Reagents and technologies to address public health & pandemic response

hello and thank you for joining the new england bio labs webinar entitled reagents and technologies to address public health and pandemic response i'm sal ricello and i direct the oem and customized solutions team at new england bio labs our group oversees the incorporation of navy agents into a variety of third-party products and services including those used in the detection treatment or prevention of human diseases including sars kobe 2. you'll hear from myself and two other speakers today dr nathan tanner and dr nicole nichols nathan one of our research scientists will talk about a number of innovations in enzyme discovery and design and how those innovations can be brought together to build new technologies important for viral rna detection you'll also hear from nicole an associate director in our applications and product development group we'll talk about how our product development strategy is influenced by the areas our customers work in including molecular diagnostics we hope you enjoyed today's webinar hopefully many of you are familiar with mimicron biolabs were founded in the early 1970s as a company of scientists for scientists nab has been hoping to drive the innovation of our customers and partners for more than 45 years from the commercialization of the first recombinant protein in the early 1980s through the incorporation of reagents in several of the early molecular diagnostics of the 90s and their adoption in new fields of genomics like clinical next gen sequencing in the early to mid 2000s through today when they're being utilized as critical raw materials and a variety of technologies useful in the detection and prevention of sars kobe 2. and throughout our history nab has ensured that our capabilities address the changing needs of our customers and the markets that they operate in ensuring that we have the right products for research users the right quality infrastructure ultimately in today's world the appropriate scale and supply chain robustness and resilience to enable our customers to utilize those reagents and molecular diagnostic technologies we'll touch on three topics today the innovations of our research group our ability to optimize and improve our reagents to ensure they're useful to customers in the mdx space and ultimately how only enables those partners throughout their product development scale up and commercial plans dr nathan tanner will start us off all right thanks sal i thought i would give a few examples of the work we do in research to innovate and build new tools for better diagnostics first example i'll use is protein ace k enzyme in a lot of workflows a molecular biology standard that's been around for a long time but it's a really sturdy protein if you're going to use it you really have to inactivate it and the standard way to do that is to boil it so that does work however that can cause some problems for different workflows if you're doing sequencing for example you may not want to damage the dna if you're looking at isothermal amplification uh the enzymes being used can't go up to those temperatures so it makes the workflow a little more complicated so what we did is we took the protein sk enzyme identified ways to make it a less thermostable protein and made a mutant version uh called thermal labile protein sk so that's the the red on the gel there so that enzyme is heated at the indicated temperature and you see that the bsa is preserved we just run it on a protein gel at lower temperatures than is needed for the wild type protein sk where you really have to go up to 90 95 degrees to really kill it so this is designing a new enzyme to enable better technology another key area where we needed to apply some innovation to improve diagnostic techniques was in carryover contamination prevention this is a commonly used workflow for molecular diagnostics particularly in pcr methods where dutp is incorporated in the production of the amplicon and then an enzyme called the uracil dna glycosylase takes out those uracils and prevents the amplicons from being re-amplified as a false positive when there's some sort of contamination in the lab workflow so when we looked at isothermal workflows in particular lamp the core polymerase the bst large fragment shown on the black curve on the graph it really doesn't incorporate dutp very well it's strongly inhibited as you go to the higher concentrations of the utp but then our engineered polymerases bst 2.0 and bst

3.0 do a much better job incorporating dutp but this is only the first half of the equation for the second half we also need a udg compatible with these workflows so here the standard enzyme e coli udg marked in blue it works great for pcr but when you look at lamp and reactions that are incubated at lower temperatures it maintains a bit of activity in the 50 to 60 degrees celsius temperature range which would be a problem for these reactions because it's still active when you would incubate for your desired reaction so rather than just preventing carryover it would actively be preventing amplification so rather than design on this one we went prospecting in nature looked at psychophyllic organisms that had homologs to e coli udg identified a number of them screened them in the lab and as a result now have antarctic thermal labial udg in our catalog enabling carryover contamination prevention for a broader range of workflows another area that required some innovation was the control of enzymes for diagnostic workflows most of you are very familiar with hot start pcr polymerases where you have some mechanism of inhibiting the enzyme until you heat it up in the denaturation temperature pcr and this works