Merck & Co., Inc. (MRK) Earnings Call Transcript & Summary

Hello, and welcome to our webinar: Multiplexing qPCR Assays for SARS-CoV-2 Variants and Beyond. My name is [ Dr. Matthew Cousins ], Manager of Product Training and Customer Engagement for Diagnostics and Regulated Materials, a part of the life science business of Merck KGaA, Darmstadt, Germany, and I will be the moderator for this webinar. Before I introduce our speaker for today's webinar, I would like to cover a few housekeeping items. Please note that this webinar is being recorded, and we'll be able to share a link with you after the event is complete. We also invite you to suit any questions you may have. Please enter your questions into the question window on your screen. You may send your questions at any time during this presentation, and we will answer them at the end. At this point, I'm pleased to introduce our speaker for today's webinar, Dr. Vikas Palhan. Dr. Palhan is a senior scientist in the Molecular Diagnostics group located in St. Louis, Missouri. He joined us in 2007 and over the last 14 years, has developed ChIP, GRIP and turk kits for genomics and epigenetic research. He is currently developing a multiplex RT-PCR assay for detection of SARS-CoV-2 variants and wild-type virus that caused COVID-19. Vikas, the audience is all yours.

Thank you very much, Matt. Welcome to all of you. Thank you for spending time to learn about multiplexing assays. And what we've been able to do while we were chasing the SaaS COVID-2 variant. So what you will learn today, I'll just break it up into 3 parts. First, we'll talk quickly about what you need to do to develop a multiplexing RT-PCR assay no matter what the application. #2, we'll tell you as a use case, how we went after SARS COVID-2 variants of concerns. For the COVID pandemic and starting from Alpha, Beta, Delta, Omicron all the way to the recombinants, XBB 1.5 and beyond. And then we'll quickly tell you what you could do beyond that in the application note slide. So there's a lot to cover. Let's get going. In the summary. The first thing is we've developed a multiplex RT-PCR assay mix, which is primers and probes to direct the SARS-CoV-2. As I told you, it's for research use only. And the main thing we've done is we've optimized the blend of the primers and probes for specific variant targets, so they do not light up the wild type or not -- if the variant doesn't have the mutation, it will not give you positive. The limited rejection is about 10 copies of RNA per reaction, in perk our GPCR reactionary, and clinical validation of this is done for the Alpha, Beta, Delta and the Omicron variants. The assay has been upgraded with the evolving virus to detect the recombinant XBB 1.5, which is the characteristic F486P mutation in the spike protein. And the final variance of concern asset mix that we have right now is Master Mix 9 that can detect 7 mutations in 1 RT-PCR reaction. We also developed a one-step multiplex RT-PCR of buffer mix containing [indiscernible], and this can be purchased on a custom order. And the multiplexing assay buffer mix can be used for infectious diseases to detect respiratory virus power, for instance, influenza flu A, flu B, RSV and SARS-CoV-2. And the multiplexing acid buffer mix can also be used for cancer panels in liquid biopsy in the future. So just to remind, this webinar is being recorded, and a manuscript describing this will be coming out soon and available as a white paper to we'll be uploading it by archive pretty sharply. All right. So let's get going. So lots to cover. So what do you need to do to design a good multiplexing assay? So the very first thing you need to worry about is to make sure you have a multiplexing qPCR machine that can do 5 channels, usually the FAM, HEX, Texas, Red, Cy 5, Cy 5.5. So you need a machine and a software that has a capability. And the best way to do this is the dual label hydrolysis probe-based chemistry, which is also known as TaqMan assay. And the amplicons that you design for each target have to be in a similar size so that they all make equivalent amount of product in each cycle. So usually, we try to keep them 80 to 100 base pay. And in the case of SARS-CoV-2 or liquid biopsy for celled mutations, you have to pick particular attention to make sure the amplicon is small because in the case of RNA viruses because of sample prep, you might have only degraded pieces of virus. And for cell-free DNA, you only have usually 160, 170 base were to look for. So don't make your amplicons too big. We try to keep them close to 80 or 90. And the primers and probes should work together. So they should not form dimers in the multiplexing setting. And the TTM of each of your primers should be between 58 to 60 degrees. So sometimes even a 1 degree difference can make a break how efficient the PCR is. And the TTM of the primer should be, as I mentioned, 1 or 2 degrees. And for the probes, it should be generally 5 to 10 degrees higher, but we have -- as you will see in a use case, we've got it down to just 5 degrees higher at 65 degrees and a short line that has still worked. So specificity of probe is first, the primes can be chosen along that. You need to determine an needle temperatures where says work efficiently. So you need to do a temperature reading to find out usually 55 to 60 degrees. And then to titrate the prime from concentrations to optimize a single-plex assays first based on the target abundance, you decide how much of each trimaran put. You need to perform standard curves with serial dilutions of your reference RNA in single-plex and multiplex setting, and you have achieved good multiplex when you've seen multiplex CDs are the same as your single-plex CDs. You amplification curve will take a little hit, but your CTs will not change. And in order to optimize the multiplexing QPCR or RT-PCR makes the components are very important. That means the Taq enzyme, the reverse transcriptase that you use, the magnesium concentration, the DMTs. So they have to be at the right level. Once it is optimized, this is the before and after situation. So in the beginning, you will see in blue, I'll show you the amplification plots of single-plex they're a little better compared to the multiplexes, which take a hit and come down in red. But afterwards, for assay 1 and assay 2 done simultaneously in multiplex, you could have a pretty good amplification profile. And in some cases, it's almost perfect. And then a single-plex and multiplex match. So what did we change? We changed the primary and probe concentrations and the mix and the buffer compound. So magnesium has to be generally higher, 7 millimolar, and your enzymes have particularly the Taq polymerase has to be higher. And because you make so much DNA for 5 reactions simultaneously, the more DNA you produce in the second half of your multiplexing, the magnesium binds to the double center DNA products also. Hence, the dNTPs also have to be around 0.8 nanomolar. All right. So let's get into the multiplexing SARS-CoV-2 variant detection assay. Here, I'd like to introduce my R&D team. We call ourselves the variant hunters. So on the left, that's -- I was the lead. Ramu, Ramara Mangena was my design guy in Haverhill. David was another intern who helped a lot in the initial standardization and followed by Gammon. My advisers are [indiscernible] and Brian from E.Leclerc in St. Louis. Snowy Food did a lot help in optimizing the primes and probe in the combinations going from single-plex to duplex, to triplex to quadruplex and then multiplex and