each kinetic profile can be obtained using less than 150 nL of solutions of reagents because this system relies on chaotic advection inside moving droplets rather than on turbulence to achieve rapid mixing. Fabrication of these devices in PDMS is straightforward and no specialized equipment, except for a standard microscope with a CCD camera, is needed to run the experiments. This microfluidic platform could serve as an inexpensive and economical complement to stopped-flow methods for a broad range of time-resolved experiments and assays in chemistry and biochemistry

Self-reinoculation with fecal flora changes microbiota density and composition leading to an altered bile-acid profile in the mouse small intestine

Background: The upper gastrointestinal tract plays a prominent role in human physiology as the primary site for enzymatic digestion and nutrient absorption, immune sampling, and drug uptake. Alterations to the small intestine microbiome have been implicated in various human diseases, such as non-alcoholic steatohepatitis and inflammatory bowel conditions. Yet, the physiological and functional roles of the small intestine microbiota in humans remain poorly characterized because of the complexities associated with its sampling. Rodent models are used extensively in microbiome research and enable the spatial, temporal, compositional, and functional interrogation of the gastrointestinal microbiota and its effects on the host physiology and disease phenotype. Classical, culture-based studies have documented that fecal microbial self-reinoculation (via coprophagy) affects the composition and abundance of microbes in the murine proximal gastrointestinal tract. This pervasive self-reinoculation behavior could be a particularly relevant study factor when investigating small intestine microbiota. Modern microbiome studies either do not take self-reinoculation into account, or assume that approaches such as single housing mice or housing on wire mesh floors eliminate it. These assumptions have not been rigorously tested with modern tools. Here, we used quantitative 16S rRNA gene amplicon sequencing, quantitative microbial functional gene content inference, and metabolomic analyses of bile acids to evaluate the effects of self-reinoculation on microbial loads, composition, and function in the murine upper gastrointestinal tract. Results: In coprophagic mice, continuous self-exposure to the fecal flora had substantial quantitative and qualitative effects on the upper gastrointestinal microbiome. These differences in microbial abundance and community composition were associated with an altered profile of the small intestine bile acid pool, and, importantly, could not be inferred from analyzing large intestine or stool samples. Overall, the patterns observed in the small intestine of non-coprophagic mice (reduced total microbial load, low abundance of anaerobic microbiota, and bile acids predominantly in the conjugated form) resemble those typically seen in the human small intestine. Conclusions: Future studies need to take self-reinoculation into account when using mouse models to evaluate gastrointestinal microbial colonization and function in relation to xenobiotic transformation and pharmacokinetics or in the context of physiological states and diseases linked to small intestine microbiome and to small intestine dysbiosis

A Quantitative Sequencing Framework for Absolute Abundance Measurements of Mucosal and Lumenal Microbial Communities

A fundamental goal in microbiome studies is determining which microbes affect host physiology. Standard methods for determining changes in microbial taxa measure relative, rather than absolute abundances. Moreover, studies often analyze only stool, despite microbial diversity differing substantially among gastrointestinal (GI) locations. Here, we develop a quantitative framework to measure absolute abundances of individual bacterial taxa by combining the precision of digital PCR with the high-throughput nature of 16S rRNA gene amplicon sequencing. In a murine ketogenic-diet study, we compare microbial loads in lumenal and mucosal samples along the GI tract. Quantitative measurements of absolute (but not relative) abundances reveal decreases in total microbial loads on the ketogenic diet and enable us to determine the differential effects of diet on each taxon in stool and small-intestine mucosa samples. This rigorous quantitative microbial analysis framework, appropriate for diverse GI locations enables mapping microbial biogeography of the mammalian GI tract and more accurate analyses of changes in microbial taxa in microbiome studies

Microfluidic cartridges preloaded with nanoliter plugs of reagents: an alternative to 96-well plates for screening