great however those antibodies or chemical modifications that you use to make that don't really translate to lower temperature regimes where you'd use a reverse transcriptase or a polymerase for an isothermal amplification method so what we've looked at instead are aptomers so we take pools of modified nucleic acid aftermers and do selects or rounds of in vitro evolution and selection to get specific sequences that bind a target of interest inactivate it or provide some inhibition until a certain release temperature that we can program in the c-lex workflow so we look for something 40 to 50 degrees where below that the abdomen is bound to the enzyme inhibits activity then we heat it up slightly after mark comes off releases and gives active enzyme so these provide a couple benefits it works at the lower temperatures most importantly but also it's reversible at the end of the reaction the aftermarket is not affected and can rebind the target of interest and it's immediate there's no activation step after my heat comes off as soon as you heat it to the temperature and by using these warm start as we refer to them aftermers you can enable control at lower temperatures and get dual enzyme control for things like rtlamp as shown here where we set up an rtlamp reaction with our bst 2.0 and rtx engineered enzymes and what happens if you run the reactions right away that's the the dark orange bars there you get a threshold time seven eight minutes or so but if you let that reaction sit on the bench for two hours and then run it now it goes out to close to 30 minutes which is a big change in relative performance the reason for this being activity by the enzymes while it sits there at room temperature and importantly both of the polymerases are contributing to this so if you look at the bst 2.0 and the rtx rows just having one of the enzymes be warm start isn't enough you still get that effect but if you go to the very top where we have both enzymes controlled by warm start aftermers now we maintain that performance sitting on the bench for two hours so it lets you do room temperature setup with dual enzyme control you can see the benefits of that dual enzyme control in rt qptr workflows as well so shown here is a commercial qpcr master mix which gives nice data on the left part of the figure here good sensitivity specificity all the milk curve products line up nice but if you let that reaction sit overnight on the bench it has a hot start attack but the reverse transcriptase is not controlled and what you see is production of non-specific products particularly in the milk curve but when you use the luna rtq pcr mix which has a dual enzyme control do that same thing run the reaction immediately or let it sit on the bench overnight you maintain performance and get the same consistent specificity no production of off-target effects due to that dual control and with these isothermal tools i've mentioned they're really designed for simple portable diagnostics but in order to do that you really need a way to read things out in the field away from the lab and your qpcr machines so the enzymes work great but they're only good if you can see the results of the reaction so what we do is utilize a natural property of the dna polymerase reaction where for every incorporated dntp you produce a pyrophosphate and a proton so if we formulate the reaction just right the reaction produces a lot of protons as it makes a lot of dna we can drop the ph of the reaction so just by building in a ph indicator here we use phenol red it's a nice pink color as a master mix but if it amplifies ph drops it turns yellow and you get a really simple visual readout of amplification that you can do just by heating the reactions in a water bath so this ph based approach was described in a paper a few years ago we have a master mix that's been a useful tool for simple diagnostics but as the covet 19 pandemic really ramped up back in the winter we saw an opportunity to demonstrate that colormetric lamp could be used in clinical settings as well so we partnered with the lab at the wuhan institute of virology where they had access to samples and in february put out the pre-print shown here these are real clinical np swabs taken then and detection by color metric lamp we just wanted to show the principle and get some attention to potentially a new way to do diagnostics you'll hear more about how this is taken off shortly another feature of molecular diagnostic techniques that we would like to bring to lamp and isothermal amplification is the ability to do multiplex target detection so it's useful in situations where you have multiple targets you want to test for in one sample where you want to include an internal control in the reaction just to verify nucleic acid material came over or to to add multiple targets for extra confirmation that the target of interest is really present so in 2012 we put out a paper describing a way to do this with lamp and then recently applied this to cova detection where we put a sars cove 2 rna target together with influenza a influenza b and an internal control you can do this real time detection on a qpcr machine or even shows a simple fluorescence plate reader that can be used for an endpoint detection so this isn't quite as simple as the color metric readouts but brings the power of real multiplex molecular diagnostics to isothermal amplification and as a final example building from the colormetric lamp pre-print with wuhan i mentioned once that came out we started getting a lot of interest for labs trying it out and one observation was the sensitivity wasn't quite where it needed to be so we went back to the lab and tried to improve that and some of the things we did are fairly obvious