Josh also helped in the beginning. The collaborators for the clinical validation in the U.S., it was Keep Gagman and Adam White here in Southern Illinois, University Carbondale, and they were helping Illinois Department of Health with the sequencing and they had an NGS platform so they could verify all the mutations that passives are detecting. And in fact, way back when Delta and Omicron first came out, they were able to detect other assays before they were able to sequence them. So I will show you the data. And in South Africa, we were able to collaborate with Dr. Flare and Sudan Kathleen, who were at the NHLS and the Wits University, the Medical Biology department, and they were able to help us identify the Beta and the Omicron samples. So thanks to all of my collaborators, we were able to send them our primers and probe mixes, and they were able to validate it initially with the [indiscernible] assay and hopefully soon with ours. So why do we care about variants because they increase the transmissibility, the variants were spreading and dominating in wave after the other because they were immune evasive. And the antigenicity has an impact on the neutralizing antibody because the virus is trying to evade our immune system and changing the surface on the spike protein. And they were giving breakthrough infections to those who are immunized to the primary Wuhan 1 wild-type vaccine. But the current vaccines kept up, the bivalent vaccines are out. They do work. They reduce the efficacy but dramatically reduce risk for severe disease and long COVID and the T cell response remains unaltered. So we all know, we passed the COVID pandemic right now, but we're not done with the virus. The virus is going to be here forever, and we need to keep updating our detection assays and our vaccines going forward as now recombinants arrive. So how did so many mutations arise in one variant? Virus has clearly replicated under different selective pressures. Initial, it was a zoonotic transfer of people thinking from bats or minks, but that wasn't a major reason. I think the current hypothesis is mainly there's a chronic infection to an immunocompromised patient, who was being treated with the canals in plasma followed by moon antibody therapy, and the virus was able to constantly select against these antibodies. And hence, it was able to pick up gain-of-function mutations and these mutations kept stacking. So that's why it's convergent evolution. We've seen the same set of mutations arise in different parts of the world is respective of the travel. And the days with international travel, it's tough to contain this. Just to show you initially how there was a surge in the U.S. and Omicron displays Delta, Delta displaced also in the beginning and what's the current stake. So the current state in the variance in the U.S. today is that XBB 1.5 and its derivatives are dominating in the U.S. right now, more than 90%, 95% are all XBB derivatives. So we've gone past the Omicron. What impact does it have on diagnostic testing? Initially, when Alpha came out first, the U.K. variant B117, there was a characteristic acronym associated to it with the GTF, which was trans for the spike gene target failure if you were using the TaqPath multiplex assay to diagnose because one of the probes was in this spike gene, which got deleted at the [indiscernible], particularly around amino acid 69/70, and they were getting a negative test. That assay had 2 other targets, which were positive, but this negative test started becoming a signature in all the labs in the U.K. and elsewhere and also when Omicron BA1 came out. So SGTF, and then, of course, they were being followed by next-gen sequencing to identify the variant and the mutation signature. We've taken that and converted that into a positive assay rather than a negative assay to rule out technical difficulties. So we actually have a deletion, which our probe will only hybridize if the division has occurred. Then subsequently, the current U.S. CDC assay, particularly the multiplex one that detects along with Flu A, Flu B, has an SC2 assay, which is in the end of the end gene. It's called the SARS-CoV-2 assay. That assay has picked up a mutation in the probe that needs to be updated. I will show you the data. Charity assays from Europe, from the Berlin Institute have also got reduced sensitivity and they need updates too. So variants are currently monitored, detected by either next-gen sequencing, which takes time or by SNP genotyping assays, which have to be done one after the other. So it's not very high throughput. And it is -- in order to be sustainable, our multiplex approach gives you the better answer. So, in one RTP reaction, and you can get many mutations. And it's very important because for the patients, it needs important to know which monoclonal antibody to administer while they're in the hospital because the current variants are very immune evasive, even the Evusheld antibody combination of Omicrons is not working right now. So the virus has mediated. It's time on TPCs catch up and get updated too. That's the theme. So what's the fit to Merck? Why should we be a player here? We are -- we have expertise in making diagnostic molecular assays for RT-PCR, and we wanted to design the multiplex assays for variants of concern. So I want to give you a use case. So we want to assay the target, we need to focus on the varying specific gain of function mutation. So we followed the literature, and we saw where the spike mutations were taking place in the receptor pining domain, RBD, that to the receptor binding motive. So you can see that where the AS2 interacts in green, I'm showing the spike RBD, the mutations are occurring predominantly at the E484K, K417, and 501Y. They were either enhancing the binding to the AS2 or they were giving resistance to certain neutralizing antibodies, whether they were Class 1 in terms of K417N or Class II E484K. And this was seen multiple variants. And these mutations started coming again and again because that's the nature. So on the left, you see the spike trimer, with the recent shape receptor mining domain and interacting with the AS2 for the Class II utilizing antibodies would fit in. And you can see in red where all the mutations are curing. So this was a 69/70 deletion that [indiscernible] in the internal NOP. And then K417N takes out Class I and then E484K takes out Class II, et cetera. So these mutations did appear repeatedly in alpha-beta and then K417, an with and Beta and Omicron variants. So monitoring the literature, monitoring where the mutations are occurring because we have only 5 assays. We had to be very careful and picky in choosing the assets and for the mutations that would be relevant as the virus abort going forward. So for instance, in Delta, if you look at the structure, In gold is the AS2 and in green is the receptor mining domain, you can see the T478K mutation where it spans. All right. The summary of comparing all the mutations that have worked in Omicron BA1 and BA2 in terms of why we chose the mutations, which were important. On top -- on the left is the amino acid which has mutated, whether it's present in BA1 or BA2, BA2 was called Stealth, Omicron because it was not picked up by the spicing target failure. It didn't have the 69/70 deletion. But K417N was common in both. And these are color-coded that either they're avoiding antibodies utilizing antibodies or they're increasing the AS2 binding. That's why they were chosen. This is a good point. Over to you, Matt, for the poll.

