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The techniques researched, developed and applied towards the measurement of radioisotope concentrations at ultra-low levels in the real-time solar neutrino experiment BOREXINO at Gran Sasso are presented and illustrated with specific results of widespread interest. We report the use of low-level germanium gamma spectrometry, low-level miniaturized gas proportional counters and low background scintillation detectors developed in solar neutrino research. Each now sets records in its field. We additionally describe our techniques of radiochemical ultra-pure, few atom manipulations and extractions. Forefront measurements also result from the powerful combination of neutron activation and low-level counting. Finally, with our techniques and commercially available mass spectrometry and atomic absorption spectroscopy, new low-level detection limits for isotopes of interest are obtained.Comment: 27 pages, 5 figures. Submitted to Astroparticle Physics (17 Sep 2001). Spokesperson of the Borexino Collaboration: G. Bellini. Corresponding author: W. Hampe

Human fecal contamination of water is a public health risk. However, inadequate testing solutions frustrate timely, actionable monitoring. Bacterial culture-based methods are simple but typically cannot distinguish fecal host source. PCR assays can identify host sources but require expertise and infrastructure. To bridge this gap we have developed a field-ready nucleic acid diagnostic platform and rapid sample preparation methods that enable on-site identification of human fecal contamination within 80 min of sampling. Our platform relies on loop-mediated isothermal amplification (LAMP) of human-associated Bacteroides HF183 genetic markers from crude samples. Oligonucleotide strand exchange (OSD) probes reduce false positives by sequence specifically transducing LAMP amplicons into visible fluorescence that can be photographed by unmodified smartphones. Our assay can detect as few as 17 copies/ml of human-associated HF183 targets in sewage-contaminated water without cross-reaction with canine or feline feces. It performs robustly with a variety of environmental water sources and with raw sewage. We have also developed lyophilized assays and inexpensive 3D-printed devices to minimize cost and facilitate field application.

bMany environments on Earth experience nutrient limitation and as a result have nongrowing or very slowly growing bacterial populations. To better understand bacterial respiration under environmentally relevant conditions, the effect of nutrient limitation on respiration rates of heterotrophic bacteria was measured. The oxygen consumption and population density of batch cultures of Escherichia coli K-12, Shewanella oneidensis MR-1, and Marinobacter aquaeolei VT8 were tracked for up to 200 days. The oxygen consumption per CFU (Q O2 ) declined by more than 2 orders of magnitude for all three strains as they transitioned from nutrient-abundant log-phase growth to the nutrient-limited early stationary phase. The large reduction in Q O2 from growth to stationary phase suggests that nutrient availability is an important factor in considering environmental respiration rates. Following the death phase, during the long-term stationary phase (LTSP), Q O2 values of the surviving population increased with time and more cells were respiring than formed colonies. Within the respiring population, a subpopulation of highly respiring cells increased in abundance with time. Apparently, as cells enter LTSP, there is a viable but not culturable population whose bulk community and per cell respiration rates are dynamic. This result has a bearing on how minimal energy requirements are met, especially in nutrient-limited environments. The minimal Q O2 rates support the extension of Kleiber's law to the mass of a bacterium (100-fg range). Heterotrophic bacterial respiration constitutes 50% to 90% of ocean community respiration (1) and plays a critical role in the recycling of organic carbon in all natural environments (2). Despite this importance, respiration remains poorly quantified in ocean models of metabolism, gas exchange, and carbon mass balances (3). This uncertainty in heterotrophy makes it difficult to predict when areas of the ocean will be sources or sinks of CO 2 (4). Microbial community ecosystem behavior is ultimately a function of auto-and heterotrophic relationships; hence, understanding the biogeochemical contribution of heterotrophic bacteria at the single-cell level is necessary for making predictions of how ecosystems will respond to global change (5). Extracting this information is difficult. Efforts are being made with single-cell observatories designed to trap cells in microwells and monitor their consumption of oxygen over time using optical techniques (6, 7). Field-based approaches involve modeling oxygen pore water profiles in marine sediments and dividing oxygen consumption rate by cell numbers (8). A more frequently employed method for analyzing cellular respiration rates is to determine bulk oxygen uptake (consumption) rates (OUR), using either batch or continuous cultures with large populations of microbes. The average cellular respiration rate is calculated by normalizing OUR to population size. Most studies of this nature determine respiration rates while cells are growing (9-14). In the rare cases wher...

Isothermal nucleic acid amplification tests (iNATs), such as loop-mediated isothermal amplification (LAMP), are good alternatives to PCR-based amplification assays, especially for point-of-care and low-resource use, in part because they can be carried out with relatively simple instrumentation. However, iNATs can often generate spurious amplicons, especially in the absence of target sequences, resulting in false-positive results. This is especially true if signals are based on non-sequence-specific probes, such as intercalating dyes or pH changes. In addition, pathogens often prove to be moving, evolving targets and can accumulate mutations that will lead to inefficient primer binding and thus false-negative results. Multiplex assays targeting different regions of the analyte and logical signal readout using sequence-specific probes can help to reduce both false negatives and false positives. Here, we describe rapid conversion of three previously described SARS-CoV-2 LAMP assays that relied on a non-sequence-specific readout into individual and multiplex one-pot assays that can be visually read using sequence-specific oligonucleotide strand exchange (OSD) probes. We describe both fluorescence-based and Boolean logic-gated colorimetric lateral flow readout methods and demonstrate detection of SARS-CoV-2 virions in crude human saliva. IMPORTANCE One of the key approaches to treatment and control of infectious diseases, such as COVID-19, is accurate and rapid diagnostics that is widely deployable in a timely and scalable manner. To achieve this, it is essential to go beyond the traditional gold standard of quantitative PCR (qPCR) that is often faced with difficulties in scaling due to the complexity of infrastructure and human resource requirements. Isothermal nucleic acid amplification methods, such as loop-mediated isothermal amplification (LAMP), have been long pursued as ideal, low-tech alternatives for rapid, portable testing. However, isothermal approaches often suffer from false signals due to employment of nonspecific readout methods. We describe general principles for rapidly converting nonspecifically read LAMP assays into assays that are read in a sequence-specific manner by using oligonucleotide strand displacement (OSD) probes. We also demonstrate that inclusion of OSD probes in LAMP assays maintains the simplicity of one-pot assays and a visual yes/no readout by using fluorescence or colorimetric lateral-flow dipsticks while providing accurate sequence-specific readout and the ability to logically query multiplex amplicons for redundancy or copresence. These principles not only yielded high-surety isothermal assays for SARS-CoV-2 but might also aid in the design of more sophisticated molecular assays for other analytes.