just new and better primer sets so we optimized our primer sets but also we took to combining primer sets together for improved sensitivity and also adding an enhancing compound quantity and hydrochloride which we described in this paper shown here and it's really strikingly seen on the image there where at the top with low copy inputs we're getting some partial positives those are the orange ones in the circle but by adding in guanidine we dramatically improve the speed and sensitivity where you get much more reliable detection at the low inputs shown by all the strong yellows so all those of the positives and this is a good example of how our innovative ideas are put into practice and that will now be described by nicole nichols in more detail showing how we take the ideas from early stage research develop them fully and turn them into novel nab products thank you nathan so i'm nicole nichols and i lead the scientists at anybody who are tasked with developing all of our amplification reagents from those that end up in our catalog to those we develop with and on behalf of our customers and collaborators as we look at the work we've done and how it can be leveraged for infectious disease diagnostics and covet in particular we can group it into three main topics the gold standard of qpcr rtqpcr the current state of rapid molecular and the promise of how that field is continuing to evolve as we move forward so first the gold standard as we looked to create a line of qpcr reagents a few years ago now we were in a position to be able to survey the field and see what was working and what could be improved upon pac is a robust enzyme and our optimal control version enables simple and fast workflows but there is clearly an opportunity to create a better rt traditional mmlv based enzymes lose activity as reaction temperatures rise but the luna rt that we engineered performs well up to 60 degrees and as nathan highlighted that optimal technology allowed us to control this enzyme decreasing non-specific activity due to the rt but in addition to the enzymes there's a significant amount of performance enhancement that can be achieved by optimizing reaction components and conditions as we took on this challenge we ran many many qpcr experiments and we quickly realized the need for a simpler evaluation tool in this graphic our dots and boxes visualization method is outlined briefly it's a way to condense 18 wells of a qpcr experiment to a single dot if the dot is large and solid and falls within the box it indicates a successful run if it's small or falls outside the box improvements need to be made we've put together a video and a white paper describing this method so i encourage you to go to our website if you'd like to learn more for now i'll use this example as an illustration where we were finding different reaction additives to improve performance i hope it's pretty obvious why we might continue to investigate additive d again but should probably not pursue b and we use this method to continue our optimization work until we have the majority of targets falling in the box and although this is a powerful development tool it also became a great way to communicate all the product comparison work we took in this we undertook with this project as you can see here for the dye based dna mix we created with all of the different uh product comparisons across two different instruments and similarly here's a comparison of rtqp cre agents again showing strong performance or lots of dots in the boxes for the linear reagents we developed and i haven't had time to go into a lot of detail here so if you're interested in learning more about any of these data or the methods behind it please go ahead and visit luna qptr.com where we have a variety of resources that help describe all of the methodology here as we turn to sars kobe 2 detection the original two tube versions of our rtqpcr reagents have been used in numerous ldt's and eua approved assays and more details can be found at the url below but one in particular that's garnered a lot of attention is the saliva direct protocol from yale where a simple pro k and heat step enable saliva samples to be taken directly into the lunar reagents recently we've launched an updated version of this product that has everything in a single 4x mix which enables higher sample input volumes and also includes our thermal label udg that nathan mentioned earlier this mix is being incorporated into a variety of sars kobe 2 assays including one from the igi at uc berkeley where luna powers a high throughput 384 well assay called igi lunar and we've been using this new 4x mix in single plus and multiplex stars cov2 assays with strong results in this and the following experiments the multiflex reactions feature the cdc designed n1 and n2 targets with the n1 probe labeled with hex and n2 with fam and a redesigned rna's p target that overlaps an exon exon boundary and can be detected in the sci-5 channel it's this it's this assay that we recently launched as an ru kit and this is the url to the product page but i'll present some of the data with the new kit in the next few slides using purified synthetic rna from twist we observe an lod of five copies across different users and instruments using this multiplex assay setup and since there have been some questions around the quantitation of these synthetic targets we've also looked at a variety of other control materials to ensure that we're seeing strong performance that will be relevant to clinical assays we've also looked at results using a pooling type workflow where results from a single positive sample are detected similarly either alone or in the context of a five sample pool you'll note that the n1 and n2 