Thank you, Vikas. So our first poll question, posing to our audience here, which multiplex SARS-CoV-2 variant detection RTP assay kit do you use? None, there's multiple individual SNP genotyping assays, the COVID variant or SARS-CoV 2 flu AB assay, CAPAs, multiplex combo kit, the TaqPath, 2019nCoV assay or the VerseNIP assays. So Vikas, I'm going to ask you which one is the most -- do you think is going to be the most popular option here?

I think most people are using the TaqPath, multiplex combo kit, I guess, that was predominantly reported in literature. That's why the GTS failure. We'll be curious to see what others have used.

Sure, sure, sure. Me, too. Do you have a particular favorite in this list here?[indiscernible] obviously.

Right, right. And I think it will help people save time. So it will be nice to see what they've been using, yes. Well, we can continue [indiscernible] or give a few seconds more.

Let's give them a few more seconds. We've just started -- now started to have our answers come in, we'll give another 30 seconds here. Anything in the past few slides you'd like to reiterate quickly mention or that's a particular importance to you?

I think I've been going really fast. I didn't have time to check out the questions. Have any questions for line. People are welcome to put them in the chat. I don't see... Yes. So I think what I would just like to remind our audience is that, yes, all of this is being written as a manuscript. So I won't have time to give you the details of the primary approach sequences, but welcome, let us know what you're interested in. We can put it together. And we're not in the business of making IVD kits. We are in the business of making the enzymes and the oligos. So that's where we can help you. And we'd like to stay on top and show that, yes, we can do our due diligence and develop customer assays for our customers.

All right. I think we've given our audience enough time to submit their answers. So let's go and see what the poll results are.

