The prototype demonstrated here is the first fully integrated sample-to-result diagnostic platform for performing nucleic acid amplification tests that requires no permanent instrument or manual sample processing. The multiplexable autonomous disposable nucleic acid amplification test (MAD NAAT) is based on two-dimensional paper networks, which enable sensitive chemical detection normally reserved for laboratories to be carried out anywhere by untrained users. All reagents are stored dry in the disposable test device and are rehydrated by stored buffer. The paper network is physically multiplexed to allow independent isothermal amplification of multiple targets; each amplification reaction is also chemically multiplexed with an internal amplification control. The total test time is less than one hour. The MAD NAAT prototype was used to characterize a set of human nasal swab specimens pre-screened for methicillin-resistant Staphylococcus aureus (MRSA) bacteria. With qPCR as the quantitative reference method, the lowest input copy number in the range where the MAD NAAT prototype consistently detected MRSA in these specimens was ∼5 × 10(3) genomic copies (∼600 genomic copies per biplexed amplification reaction).
VWF is required for platelet adhesion to sites of vessel injury, a process vital for both hemostasis and thrombosis. Enhanced VWF secretion and oxidative stress are both hallmarks of inflammation. We recently showed that the neutrophil oxidant hypochlorous acid (HOCl) inhibits VWF proteolysis by ADAMTS13 by oxidizing VWF methionine 1606 (M1606) in the A2 domain. M1606 was readily oxidized in a substrate peptide, but required urea in multimeric plasma VWF. In the present study, we examined whether shear stress enhances VWF oxidation. With an HOCl-generating system containing myeloperoxidase (MPO) and H 2 O 2 , we found that shear stress accelerated M1606 oxidation, with 56% becoming oxidized within 1 hour. Seven other methionine residues in the VWF A1A2A3 region (containing the sites for platelet and collagen binding and ADAMTS13 cleavage) were variably oxidized, one completely. Oxidized methionines accumulated preferentially in the largest VWF multimers. HOCl-oxidized VWF was hyperfunctional, agglutinating platelets at ristocetin concentrations that induced minimal agglutination using unoxidized VWF and binding more of the nanobody AU/VWFa-11, which detects a gain-offunction conformation of the A1 domain. These findings suggest that neutrophil oxidants will both render newly secreted VWF uncleavable and alter the largest plasma VWF forms such that they become hyperfunctional and resistant to proteolysis by ADAMTS13. (Blood. 2011; 118(19):5283-5291) IntroductionVWF is an enormous plasma glycoprotein synthesized in endothelial cells and megakaryocytes, the primary function of which is to attach platelets to sites of blood vessel injury. 1 VWF also chaperones coagulation factor VIII in plasma, protecting it from degradation and delivering it to the platelet surface, where it participates in blood coagulation. 2 VWF comprises homopolymers of a 2050-amino acid polypeptide synthesized through N-terminal disulfide bonding of C-terminal disulfide-bonded dimers. 3 Each monomer has many domains, including 3 tandem A domains (A1A2A3) containing, respectively, the platelet glycoprotein (GP) Ib-binding site, the cleavage site for ADAMTS13, and a collagen-binding site. 3 The polymers can reach a mass of Ͼ 20 million Da 4 and are either constitutively secreted or stored in granules, the Weibel-Palade bodies of endothelial cells, or the ␣-granules of platelets, from which they are released upon stimulation. 5 Newly released VWF multimers (termed ultra-large VWF or ULVWF) are hyperadhesive for platelets compared with the VWF normally found in plasma, 6 but are rapidly converted to smaller, less reactive plasma forms by cleavage by the metalloprotease ADAMTS13. 1 In addition to its hemostatic roles, VWF is also involved in thrombosis. The most obvious example is when ADAMTS13 fails to process ULVWF, resulting in the sometimes fatal microvascular clotting syndrome, thrombotic thrombocytopenic purpura. 7 VWF has also been implicated in thrombosis associated with disorders such as HELLP syndrome (hemolysis, elevated liver enzymes, a...
The rapid onset of the global COVID-19 pandemic has led to challenges for accurately diagnosing the disease, including supply shortages for sample collection, preservation, and purification. Currently, most diagnostic tests require RNA extraction and detection by RT-PCR; however, extraction is expensive and time-consuming and requires technical expertise. With these challenges in mind, we report extraction-free, multiplexed amplification of SARS-CoV-2 RNA from 246 clinical samples, resulting in 86% sensitivity and 100% specificity. The multiplex RT-PCR uses the CDC singleplex targets and has an LoD of 2 c/μL. We also report on amplification using a range of master mixes in different transport media. This work can help guide which combinations of reagents will enable accurate results when availability of supplies changes throughout the pandemic. Implementing these methods can reduce complexity and cost, minimize reagent usage, expedite time to results, and increase testing capacity.
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