![]() "Baḳbuḳ" ( Jeremiah 19:1,10 1 Kings 14:3), also meaning an earthen vessel, was perhaps used for drinking purposes. You can easily improve your search by specifying the number of letters in the answer. "Nehel," which has a similar meaning, may have originally designated a waterskin ( 1 Samuel 1:24, 10:3, etc.), but later it undoubtedly signified an earthen vessel ( Isaiah 30:14 Lamentations 4:2). Crossword Clue The crossword clue Drinking vessel with 7 letters was last seen on the April 17, 2022.We think the likely answer to this clue is TANKARD.Below are all possible answers to this clue ordered by its rank. Jugs were also used as drinking-vessels in 1 Samuel 26:12,16 a "ẓappaḥat" (cruse) is mentioned, probably a bulging jug carried on journeys as a drinking-vessel. Ecclesiastes 12:6) rather than as a drinking-vessel (comp. The "kad"-mentioned in Genesis 24:14 et seq., which was carried on the shoulder, and from which Rebekah gave Eliezer water ( Genesis 24:18)-was used for drawing water (comp. The "aggan" mentioned in Song of Solomon 7:3 is not a bowl for drinking, but rather for mixing wine with spices hence κρατήρ in Septuagint. In Exodus 12:22 and Zechariah 12:2 a saf is used at the sacrifice. The word "saf" mentioned in 1 Kings 7:50 2 Kings 12:14 and Jeremiah 52:19 probably refers to a bowl also. ![]() Judges 5:25 shows that in addition to the bowls of ordinary size there were larger ones, evidently designed for guests of honor, who were served with double portions ( Genesis 43:34 1 Samuel 9:23 et seq.), not only of meat, but also of drink hence the use of the phrase "sefel addirim" (lordly dish). The bowl, which was called "sefel," was used for holding milk ( Judges 5:25) and for drawing water ( Judges 6:38). "Kefor" ( 1 Chronicles 28:17 Ezra 1:10, 8:27) means "cup," as is evident from the Assyrian "kapru," and from the Neo-Hebraic and Judæo-Aramaic "kefor" (compare Euting's combination with = "bulging," in Nabatæan Inscription No. The same applies perhaps to "ḳubba'at" ( Isaiah 51:17), to which the accompanying word "kos" is probably a gloss. the term "gabi'a" is used to designate "Joseph's cup," which, according to Jeremiah 35:5, seems to have been larger than a kos, and was probably a chalice or a goblet. 1 Kings 7:26 shows that the rim was often bent, and Isaiah 51:17,22 indicates that the sides were bulging. A cup was usually called "kos," a designation applied both to the cup of the poor man ( 2 Samuel 12:3) and to that of the king (Gen. In regard to form the vessels may be divided into two groups viz., (1) cups and (2) bowls. ) hence it is probable that the drinking-vessels of the Israelites resembled very closely those in use among the Phenicians. It may be safely assumed that these metal vessels were first imported by the Phenicians, and that the Israelites learned from them how to work the metals (compare 1 Kings 7:12 et seq. The wealthy had metal-usually silver-ones ( Genesis 44:2), while those of the kings were of gold ( 1 Kings 10:21 2 Chronicles 9:21 ) or probably of bronze. The Israelites probably first saw earthen drinking-vessels in Palestine, where they were used by the common people. It served both as a receptacle for water and for milk and as a drinking-vessel. The water-skin ("hemet," Genesis 21:15,19 "ob," Job 32:19 and "nod," Judges 4:19), made of the hide of the goat and the kid, and still used among the Bedouins, certainly dates from very early times. Less is known of the form and material of the drinking-vessels of the Hebrews than of those of the Greeks and the Romans. A drinking vessel of sanyāsis, kamandalu s. Kettuka to prevent the milk from flowing (by charms). A female screw the part of an instrument which receives the handle, so. Naattu - cucurbita lagenaria, the gourd serves as drinking vessel (churangaatthondu) paalcchuranga (kutthi unakki a med. kinds: kaattu - also kyppacchura, from the gourd of which the chirappakkurari is made - kaattuchu ranga theerththatthil mukkiyavan n. , churanga the fruit, used as vessel by toddy-drawers (also churatthondu no. Pumpkin, water-gourd churavalli, churattha ndu med. Enre chu kkiri tharane (the customary gratis-drink to drunkards). A vessel intended for drinking Related definition of Drinking vesselĬhukkiri čukkiri a drinking vessel in toddy- & arrack-shops (a small chiratta).