signals remain consistent while the rna's p signal increases as the sample number is increased and as many labs are thinking about increasing throughput we've heard from our customers that our reagents have been consistent even when they run into automation bottlenecks for example when a delay happens between sample setup and thermocycling what you can see here is our own data supporting that feedback where consistent cqs are observed for reactions set up and held at room temperature for up to 24 hours and consistent with that feedback we also observed that this isn't the case with all reagents and the results aren't impacted whether the lunar reagents are tested in a singleplex or multiplex assay so before i switch topics here's that url again if you'd like to learn more about where rv agents are being used now onto the current state of rapid molecular assays and what's next one of the tremendous impacts that this pandemic has had is on the state of molecular assays and where those assays are being run they're not just being run in hospitals and clia labs but they're also being run in academia and in low resource settings and for those settings simple isothermal assays are particularly useful as nathan outlined we've had an interest in isothermal techniques for some time now and previously this year we gathered up the various products and educational materials under a single url listed here we have a really helpful video demonstrating how lamp works but we also have cartoons and diagrams outlining a wide variety of isothermal methods and really providing a variety of materials that will help you understand how and when and why some of these might be chosen over others and for lamps specifically what we've heard over the years is that primer design can represent a real barrier to further exploration of this powerful technique so earlier this year we launched a new lamp primer design tool to make that process much simpler it has an easy to use interface and greatly simplifies the design of loop primers which help to speed up the lamp reaction it also includes visual aspects like consistent color coding to help orient new users to the intricacies of lamp primer design the neb developed variant of lamp the ph dependent colorimetric lamp that nathan described has been used in a variety of different diagnostic assays and settings before stars cov2 but this technique is getting even more attention now given the widespread need for rapid and simple molecular assays with the increased interest in conducting assays in a variety of settings we upgraded our original color metric mix to include the thermal label udg to help prevent carryover contamination this is just one aspect of a larger strategy that should be undertaken to reduce the possibility of generating false positive results due to contamination issues and we've incorporated this mix into a full kit for stars cobie 2 detection where we've been able to leverage the improvements highlighted earlier to create a strong but simple assay you can see the kit components here and examples of the color changes in the different reactions the kit targets two portions of the sars kobe 2 genome but the primers are mixed into a single reaction actually increasing the chance of detecting a positive sample the control provided in the kit is a plasma that contains the full n gene it also has a t7 rna promoter sequence for generation of rna transcripts if desired so what you can see here on the top panel are representative replicate tests done with synthetic rna used to determine lod in the bottom image multiple negative controls are shown and in the table to the right is a summary of a much larger set of reactions conducted by multiple users to help support these conclusions the colormetric lamp kit can be used in a multiple different types of workflows from on-demand testing to a more high-throughput assay setup in 96 well plates for example and although we've mainly been discussing the products used for color metric lamp we shouldn't forget other useful types of lamp assays all the lamp mixes we provide include the warm start engineered bsd 2.0 and

rtx enzymes but they've been specifically formulated to enable the different lamp techniques the color metric mix has a lightly buffered solution and should be used for ph based color metric lamp and although we typically recommend fluorescent lamp as a more high throughput friendly technique the color metric lamp technology is being used in the color genomics eua approved high throughput assay the e 1700 product and its new version with udg that will be coming out soon are better suited for fluorescent lamp where an intercalating dye is added four other versions of colorimetric lamp such as those involving hydroxynapal blue if you want to learn more information about where our lamp based products are being used you can find additional information at this url below so i hope i've given you a better understanding of the product development work we've been engaged with and how passionate we all are to work together to do good science and enable others with our reagents i don't know when i first encountered this idea from stephen covey about the speed of trust but it's one that positively impacts not only the work we do together at biolabs but also how we work with our customers and collaborators as sal will highlight next thank you nicole i'd like to spend the concluding few minutes talking about any of these facilities and how we enable innovators in the molecular diagnostic space we enable diagnostic development by first and foremost focusing on what we do well namely the design development and production of high quality electrobiology reagents additionally we have no competing interests with