There you go. Thanks, [indiscernible] so that's good because it does allow us to benchmark against them, which we did, and we can show you that we were able to outperform. So in the beginning, when we didn't have our own mix ready sorted out, we did rely on the TaqPath mix. So just to get into the data now to tell you how we did, what we did. So the multiplex RT-PCR assets set up, the cocktail was using the 4X Master Mix from TaqPath which is very good to begin with, and this is the No ROX mix. It has the DOT, so allows people to do the PCR contamination crossover step. And the 28 multiplex assay, whatever mix of primary probes we have, depending on which variants you're looking at. You will notice extra magnesium was required, and then the rest was water to microlitre15, 5-milimeter of your sample. And then the 20 microliter reaction has goes for cycling. The first is the UDG after 2 minutes at 25. The reverse transcription is in a thermal stable 53 degrees for 10 minutes and then you quickly denature for 2 minutes, and then you go for 40 cycles of quick denitration followed by 60 degrees for 30 seconds, and you read. We use the bioADFX PCR machine. This will work on other instruments also, and we use their software for our data analysis. So here's how we were doing the Twist RNA that was our positive control templates, thanks to them from providing right from the wild-type on Wuhan 1, all the way to the Omicron, but XBB, we had to make our own. And we would take their RNA and add a universal human reference RNA to stabilize the RNA in the lower copies, which were 10 and below as we were doing serial dilutions. So we would go dilutions tenfold starting from 15 to 40 to 350 in 10 and sometimes we would do 5.2 and triplicate to make sure everything is okay. And this was done in our PC grade water, which is RNA free. So let's see what the data look like. The very first thing I want to show you is how much attention we paid to show that our probes are specific. So we were very careful in where we designed -- where we put the LNA, the lock nucleic acid, which stabilizes and increases the TM of the probe. So these are 5 probe choices, Probe 1 TO 5. They're all targeting the E484K mutation, in this 5G. And for this SNP detection, the convention was to have your probes pretty long, '19/20 have the PM 5 degrees higher than our primers. The primers are 60, the probes are going from 67 to down to 65. And you can see when we tested them with no temporary controls, everything is quiet. But when we do wild-type, we did see signal come up from probe 1 to 5. So that means they were not specific. Of course, the beta RNA was positive, but then probe 5 really work. So what is difference in probe 5? You can see the SNP is in bold, right, in the middle. We have -- Yes. So on the nucleotide and this was a shorter probe. So reducing the probe from '19 to '16 so that the neighboring -- sorry, nucleotides do not bias it towards the wild type. The hybridization is dependent right on the SNP in the middle and which trend you are targeting is very important because it's a G2 transition. And we know that there can be some GT single bond, whereas , there is no single bond on the A nucleotide. We also tried a super base, which is di-amine-purine in probe 3,but that did not show the specificity and targeting the right trend with the right nucleotide and the lock nucleic acids positioned appropriately 3 in a row rather than spacing to and placing them as the convention was worked out for us. So all these learnings and less things will be in the mass script. And here, you can see Probe 5 is showing a beautiful red amplification plot, whereas on wild type, it's not showing any. And this is both N405. We challenge it with the highest amount of nonspecific template to show probe specificity. So David did a great job and then Snowy and Gavin and others followed the top. And what lessons we learned in this were transferred to all our assays going forward. And once you have a specific probe, then you play around with the primers, the forward and reverse and you find the best combination, which gives you the best efficiency. And that's how we went fast. So I'll be going fast. I will just show you our first master mix. And these are the targets. And here are the variance that will pick up in this chart. So internal control is the SC2, which targets the end of the engine. We had that in Texas red. All viruses will pick this up except when you don't have coat. And despite deletion, 69/70, we'll actually hybridize only to the deletion. We put that in the today, which gives a brighter signal. It's in the Sifi channel. So the alpha variant would pick that up. And then the K417N mutation was in the ATO532, which is stronger than HEX, and that give us the signal Beta, the Delta+, and the Omicron. And then the T478K was in the FAM channel, which is a mutation characteristic of Delta and the Delta+. And this was the version that was given to Southern Illinois University for the first clinical validation also. So CT below 40 was considerably positive based on CDC guidelines. So I go through this data really quick. This is just to show you that the alpha assay is working in a multiplex setting. You can see the signal right up to 10 copies with good efficiency in our square and not detected on the A1 or the beta of the delta strength on the top in B. And in CMD, I show you the K417 assay, which is characteristic beta in the single plex in blue and the multiplex amplification in red. And on the right, that you can detect up to 10 copies for both of these with good efficiencies. The next slide shows you the Delta assay, which is the T478K in single-plex and Duplex. And on the right, you can see we challenged it versus Alpha and Beta. You do not detect them. The serial dilution goes all the way from 5 copies down to 10 copies and 10 copies can be detected. This is 10 copies of the Twist RNA in one reaction. And remember, I'm