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![]() GIMP even has a way to animate your pixel art and you can get onion skinning to work too. Doing straight up pixel art, you really need to know how to set GIMP up for it. If you're really good at drawing or sketching out your art, you can use GIMP to pixelize it. ![]() Those were just some of the things I found in the short time I used this software. Also, if you want to always start off with a blank palette, you first would have to create a blank palette, but the good thing is that you can save and set that palette to default. One thing I didn't quite like about it is that if you change one of your colors on your palette, it doesn't change that color where you used it on your canvas. It might have a steep learning curve though. If you can get used to the hotkeys, I'd say this offers the fastest possible workflow along with every tool or function you could possibly want when it comes to pixel art. I'm currently learning how to use this software and I'm really impressed so far. So yeah, Photoshop is still the best tool out there, the only problem is a little slow startup and resource consumption on older PCs, but then you can probably find older Photoshop version, for say Windows XP - it would still be order of magnitude better than GIMP. I asked on their mailing list why don't they have proper spectral editing, and they answered something about it being implemented long ago, but patches were rejected, due to the development organization. I'm surprised that for several decades GIMP community has failed to implement something as simple as proper animation support.īut well, same is true for Audacity vs Adobe Audition, which has nice pitch filters following envelope and spectral editing capabilities, surpassed only by SpectraLayers. After installing GAP on a 64-bit system, there are a number of DLLs that fail to load and startup time becomes even longer. I'm currently using GIMP, but, it has the worst shortcuts of all drawing programs in existence: there is no way you can bind color picking (the most important operation after painting itself) to the right mouse button and eraser is bound to SHIFT-E! Why, God?!! Why adding shift? But truly the worst is animating with GIMP: compared to Adobe Photoshop, GIMP doesn't have layered animation or onion skins, and you have to install this glitchy barely maintained GAP toolkit and split your project into a mess of *.XCF files - one for each frame (yes, separate XCF files, instead of frames sharing a set of layers). Other features which stand out are selection tools, and it's constant updates.Īseprite is open source and you can compile your own copy for free, but a precompiled copy costs $20įor more software recommendations, check out the lists at: OS: Windows, Linux, and various ports including for OS XĪseprite is trying very hard to be the most complete pixel art editor, and has great support for layers and animations. Support for layers and animation is a bit clunky, but it has tile editing features, great palette editing tools, and you can install DawnBringers toolbox on it! It's open source software The interface is very different from modern software which might be confusing at first, but it has all of the essential tools for pixel art. GrafX2 is based on the old school DeluxePaint. It supports layers, onion skinning, indexed palettes, sprite sheets and has many more nifty tools. GraphicsGale has recently become freeware! This wonderful software is ideal for animating pixel art. Here below I will write about the ones I recommend, feel free to add your favorite, or ask software related questions!įree does not mean bad. Some software makes it easier than others. Not only is it ideal for artists who create detailed and pixel-accurate graphics for mobile games and portable game consoles, it is also ideal for creating lightweight graphics for Web or Web-based applications and games.Making pixel art requires graphical software. Pro Motion NG is an excellent pixel drawing and animation software developed by cosmigo from Germany, whose design is similar to the famous Deluxe Paint (DPaint) on Amiga OS. This work is licensed under a Creative Commons Attribution-NonCommercial-ShareAlike 4.0 International License. Otherwise, please bear all the consequences by yourself. Otherwise, you may receive a variety of copyright complaints and have to deal with them by yourself.īefore using (especially downloading) any resources shared by AppNee, please first go to read our F.A.Q. page more or less. To repost or reproduce, you must add an explicit footnote along with the URL to this article!Īny manual or automated whole-website collecting/crawling behaviors are strictly prohibited.