our customers and partners as we don't manufacture or sell our own diagnostic products our full production capacity and devoted to meeting the needs of customers whether they be among the largest diagnostic centralized core facilities or early stage platform technology companies developing groundbreaking point of care detection technologies lastly our products are manufactured to stringent quality standards to ensure that they function reproducibly in the hands of our customers and we pride ourselves on the fact that those products and the scientists that discover develop produce quality control and support them are trusted worldwide though nab has a global footprint and reach around the world our manufacturing infrastructure is located entirely in the u.s with facilities in ipswich rowley and beverly massachusetts this is an aerial photo of our ipswich facility where the manufacturer of reagents useful in the development and production of our customers molecular diagnostic reagents takes place the facility is roughly 180 000 square feet and houses research production development and manufacturing infrastructure furthermore our ipswich facility houses scalable high-density fermentation equipment and downstream processing infrastructure to ensure that our scale effectively matches the demands we see for our products the ability to flex our production capacity help position nab to maintain the supply of reagents required to help address the demand for molecular diagnostic products created by the recent sars cov2 pandemic we'll discuss this further in a few moments our rowley facility located just a few miles from our corporate headquarters in ipswich was purpose-built to address the reagent needs of the nucleic acid production market a rather new area yielding vaccine and therapeutic modalities based on dna rna and mrna this facility effectively doubled our manufacturing capacity for selected proteins and enabled nab to focus our efforts on scaling the ipswich facility for those products crucial for molecular diagnostic development all of nab's facilities maintain iso 9001 and 1345 certifications demonstrating our commitment to quality by design and ensuring our customers receive not only a consistent product in the tube but the necessary documentation and support infrastructure around it products produced in the rally facility while not see gmp themselves are useful in the production of apis and other components that are required to be as my colleagues discussed earlier any of these technologies and products are important components of the diagnostic supply chain i'd like to take a few moments to talk about any of these efforts to prepare for and scale up our production capacity during the pandemic briefly back in january nfb's focus was on ensuring that the impact of scars kobe 2 on our supply chain would not adversely affect our ability to produce components that might be useful in the response to the pandemic this involved investing in inventory and raw materials important for making our products as well as dual sourcing wherever risks reside nad quickly transitioned to a work from home environment for as many of us as possible and implemented a number of practical measures to reduce the density of our people on site to both ensure their health and to protect our ability to serve as a supply chain partner to the diagnostic community in march we began to see surge demand for a subset of our products many of which my colleagues touched on earlier we quickly assessed any potential production bottlenecks and fast track the scale up of any processes that were of concern many of these scale-up efforts were planned for in connection with a sizable expansion of our ipswich campus or implemented in a few short months to address the unmet reagent needs created by the pandemic and just a few short months later we're in a position to produce components including rtq pcr and lamprey agents and master mixes at a scale appropriate to de-risk the supply chains of our customers who are anticipating the need to test millions of people routinely the impact of our efforts to scale up across the organization resulted in an increase in production output that in some cases facilitated orders of magnitude increases in capacity i touched earlier on our plans to expand the ipswich campus as part of that site plan we're in the midst of construction on a 90 000 square foot addition to our manufacturing plant that would house an expanded shipping and receiving area expanded warehousing infrastructure and more space for filling as well as expanded footprint for our quality and regulatory affairs group and our oem customized solutions team this expansion will enable nap to increase the scale of certain production activities by an additional order of magnitude for diagnostic reagent supply in addition to a commitment to scale and product development in support of pandemic response nab introduced a variety of online tools and resources intended to inform from fundamental research on new detection modalities performed by nab scientists through informational tools about isothermal amplification rtq pcr and lamp nab strives to be a destination for good responsible science both performed at nab and by our customers we've also been aggregating information about the use of our products in both sarsko v2 publications as well as those ewas in which our reagents have been publicly disclosed while nab is an innovator in the field of molecular biology reagents in their applications our customers are doing the hard work of translating those innovations to research insights and new technologies we've been highlighting their endeavors to a series of copic 19 focused researcher spotlights featuring a number of