showing you these separately but all of these assays are happening simultaneously in the same cube. That's why it's breaking it up so that you can appreciate the data really well. And the FC2, the [indiscernible] assay serves as an internal control to make sure -- because this is a highly conserved region of the virus, it doesn't change, so it will work for any variant in future also. All right. So having done this, we went on to Mastek 2 because we realized that there was another mutation in the spike region, right next to 697, which is A67B, which is showing up in the Omicron V1. So we had to update our assay. And this just happened before we send our samples over to South Africa. And we also send these back to [indiscernible] for validation. This was called Master Mix 2. And then the other thing we included was we added the human RNase P is another target for sample prep to make sure if there was any human material that came through from the swab in the virus transport medium, it would light up. And the biggest advantage it gave is if there was any PCR inhibitor present from your clinical sample, all the assays will be negative and then you would have a fall negative prediction. But if you include the human RNA or you have RNA coming from your sample, that will be positive. That will tell you that the negative result that you're seeing is not because of a PCR inhibitor. It is a genuine COVID-negative sample. So those are the 2 improvements in the Master Mix 2. And here's the data. I will not go individually. I'll just summarize for you that we tested it on Omicron BA1 RNA from Twist. This is Master Mix 2. And all the assays worked really well, and we were able to detect up to 10 copies. And you will notice that the primary probe titration as done in such a way that the human RNase P assay only lights up and you have limiting amounts of virus RNA and that does not change because we spiked it into our mix. And if you put too much of this, it will start dominating and overtaking the other assays. So we had taken pain staking efforts in how much primary and proto put so that the RNase P only lights up when you don't have any of the very other RNAs present that is the thinking. So that will help our customers to rule out any false negatives. When we send the samples for clinical validation in the U.S., I'm showing you Adam White data, who's in cube Diagnose lab and SIU here in the U.S. They tested over 400 samples. We tested with 44 of alpha with very good concordance and they had sequences to confirm the mutation. So it was very high in almost 95% positive concordance between RT-PCR positive and sequencing positive by NGS. It doesn't matter whether it is also and then it was -- there was changes of hearing. It was a non-variant of concern or whether it was delta. And then with Omicron at that point, our assay had not caught up. So we were missing a few of the mutations because right next to T478K, there was T477 and mutation. So that was the reason why we were missing catching some of the micron variants, but we did update that subsequently. But the most interesting part was there was a new lineage appearing in New York, which had the T478K characteristic of delta along with the 69/70 deletion in alpha combination, and they were able to detect this very well, both the assets are positive. So we know that the virus is coming up with unique combinations and having RT-PCR assays focusing on key mutation gives you the advantage of identifying these. Of course, you'll have to do NGS to confirm the entire variant. But at least if you know 4 or 5 mutations right away by our PCR, the answer is immediate, whereas the NGS takes about 2 more days. So it will help the doctors from antibody treatment choices in the hospitals. Here is one representative of the several 96 well plates that Adam ran and SIU, just to tell you that there were 5 assays happening simultaneously in several patients. And you can see all the ones in blue were delta and the internal control was positive all across. And the ones that had the spike deletion and K417, were all Omicron D1. And the -- the human assay was only coming in some samples, not all. These are all clinical samples. They were doing RNA prep using another -- so our assay comes downstream to whatever RNA prep you'd use, they were using an automated system for RNA prep. All right. So by the time we were about to send the samples to South Africa, we had already upgraded to Master Mix 2 to incorporate the A69/70 mutation right here in by deletion. We also added the human RNase P. And then we subsequently realized that the beta, the FAM assay, T478K was having issues. So we added the S477, T478K in our probe. And then we also gave them E484K in to 532 as a single flex, they combine and use them as a duplex along with the multiplex to be able to identify whether it is an Omicron BA1, BA2 or is it a beta sample. And here's their data summary. So they had done about 181 samples with almost 99% concordance, so that is very satisfying to see that in clinical samples, whatever mutations were called by DPCR were confirmed by NGS. Thank you very much, Katherine, and Floretor your help with this. And they also did find some unique novel combinations. And of course, all their COVID samples were negative. And there were some that were unassigned that means they didn't have the characteristic mutations we were looking for. There was -- or there was something else close by, but they were always positive by the internal control SC2 assay. And there were some novel combinations. So I just wanted to highlight, they did see T477K with the 69/70 deletion, the New York lineage type and that was confirmed by NGS. So that was very satisfying. Here's just one sample of their play to tell you how the assay work. They could clearly see all 4 channels lighting up positive. And here's a good time to go in for the poll to check which mix our customers have been using.