Īny resources shared on AppNee are limited to personal study and research only, any form of commercial behaviors are strictly prohibited. This article along with all titles and tags are the original content of AppNee. Classifications can be transferred between projects via XML file format. In addition, classifications are core to intelligent OPEN BIM workflows, providing unprecedented interoperability among disciplines. While previous ARCHICAD versions provided a fixed set of Element Classifications, ARCHICAD 21 introduces a flexible way to classify elements supporting any national or company-standard classification system. Element Classification SystemĪn ARCHICAD model can be described as a central BIM database that stores all project data and makes it accessible to any project stakeholder. The latest version introduces Light Mapping and Secondary GI methods for more realistic, yet fast rendering. Updated CineRender EngineĬineRender by MAXON offers architects integrated, photo-realistic rendering options in the BIM context. As for the Railing Tool, one-click input creates an instant associative railing along stairs or other building elements, whose posts and panels can be assigned as patterns, or customized individually. Intuitive graphical methods allow users to easily tweak the stair’s shape and to customize structural and finish components. If a problem can be solved in a variety of ways, the software offers a selection of the best options that fit both the user’s graphical input and the chosen standards. With ARCHICAD 21’s next-gen Stair Tool, architects can jump right into the creative process: placing stairs with simple polyline input and choosing from various design options. In other words, ARCHICAD’s algorithms validate thousands of design options in the background and offer architects the most optimal stair designs to choose from within the context of the specific building. The new Stair Tool in ARCHICAD 21 extends architects’ creativity with automatic validation against human ergonomics - on the fly. The countless iterations required to sync design intent with these standards in a specific building has meant tedious, manual work – up until now. What’s new in ARCHICAD 21 New Stair and Railing Toolsĭesigning stairs is one of the most complex tasks in architecture, since the stair must comply with stringent standards. We also further refined ARCHICAD’s best-in-class OPEN BIM workflows in version 21, making interdisciplinary collaboration a very smooth process.” said Peter Temesvari, Director of Product Management at GRAPHISOFT. ![]() Our next-gen Stair Tool provides a creative, productive and enjoyable user experience – the reason why architects love ARCHICAD, ARCHICAD 21’s Stair Tool does the heavy lifting in the background – making sure the architect’s stair design complies with relevant global and local design standards – turning what is usually a painful task into something fun. Intuitiveness has been a key differentiator for ARCHICAD since the beginning. The latest version of ARCHICAD introduces the highly-anticipated Stair Tool, featuring GRAPHISOFT’s patent-pending Predictive Design™ technology. GRAPHISOFT® today announced ARCHICAD 21, the upcoming release of its industry-leading BIM software solution. ![]() We can also offer you a demo or assist you in the implementation. Should you have any questions please feel free to contact us. You will find users guide and deployment guides for administrator. Please take a couple of minutes to watch this YouTube playlist. The App Award winning App, iGlobe Outlook iPlanner Pro Add-in, provided by iGlobe, brings you the Planner to Outlook. Planner can be used to manage a marketing event, brainstorm new product ideas, track a school project, prepare for a customer visit, or just organize your team more effectively. Planner makes it easy for your team to create new plans, organize and assign tasks, share files, chat about what you’re working on, and get updates on progress. The Planner offers people a simple and highly visual way to organize teamwork. From 50 users, US $1 (Euro 1,-) per user per month based on a yearly subscription.Īs a member of the Office 365 suite, Planner is integrated with other Office 365 services, such as Groups and Microsoft Teams. Price, up to 50 users, US $2 (Euro 2,-) per user per month based on a yearly subscription and a minimum of 5 user per order. To purchase from iGlobe send and email to with the number of required users and invoicing information. After the FREE trial period you can purchase a subscription by contacting iGlobe, your Microsoft Partner or at Microsoft Store. When iPlanner Pro is deployed, you will automatically get a FREE 30 days’ trail for 3 users. Congratulations to all of the winners.’ – Rob Howard It’s great to see so many apps making a positive impact through well-crafted integrations with Microsoft 365. ‘The Microsoft 365 App Awards recognize apps that provide an outstanding productivity experience, as nominated by its users. IPlanner Pro winner of Best Integration 1st place Microsoft 365 App Award Winners 2019. Get Planner tasks from Office 365 Planner & Teams at the tip of your hand, in Outlook for Windows, MAC, IOS or Web mail. Managing and creating tasks has never been easier. ![]() It's a type of Auto Clicking tool, that can make clicks at a very fast speed. Here are some best auto clicker for windows also you can find perfect auto clicker for games:įast Mouse Clicker Pro is one of the best Auto clicker software. Best Auto Clickers for windows and games: Mostly used the software just for fun, also helpful as well as very interesting. So, according to the medical point of view, such programs are very beneficial to avoid any type of physical injuries. In these circumstances, users have to face many more physical injuries. Moreover, this software helps gamers to avoid physical injuries because in most cases, players use the different methods of clicking and click the mouse many times in one breathe to beat the score. So, players must use this program to make their ideal score, because it makes a lot of clicks automatically without any effort and provides you with an incredible number of clicks. Some are unable to get their dream score even after many attempts. Players need to put a great strength or stamina to play and win the game within the given timeframe. Auto Clicker also makes your clicks faster and gave you the exact and more accurate number of clicks. Auto Clicker Benefitsīy using this program, you can get your high CPS in click test games. Hotkeys work in the background for convenience. The maximum amounts of clicked can also be set (or left as infinite). A full-fledged autoclicker with two modes of autoclicking, at your dynamic cursor location or at a prespecified location. Press Windows key + X on the keyboard at once. The tool allows you to auto click your mouse, no need to exert any effort on clicking the mouse button or space bar, very helpful for those who don't want to put their energy into continuous clicking. Once the installation is complete, open the auto clicker. Double click on the downloaded file and follow on-screen instructions to install the auto clicker. Click the Download button above to download the installer. How to use Auto Clicker for Roblox? Here are the steps to use an auto clicker in your favorite Roblox games.And we also have one free auto clicker tool named 'Free Mouse Auto Clicker'.It is absolutely free for all.' Free Mouse Auto Clicker' can automate click mouse at the current mouse point for you.It is simple but very useful.It support left/right mouse button and single/double click.And it can set interval between every clicks.Basically, auto clicker for windows, auto clicker for mac, and auto clicker for games are most commonly used. The program is very useful among players or gamers. Auto-Clicker is software that helps you to automate the clicking process. ![]() →You cannot use “For Reference Only” content in any manner that entails advertising, marketing or commercialization of any product or service.įor Full Edition of Pikbest License Agreement, please click here to the Pikbest License page. →You cannot imply that the content is created or claimed to be the copyright of the artwork by you or someone other than the copyright holder of the Pikbest content. ![]() You’ll see a variety of envelope templates. Type envelope in the search box at the top of the screen, then press Enter. Select Public Templates to explore all available templates, and Documents to return templates for Google Docs. →You can only use an Item for lawful purposes. Google Docs provides envelope templates, but they’re a bit hidden. You can’tĬlaim trademark or service mark rights over an Item within the End Product created using that Item. →You cannot use the content in the Pikbest Content (totally or partially) in any trademark, logo or part of the same. 10+ Envelope Templates in Word Beauty Parlor Envelope Template in Word. →You cannot use the content in the Pikbest Content in printed or electronic items aimed to be resold. Here, we have provided you the step-by-step instructions to help you create envelopes in any version of Microsoft Word as you utilize the best printable. →You cannot resell, redistribute, access, share or transfer Pikbest Content. DefaultHeight: Returns or sets the default envelope height, in points. DefaultFaceUp: Determines if envelopes are fed face up by default. Creator: Returns a 32-bit integer that indicates the application in which the specified object was created. While privileges are different upon different user types, there are some restrictions which are applicable to all Pikbest users. Returns a Application object that represents the Microsoft Word application. The Pikbest Authorization differs upon different situations. The playful summer Santa design envelope template in Word could be great for your next holiday letter.