notable thought leaders publishing in the cobin 19 space in conclusion nab is well positioned to partner with companies throughout the life cycle of their product or technology and it's our intent to work collaboratively across our organization at each stage of your growth and development whether during the research phase where we can help provide unique insights into the performance of our products in your technology or through the optimization of our reagents to make them better integrate into your product and ultimately through the scale of that product and its global rollout anybody wants to be your partner as you grow thank you for listening and we look forward to working with you thank you all for joining our webinar we now have some questions that nathan nicole and sal are going to address nathan do you want to take one of the first questions sure i'll go ahead uh hello everyone this is nathan tanner uh you heard me earlier talking about lamp and innovative enzymes a few questions coming in that i'll start off with uh one is about using direct sample inputs for lamp and and potentially qpcr as well you heard some good data about this from nicole on the saliva direct front but there's obviously a variety of samples that can be used the web resources that have been mentioned a couple times we have a good collection of methods and protocols for use of saliva swab other types of samples directly with lamp qpcr color metric lamp a whole variety of things and in general most samples are tolerated pretty well you got to consider things like color metric effects so if you're going to use color metric lamp you got to watch out for things that are strongly buffered alkaline or basic because of the the the ph effects but again there's plenty of protocols available at preprints use of wastewater has become a popular topic and that's a nice advantage of these molecular tests you can screen for things in wastewater samples there's been a pre-plant or two using just direct raw sewage right into lamp so you can even go that far if you like usually people prefer a concentration step where the waste water is put through a filter of some kind to enrich for nucleic acids just to get enough copies to be detected in the reaction so all of these things are possible i again suggest checking our web resource list and for protocols and things there or if there's any specific sample of interest let us know and we can offer some advice great and i'll go next so we have a question here about whether we've seen any compatibility issues with the primers in our kit and the new variants that are out there um so we take a look at this actually uh quite often um and uh we've been fortunate enough to have access to the sequences that are in gised which has been fabulous and so we've been taking a look at the primers in both our qpcr and lamp kits and and to date uh don't see any issues with the major variants that are out there the b117 the one three five one p1 and the the newer b142n from california so none of those are predicted to be problematic with either of our kits which is great but we continue to monitor those and we're in the process of developing a tool um that we can share more broadly so that people can watch their primers of interest as well and take a look at that same evaluation to be able to determine whether they might see or predict any issues with the primers that they might be interested in so so keep keep your eyes open for that tool all right i'll take another one why would you use lamp instead of qpcr both methods are great sensitive molecular diagnostic tools they just have a few different advantages and considerations if you're choosing between the two or trying to build the assay you want lamp is really good in situations where you want to do things in the field at point of care it's faster you can do 20 30 minute reactions and as we've shown a couple times you can even use color metric simple readouts where you just look at things by eye qpcr does have a higher sensitivity it's more standard for clinical labs a lot of automated workflows are built around using qpcr uh it's a bit easier to multiplex i mentioned our lamp multiplexing but that's sort of a novel thing not not quite as as widely used as something like tacman or probe based qpcr so i'd say depending on your intended application either method can work but they both provide a very sensitive detection lamp just offers you another level of simplicity if you really want to do things directly powering things like testing at home for example you may all have seen the lucira health test for example that uses lamp and it's the first fda authorized at-home molecular test so just a good level of simplicity and speed that comes with the isothermal methods great and i have another question here around biocompatible and freeze drying reactions so the products that we have available through the catalog are not appropriate for free drying as is generally they have components like glycerol and other things that are just incompatible with biopolization but we do have versions of most of our master mixes that we've discussed in this presentation that are available for freeze drawing that's something that we can offer through our custom or oem channel that sal discussed so certainly please reach out if those are of interest generally yes you can freeze drive both pcr and lamprey options we do see some some better performance with the fluorescent lamp than we do with some of the ph based the color metric uh lamp reactions so generally for freeze drying we recommend going with the fluorescent based approach but that's something that we continue to work on going forward so please feel free to reach out if those topics are of interest