AutoCAD Plant 3D 2011 Portable
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Autodesk has named Weatherhaven as the Autodesk Inventor of the Month for March 2011. Weatherhaven has been using Autodesk Inventor software to more effectively create portable shelters designed for use in the most rugged and remote locations around the world.
Nucleic acid-based tests (NATs) offer advantages over immunoassays, cell culture, microscopy, and other techniques to become the gold standard for the accurate diagnosis of many infectious diseases involved in plants, animals, and humans (Rougemont et al., 2004; Zhou et al., 2011). The sequence variation in the internally transcribed spacer (ITS) region genes of Leptosphaeria is often used to discriminate the species using Polymerase Chain Reaction (PCR)-based assays (Bailey et al., 2009; Zhou et al., 2010; Ashfield et al., 2012), recombinase polymerase amplification (RPA) (Lei et al., 2019), and Loop-mediated Isothermal AMPlification (LAMP) (Jedryczka et al., 2013; Zhou et al., 2016), but the conserved sequences among closely related species or isolates are limited (Mahuku et al., 1996). CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) and CRISPR-associated enzyme (Cas) systems can recognize and cleave specific nucleic acid sequences (namely cis-cleavage), thus increasing further selectivity and specificity (Jinek et al., 2012; Cong et al., 2013; Kellner et al., 2019). Cas12a, an RNA-guided enzymes, recognizes dsDNA, and exhibits its non-specific cleavage activities for single-stranded DNA (ssDNA) upon activation (Chen et al., 2018; Swarts and Jinek, 2019). Combining the cleavage effect of Cas12a with isothermal amplification techniques has created a versatile rapid and specific platform, such as DETECTR (DNA Endonuclease Targeted CRISPR Trans Reporter) with fluorescence readout (Chen et al., 2018), Cas12VDet (Cas12a-based Visual Detection) in a one-pot reaction (Wang et al., 2019), lateral flow strips for visual readout (Gootenberg et al., 2017; Myhrvold et al., 2018), as well as colorimetric detection with AuNPs-DNA probe (Li et al., 2019; Yuan et al., 2020). In contrast, the visual readout mode is more suitable for on-site detection needs than the fluorescence signal requiring lab instruments.
Principle of the RPA/Cas12a-based portable platform for the detection of plant fungi. (1) Plant tissues, such as stems with black canker symptoms, leaves with phoma leaf spotting, wizened oilseed rape seeds or residues with black spots, or fungi hyphae were collected from samples in field or at port. (2) Plant tissues or fungal hyphae were ground with quartz sands and plastic pestle to mud or powder, and the DNA was extracted with fast nucleic acid release reagents. (3) Lyophilized RPA pre-amplification reagents (containing the enzymes, primers, probe, and buffers), and Cas12a reagent mixture (containing crRNA, cas12a enzyme, and reaction buffer) were pre-stored in one tube or the chambers of the chip. (4) The fungi genomic DNA was pre-amplified with RPA enzymes and primers at 37°C for 20 min. (5) The RPA pre-amplification products were detected with CRISPR/Cas12a system at 37°C for 20 min. When the RPA products contained the target DNA, the binding of Cas12a/crRNA to target DNA would initiate its ability to digest reporter ssDNA with biotin and FAM group. (6) The intact ssDNA reporter was captured by the streptavidin on lateral flow strips to form control band, or else the cleaved FAM group binding with the FITC antibody conjugated gold particles moved to the test band. The lateral flow assay was complete within 5 min, and the bands could be immediately visually observed. N, negative sample; P, positive sample; SP, strong positive sample. 2b1af7f3a8