Ĭheck out all the templates for envelope design to see what works for you.Pikbest authorizes the User in a non-transferable, non-exclusive manner and on a worldwide basis for the duration of the relevant rights to download, use and modify the Pikbest Content, as expressly permitted by the applicable license and subject to this document. Templates for envelope creation are useful for wedding invitations, business announcements, and other mass mailings. In addition, customized designs can also be made using programs such as MS Word. Choose a standard 5x7 envelope template to easily create the perfect sleeve for your next correspondence. There are many templates available online as well as on different programs for making envelopes. In the Stencils view in the Shape window, right-click the shape, and point to Add to My Shapes. Save an individual shape to your Favorites stencil or a different stencil. To produce templates for envelope, simply choose your design, customize with your name and contact information, type in your recipient’s address, and print on the size of envelope indicated in the instructions. Right-click the title of a stencil in the search results, and then click Save As.Type a name for the stencil, and save it in your My Shapes folder. You can also create your business stationery using a matching letterhead and Word envelope template. The green wave design envelope template in Word is a particularly attractive option to catch your customers’ eye. A standard number 10 envelope template can complete your collection of business identity documents-just add your company name, logo, and address. Give your professional and personal correspondence an edge with Microsoft envelope templates. Envelope templates make your mailings look marvelousĪ well-designed printed envelope makes an impression, no matter what it contains inside. Visualize audio in astonishing new ways, including 3D. For the first time you can directly edit spectral data, opening advanced sonic worlds, and use innovative enhancements to traditional techniques. SpectraLayers is a new approach to audio editing, with new concepts to transform the way you work. Read included R2R.txt to see how to install.Steinberg SpectraLayers Pro 8.0.20 (macOS) | 400 MB * No compatiblity issue with other legit Silk and Silk powered apps. * No SteinbergActivationManager is required. * No SteinbergLibraryMangager is required (optional). Invert the process to audition problem sounds in isolation, then execute the repair after you’ve dialed in the fix you need.īetter interpolation and faster performance, now assisted by AI, looks more closely at noise, tones and transients, making this easy repair process more versatile and precise than ever. The AI in SpectraLayers Pro 7 has seen and evaluated 32,000 of them, so it knows exactly what to look for and how to automatically apply the best repair.Īutomatically detect and repair clicks, crackles and pops. Hum harmonics can be independently attenuated to the level you choose.Ĭlipped peaks come in many shapes and sizes. This automatic process detects and eliminates unwanted hum, as well as higher harmonics generated by the fundamental tone. Manual or automatic - either approach is far more accurate than typical waveform-based plug-in processing. Now, the new automatic de-essing feature makes it even easier. Manual de-essing in SpectraLayers Pro has always been quick and easy. Spectral de-essing is the most precise and least invasive way to tame and shape vocal sibilants. ![]() You can automatically attenuate everything but the voice for maximum intelligibility. Perfect for cleaning up location interview recordings, the Voice Denoiser algorithm is trained to recognize and isolate the human voice. SpectraLayers Pro 7 allows you to select a target sound and then trigger a search to automatically locate and select every other similar sound in the spectrum. Significant advances in pattern recognition are a hallmark of today’s rapidly evolving AI technology. Comprehensive learning algorithms clear the path to clean extraction. Reconstruct missing noise hidden by tones and transients. Unmix stems into tone, noise and transient component layers. (SpectraLayers Elements allows extraction of vocals only.) This AI-assisted process in SpectraLayers Pro scans the finished file of a song, allowing you to “unmix” it and extract the different instruments to separate layers, with default settings for vocals, piano, bass, drums, and more. ![]() AI is changing faster than its history can be written - be at the leading edge with SpectraLayers Pro 7. In the same way that these algorithms can now accurately determine how to apply color to a black and white photograph, the AI in SpectraLayers Pro can sense discrete events in a sound field and isolate them on independent, colorful layers for processing. Just as audio engineers focus on separating increasingly fine details in sound, the