to you i can take another one here there's another question about variance and primer design specifically for variants um obviously there's um some interesting qpcr primers that are out there people are probably aware of the use of some of the s gene dropouts um you know those are um those are certainly available and there are more options coming online uh sort of routinely uh in terms of being able to monitor specifically variants through qpcr there are there's a few versions in protocols.i o from the grubel lab in particular to be able to monitor a number of those variants and i will say it's pretty easy to design primers for qpcr for variant detection although certainly you will then have to take into account the fact that the variance of interest may change in your demographic um quite quite quickly but maybe nathan can speak to designing variants or primers for variant detection with lamp and considerations that need to be thought of there uh sure i'll take that um layup generally speaking is a little more tolerant of mutations and things which is good or bad depending on how you look at it so just a mismatch or two in one of the six primers is unlikely to cause much trouble and things like the s drop out that people have seen as a sort of phenotype marker for picking up b117 and other variants that's usually those deletions happen in the taqman probe and you can easily target that for for pickup or not lamps a little bit different you don't tend to do probe based detection so that approach doesn't really work it's usually based around the amplification itself and you go for designing the primers to target the mutation of interest and you get amplification or not depending on if it's wild type or mutant so the standard approach is to target the mutations right at the ends of the the looping primers the fip and bip if you know your lamp nomenclature if not it's just two of the key primers you design in the reaction so in the primer design software you can specify where you want the primers to be and then adjust manually based on mutation versus the wild type sequence uh so that there's a few ways to do it but that's that's the most common uh and you tend to get pretty good discrimination if you can put the mismatch right at the primers for things like uh the the s dropout or there's a north one deletion of nine bases it does get a little bit easier because it's a bigger area to target so you still want to put those regions right at the primers and again you can do this in our design tool available on our website perfect thank you nathan so there's a question here around whether we're looking at any additional changes to the luna forex mix the one step rtq bcr mix um i described how we had started with the 2x mix that was in two tubes and we've now updated that to a 4x mix that is certainly um when we took that project on to update that mix we did open it back up again in terms of formulation and see if we could make any further improvements to it um and both you know from a positive and negative perspective i guess we we found that we had already maxed out um a lot of the performance uh capabilities of that mix we spent a long time as i described developing that mix so um again that was sort of a good day bad day kind of thing uh when we figured out that it's it's looking really good and it's really hard to continue to make improvements there i will say though that um we continue to offer different versions uh particularly for customers who have specific needs uh so if that's your interest please feel free to reach out to us and and we're happy to look at what your needs are and make versions that could work for your specific workflows all right i'll answer one here about measuring the color metric lamp reactions uh so obviously we show lots of pictures you can look at it by eye and that's a very simple way to tell if things amplify or not but a lot of people prefer something more quantitative or if you're going to do a higher throughput or any sort of regulated test you're going to want more than just an eyeball subjective yes or no it's a very clear color pink and yellow but still it's nice to have something that can be run against an algorithm or a metric of some kind and there are definitely ways to do this said nicole mentioned color genomics if you look at their eua documentation you can see this they take the color metric lamp and measure real-time absorbance so you can use a spectrophotometer or a plate reader where you measure absorbance of two specific wavelengths to get a quantitative measurement of the color change there are simple ways to take a photo and do image analysis there's even an app in the in the app store called lampreader that'll take a picture of your pink and yellow reactions and give you a judgment if you prefer doing something like that or just honestly if you have a 96 well played it's a lot easier to do that than judge them all one at a time by eye so they're definitely ways to do the color metric lamp relatively simply but as nicole also highlighted there's plenty of fluorescence and alternative base measurements to go into lamp to do that as well thank you everyone so much um if we didn't address your question during this q a we will be sure to follow up with you offline additionally if you have any other questions or you'd like to reach out to us please do so at webinars neb.com and we'll be sure to get right back to you the webinar will also be available on demand after today's presentation so feel free to watch again or share with your colleagues and again big thank you to our speakers today and hope that you are all staying well and safe thanks again you

2021-04-22 22:03

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