Low- and middle-income countries (LMICs) are significantly affected by SARS-CoV-2, partially due to their limited capacity for local production and implementation of molecular testing. Here, we provide detailed methods and validation of a molecular toolkit that can be readily produced and deployed using laboratory equipment available in LMICs. Our results show that lab-scale production of enzymes and nucleic acids can supply over 50,000 tests per production batch. The optimized one-step RT-PCR coupled to CRISPR-Cas12a-mediated detection showed a limit of detection of 10 2 ge/μL in a turnaround time of 2 h. The clinical validation indicated an overall sensitivity of 80%–88%, while for middle and high viral load samples (Cq ≤ 31) the sensitivity was 92%–100%. The specificity was 96%–100% regardless of viral load. Furthermore, we show that the toolkit can be used with the mobile laboratory Bento Lab, potentially enabling LMICs to implement detection services in unattended remote regions.

Here, we describe a detailed step-by-step protocol to detect SARS-CoV-2 RNA using RT-PCR-mediated amplification and Crispr/Cas-based visualization. The optimized assay uses basic molecular biology equipment such as conventional thermocyclers and transilluminators for qualitative detection. Alternatively, a fluorescence plate reader can be used for quantitative measurements. The protocol detects two regions of the SARS-CoV-2 genome in addition to the human RNaseP sample control. Aiming to reach remote regions, this work was developed to use the portable molecular workstation from BentoLab.

Rapid Diagnostic Tests (RDTs) for malaria are restricted to a few biomarkers and antibody-mediated detection. However, the expression of commonly used biomarkers varies geographically and the sensibility of immunodetection can be affected by batch-to-batch differences or limited thermal stability. In this study we aimed to overcome these limitations by identifying a potential biomarker and by developing molecular sensors based on aptamer technology. Using gene expression databases, ribosome profiling analysis, and structural modeling, we find that the High Mobility Group Box 1 protein (HMGB1) of Plasmodium falciparum is highly expressed, structurally stable, and present along all blood-stages of P . falciparum infection. To develop biosensors, we used in vitro evolution techniques to produce DNA aptamers for the recombinantly expressed HMG-box, the conserved domain of HMGB1. An evolutionary approach for evaluating the dynamics of aptamer populations suggested three predominant aptamer motifs. Representatives of the aptamer families were tested for binding parameters to the HMG-box domain using microscale thermophoresis and rapid kinetics. Dissociation constants of the aptamers varied over two orders of magnitude between nano- and micromolar ranges while the aptamer-HMG-box interaction occurred in a few seconds. The specificity of aptamer binding to the HMG-box of P . falciparum compared to its human homolog depended on pH conditions. Altogether, our study proposes HMGB1 as a candidate biomarker and a set of sensing aptamers that can be further developed into rapid diagnostic tests for P . falciparum detection.

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21 Rapid Diagnostic Tests (RDTs) for malaria are restricted to a few biomarkers and antibody-22 mediated detection. However, the expression of commonly used biomarkers varies 23 geographically and the sensibility of immunodetection can be affected by batch-to-batch 24 differences or limited thermal stability. In this study we aimed to overcome these limitations 25 by identifying a potential biomarker and by developing molecular sensors based on aptamer 26 technology. Using gene expression databases, ribosome profiling analysis, and structural 27 modeling, we find that the High Mobility Group Box 1 protein (HMGB1) of Plasmodium 28 falciparum is highly expressed, structurally stable and steadily present along all blood-stages 29 of P. falciparum infection. To develop biosensors, we used in vitro evolution techniques to 30 produce DNA aptamers for the recombinantly expressed HMG-box, the conserved domain 31 of HMGB1. An evolutionary approach for evaluating the dynamics of aptamer populations 32 suggested three predominant aptamer motifs. Representatives of the aptamer families were 33 tested for binding parameters to the HMG-box domain using microscale thermophoresis and 34 rapid kinetics. Dissociation constants of the aptamers varied over two orders of magnitude 35 between nano-and micromolar ranges while the aptamer-HMG-box interaction occurred in 36 less than 30 seconds. The specificity of aptamer binding to the HMG-box of P. falciparum 37 compared to its human homolog depended on pH conditions. Altogether, our study proposes 38 HMGB1 as a potential biomarker and a set of sensing aptamers that can be further developed 39 into rapid diagnostic tests for P. falciparum detection. 40 41 42 3 43 44 45 46 47 Malaria is an infectious disease that affects animals and humans, caused by protozoans of the 48 genus Plasmodium. Malaria remains the cause of 435,000 deaths worldwide, with 219 49 million cases reported during 2017 [1]. Accurate treatment requires the identification of the 50 parasite of the genus Plasmodium, in addition to the species causing the disease. Currently, 51 there are several methods to diagnose malaria, such as PCR-based, Giemsa microscopy, and 52 Rapid Diagnostic Tests (RDTs). Among these options, the last two appear to be suitable for 53 low-income and mostly affected countries; nonetheless, they also present certain limitations 54 [2]. 55 Giemsa microscopy is inexpensive to perform, can differentiate malaria species and stages, 56 and can quantify the parasites [2]. However, well-trained personnel is required and, 57 frequently unavailable in low-income countries [2]. RDTs, on the other hand, 58 detect Plasmodium spp. biomarker antigens in a small amount of blood using immobilized 59 antibodies. The two most used biomarkers are histidine-rich protein 2 (HRP-2) and lactate 60 dehydrogenase (LDH), which have been shown of high sensitivity and specificity for 61 detecting P. falciparum [3]. However, P. falciparum strains lacking the HRP-2 and HRP-3 62 genes have appeared, increasing false negative re...