new AI in SpectraLayers Pro 7 uses algorithms to learn from the data flowing through its built-in neural network. So, despite its advanced technology, SpectraLayers is immediately familiar and instantly powerful. To make this approach intuitive, SpectraLayers employs the model of high-end image editors - not just the interface, but what you can do with sound and how you do it. It extends the traditional visualization of spectral editors to every aspect of editing sounds, including how they mix together. SpectraLayers is the first completely visual editing system for audio. ![]() And all in a familiar interface that you didn’t realize you already knew. Integrate these abilities into your DAW and other tools. INTRODUCING THE WORLD'S FIRST ADVANCED SPECTRAL EDITOR ![]() These flower heads are available in different colors, so you can easily adapt the color scheme to your own wedding palette. I did create a silk flower page, so you can find the head sizes you need in silk. ![]() If you look closely at the photo, you'll see the upper left corner is filled in with Baby's Breath (filler) and greenery. Find suppliers of artificial flowers wedding. CAMBREL S Notches on it, on which CAMPANULATE Flower ( among HerButchers hang. 32 stems of Medium Flowers (4" - 5" heads) Get artificial flowers wedding arch from when you need beautiful decor that will last a long time.Whats more, wedding artificial flower arch, made of high quality materials, durable and easy to. 28 stems of Large Flowers (6" - 7" heads) Shop wedding artificial flower arch at great deals online.14 stems of X-Large Flowers (8" - 9" heads).Choose from Same Day Delivery, Drive Up or Order Pickup plus free shipping on. White Craft Glue (Silk) or 2 cans Floralock Stem Adhesive (Fresh) Shop Target for silk flower wall decor you will love at great low prices.24 Brick Sahara (silk) Foam Bricks or 24 Oasis (Fresh) Foam Bricks.So how do you create this look? Let me give you an estimate of the number of flowers used to create this design: Follow the link, as I explain it in detail. For this, I have used my estimate (calculating flowers) sheet for years. The tie is Direct Importers and Manufacturers entirely made of silk, and is joined. ![]() ![]() Why is this important? For one thing, you'll need to know how many flowers to order. The South's Largest Millinery House chic novelty in floral trimmings. I alway make up a estimation of what flowers I will use, breaking it down by size. Many artificial flowers wedding arch have features and prices that will help you serve your personal or business needs while keeping within a budget.So how do you calculate how many flowers you need? 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Many come in glasses or boxes for easy storage and display for your convenience. Certain types work best as everyday home decorations, while others are best used for parties and weddings. Everything is set on a felt base to protect. Others come still attached to their branches and their stems for a more natural appearance. The flowers are an assortment of handcrafted paper flowers and silks. Some artificial flowers wedding arch come in large bunches with bright colors. Browse a wide selection of colors and flower types. Many varieties are suitable for use in bouquets or arrangements to bring festivity to a special occasion. Decorators and home designers can make good use of these artificial flowers wedding arch. All varieties have been preserved to ensure they will not wilt in the same way living plants would. About products and suppliers: Find artificial flowers wedding arch on when making attractive decorations that last a long time. If you do have accelerator pack, you will see the Unlock everything button option on the purchasables section. The accelerator in the game will unlock everything, all of WWE 2K20’s unlockable superstars, arenas, as well as all of the Daniel Bryan 2K Showcase mode unlockable. You can unlock easily by the Accelerator, which is part of the WWE 2K20 Deluxe Edition. WWE 2K20 has a lot of unlockables of the superstars and championships. You could access the store after going to the game on your console’s home screen and scrolling down to the ‘Store’ option which will be right at the bottom of the list. You can buy these unlockables on your consoles. Josie Jane, Unlocked in M圜areer Kassius Ohno (Zombie) (DLC), Complete the STORY TOWER: All Fed-Up Kevin Nash, 3,000 VC Kurt Angle 01, 5,000. These are the list of characters you can unlock and play with, and for how much. Let’s discuss those and what it could mean for the game. Unlock all 2K Showcase Videos/Story Missions etc. Unlock Everything Unlock all outfits, moves, characters belts, etc. And now the fans would love to get their hands on some cheat codes of the