Yes. So which one step, RT-PCR mix, you use for your diagnostic multiplex assays. TaqPath, one step, the QIAGEN one step the Luna probe, One-Step, GoTek or the prime script, the CAS, how come there's no 2-step options in here?

Nobody has the time, you want the result before the patient dies. So everybody wants it done as soon as possible and as sensitive as possible. So I think the key is to be able to detect 10 copies of RNA or below and get an answer very quickly. And the sooner you know which mutation it is, the sooner the antibody can be administered So I think that path was pretty popular. So was the QIAGEN mix and A lot of people were using the Luna mix also initially. And we did use benchmark against them. And in our hands, the Luna mix did not perform so well, but the TaqPath did, so that was the benchmark. And now I'll be showing you in the next half of the presentation how well our kit performed compared to TaqPath and how we can outperform it now.

Okay. Great. We'll give them a few more seconds to submit some answers. While we're waiting, Gladly, remind people to submit your questions to the question box. Also, the learning doesn't have to stop here. We've attached several resources to this webinar that we would encourage you to browse and look through on your own after we conclude today's event. I think with that, I think we can go ahead and see what the audience is that's chosen here.

Sure. They go -- 60% of the [indiscernible]. That was the very popular mix all over the world. And I'm glad we chose that to benchmark our assay all the results are based on the TaqPath mix until now. Now I think I would sway by our own mix going forward, and I love to send it to other people who are willing to test it. Just remind you that it is available for custom only. So shall I proceed?

Yes. Go ahead.