In traditional screening with 96-well plates, microliters of substrates are consumed for each reaction. Further miniaturization is limited by the special equipment and techniques required to dispense nanoliter volumes of fluid. Plug-based microfluidics confines reagents in nanoliter plugs (droplets surrounded by fluorinated carrier fluid), and uses simple pumps to control the flow of plugs. By using cartridges pre-loaded with nanoliter plugs of reagents, only two pumps and a merging junction are needed to set up a screen. Screening with preloaded cartridges uses only nanoliters of substrate per reaction, and requires no microfabrication. The low cost and simplicity of this method has the potential of replacing 96-well and other multi-well plates, and has been applied to enzymatic assays, protein crystallization and optimization of organic reactions

Quantitative microbiome profiling in lumenal and tissue samples with broad coverage and dynamic range via a single-step 16S rRNA gene DNA copy quantification and amplicon barcoding

Current methods for detecting, accurately quantifying, and profiling complex microbial communities based on the microbial 16S rRNA marker genes are limited by a number of factors, including inconsistent extraction of microbial nucleic acids, amplification interference from contaminants and host DNA, different coverage of PCR primers utilized for quantification and sequencing, and potentially biases in PCR amplification rates among microbial taxa during amplicon barcoding. Here, we describe a single-step method that enables the quantification of microbial 16S rRNA gene DNA copies with wide dynamic range and broad microbial diversity, and simultaneous amplicon barcoding for quantitative 16S rRNA gene amplicon profiling of microbiota. The method is suitable for a variety of sample types and is robust in samples with low microbial abundance, including samples containing high levels of host mammalian DNA, as is common in human clinical samples. We demonstrate that our modification to the Earth Microbiome Project (EMP) V4 16S rRNA gene primers expands their microbial coverage while dramatically reducing non-specific mammalian mitochondrial DNA amplification, thus achieving wide dynamic range in microbial quantification and broad coverage for capturing high microbial diversity in samples with or without high host DNA background. The approach relies only on broadly available hardware (real-time PCR instruments) and standard reagents utilized for conventional 16S rRNA gene amplicon library preparation both of which make it amenable for immediate and widespread adoption. Simultaneous 16S rRNA gene DNA copy quantification and amplicon barcoding for multiplexed next-generation sequencing from the same analyzed sample, performed in a combined workflow, reduces the amount of sample needed and reduces time and reagent costs. Additionally, we demonstrate that using our modified 16S rRNA gene primers in a digital PCR (dPCR) format enables precise and exact microbial quantification in samples with very high host DNA background levels without the need for quantification standards. Potential future applications of this approach include: (1) quantitative microbiome profiling in human and animal microbiome research; (2) detection of monoinfections and profiling of polymicrobial infections in tissues, stool, and bodily fluids in human and veterinary medicine; (3) environmental sample analyses (e.g., soil and water); and (4) broad-coverage detection of microbial food contamination in products high in mammalian DNA, such as meat products. We predict that utilization of this approach primarily for quantitative microbiome profiling will be invaluable to microbiome studies, which have historically been limited to analysis of relative abundances of microbes

Real-time kinetics and high-resolution melt curves in single-molecule digital LAMP to differentiate and study specific and non-specific amplification

Isothermal amplification assays, such as loop-mediated isothermal amplification (LAMP), show great utility for the development of rapid diagnostics for infectious diseases because they have high sensitivity, pathogen-specificity and potential for implementation at the point of care. However, elimination of non-specific amplification remains a key challenge for the optimization of LAMP assays. Here, using chlamydia DNA as a clinically relevant target and high-throughput sequencing as an analytical tool, we investigate a potential mechanism of non-specific amplification. We then develop a real-time digital LAMP (dLAMP) with high-resolution melting temperature (HRM) analysis and use this single-molecule approach to analyze approximately 1.2 million amplification events. We show that single-molecule HRM provides insight into specific and non-specific amplification in LAMP that are difficult to deduce from bulk measurements. We use real-time dLAMP with HRM to evaluate differences between polymerase enzymes, the impact of assay parameters (e.g. time, rate or florescence intensity), and the effect background human DNA. By differentiating true and false positives, HRM enables determination of the optimal assay and analysis parameters that leads to the lowest limit of detection (LOD) in a digital isothermal amplification assay