game. It works for Current Roster (Play) Mode & WWE Universe Mode (All 3 Slots). ![]() The game did however, start working properly, eight months after its debut. If you’ve got the Season Pass for WWE 2K20, you’ll already have the Accelerator. Once you’ve purchased it, simply make your way to the same menu as before and press Square (X on Xbox One) to unlock everything at once. The company has decided to not release any more WWE games for the foreseeable future. DLC that essentially unlocks everything in the game for you from the beginning. The horrible graphics and full of lags gameplay was too poor. It was regarded as one of the worst games of 2019 and one of the worst wrestling games of all time. ![]() WWE 2k20 came out at the end of October last fall and fans were expecting so much from it but it just disappointed. ![]() Among the benefits of this design, a chimera allows concurrent testing of disparate target regions of a single pathogen or even different organisms and splitting targets between standards A and B enables control cross-validation, facilitating the distinction of control failure from test failure or success. Furthermore, the equally partitioning of target sites between standards A and B enables cross-validation of positive and negative controls, increasing the confidence in test results. This means that for each target used in the molecular test, standard A would act as positive control, while standard B would act as negative control, or vice-versa. In this design, all the target sequences of a molecular test are retrieved and split between groups A and B, which are then joined in tandem to form single chimeric sequences A and B. ![]() Here, we propose a new design strategy for reference standards that uses matched chimeric synthetic standards in accordance with the principle of A/B testing. The use of in vitro synthesis of RNA and DNA standards allows flexibility in control design and tailoring of controls to the diagnostic test and targets. This leads to delays in diagnosis, missed diagnoses and invalidation of correct test results. For example, if the test returns a negative result from the positive control, it could be because (i) the test failed, (2) the reference control failed or (3) a technical issue with the testing platform. However, this failure of either positive or negative controls is difficult to distinguish from the failure of the diagnostic test itself. In the case of RNA standards, the synthetic controls can also undergo degradation over time, and can be contaminated, confounding the interpretation of test results. Reference standards must be validated and proven fit-for-purpose before used in diagnostic tests. However, separate negative controls, without the target sequences, are also required to ensure the specificity of the diagnostic test. These can be used as positive controls to assess sensitivity of the molecular test undergoing evaluation. The recent advent of DNA synthesis enables the rapid development of reference standards as synthetic constructs representing target genetic material. Reference standards are required to validate the performance of any diagnostic test 1. This chimeric reference standard design approach offers extensive flexibility, allowing representation of diverse genetic features and distantly related sequences, even from different organisms. The chimeric A/B standards were assessed using the US Centres for Disease Control real-time RT-PCR protocol, and showed results congruent with other commercial controls in detecting SARS-CoV-2 in patient samples. This enables control and test failures to be distinguished, increasing confidence in the accuracy of results. This design enables cross-validation of positive and negative controls between the paired standards in the same reaction, with identical conditions. Accordingly, a target region that is present in standard A provides a positive control, whilst being absent in standard B, thereby providing a negative control. The chimeric standards constituted target regions for RT-PCR primer/probe sets that are joined in tandem across two separate synthetic molecules. Using a pair of chimeric A/B RNA standards, this allowed incorporation of positive and negative controls into diagnostic testing for the Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2). However, negative controls are also required to evaluate test specificity. Current reference standards typically represent target genetic material, and act only as positive controls to assess test sensitivity. These are used as controls, and allow measurement and improvement of the accuracy and quality of diagnostic tests. DNA synthesis in vitro has enabled the rapid production of reference standards. |
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