Right. So the next part of the presentation, I'll tell you our multiplex one-step RT-PCR mix with the dUTP and UDG . It's a custom formulation. So if you're interested, we can make it for you with recombinant enzyme. So -- we're very glad that with the support of our molecular diagnostic team with the Tracy's leadership and Josh Bettman and all the others who have helped in making these enzymes. We now have an offering for the ultra-pure high-temp reverse transcriptase, which is not only RNase, nickase DNA in free, but it's also DNA free. So people who are interested in NGS could use these along with our ultrapure Jump start Taq, which is also DNA free besides the usual nickase RNase, DNase. So combine that with the murine RNS inhibitor, heat-labile UNG and the 5x buffer, the DNP mix and the magnetite, everything is optimized to perform really well. And the use case was our SARS-CoV-2 multiplexing primary probe mix that we've been using. So let's see how it performs in that. So we tested it first with the Master Mix 5, which was meant for checking Omicron at that point. And this list tells you how it fares. So the internal control SC2 was in Texas, right, that will detect all the viruses, including those that do not have these mutations but are on a site, but negative for no COVID. The 69/70 deletion is combined with A67B in this situation. So only BA1 will be positive, not the BA2 onwards. Whereas the K477N, we had to optimize our forward primer to have it on Omicron specific because we realize from our South Africa colleagues and our collaborators that this assay was struggling because a SNP had come in to a forward primer. So one itty difference also compromises the performance of the probe. So that will be positive for all of these viruses. The T478K is combined with K477N at this stage, so it would be positive for Omicrons, all of them. And the human NSP could be plus or minus, but the bottom line is, it will be positive and everything else is negative to tell you that there's no PCR inhibitor. So how did this asset perform? Here we go. So this is the first assay, we were comparing it in the top half versus TaqPath mix and the bottom half or dUTP mix. And this is done on Omicron RNA. So all 5 assays are happening simultaneously. I would just like to highlight your attention to the fact that you see all these in red. So the TaqPath assays starts struggling, particularly with this S477N, T478K AT-rich assay and can't detect less than 5 copies for BA1 and 2. And then in BA4, it was struggling to detect even 100 copies and below, whereas our mix could detect all the way, which contains the dUTP up to 10 copy. So it was very satisfying across BA 1, 2, 4 and 5, we could detect all of them. And this is for one assay here the purification profile. The TaqPath is in blue and the -- our diagnostic mix is indirect. You can see it performed really well. And then we went through the other assays. So this is a second assay, K417N, both the mixes performed equally well. And the third one is the Spike deletion assay, 67B, as expected, only BA shows, BA2 does not put 4 and 5 do with both mixes, so very satisfying results. And then the internal control FC2, both the kits performed equally well all the way up to 10 copies. And then finally was the human RNP assay. So as I did tell you, it looks like our assay was able to detect no matter which Omicron in the absence. As long as you had 10 copies and below, I mean, of the virus, the human RNAse P has kicked in and there's something limiting in the tack pet mix that could not have enough juice left for the fifth assay. We saw this consistently. So I think the way we've optimized our primers and probes and our mixes, they work really well even to give you human RNase. There is some material left in terms of the enzymes and the NPs and the buffers to even detect this. So I mean used all 5. It is also [indiscernible] to remind you that the SC2 assay from CDC needs an upgrade. So this is where the probe lands, the forward primer and the reverse primer on the top is the wild type and the Omicron BA2RNA and below. There is this A to C transition, which changes the S13 --N41 to R and minor assay level. So this is right in the middle of the probe. Our probe is much shorter than the CDC probe, and this compromises the performance of the probe and the sensitivity of the assay. The limited detection falls by 2 low orders. So I would highly recommend who was watching to upgrade the CDC, SC2 assay in the multiplexing that CDC is currently offering and now most of the people are using. So here's the performance difference. You can see very clearly, you pick up at least about 2 to 3 cities. And this is using our optimized mix and all subsequent assays have been internal control SC2 NS-483R incorporated in them. Now I'd like to quickly give you an update about XBB 1.5 and why it's important, and we have upgraded our asset to that. As most of you must be aware, it's the combination between 2 Omicron strains. And it's the most transmissible variant. It's most immune evasive. And it was a combination of the [indiscernible], the BGA1 and then subsequently picked up the S486P mutation to be XBB 1.5. So you can see it's pretty far away from all the other mutations and all the other strains that were dominating in the past. This is a nice polygenetic tree. It was causing better virus entry because of better binding to a 2, increasing hospitalization wherever it dominated in the East Coast in the U.S. and rapid replication in nasal epithelium cells. Fortunately, it was not that deleterious as delta because it lost the interiors to because the spike and meet it so much. Just a quick summary of what all has mutated and lined up all the Omicron sequences in outbreak website, and you can see where the mutations are lining up on the spike protein and which variant has got which mutations lighted up. What is important for you to remember is Omicron on the top versus the XBB 1.5 right below it, the deletion for 69/70 is only in Omicron, not in XBB, whereas K417 is present in both the S477P, 478K are close by. And right in its proximity of our amplicon is the 486p and 490S. These 2 have been updated and corporate in this assay. So what I wanted to tell you is that now we've got a 4 in 1 assay for the very first time that we've got 2 mutations in a probe and 2 mutations in our primer. And we've designed a reverse prime so carefully that it lands right on this for 6 mutation, and it will only fire if the mutation is present. So it will be a negative on BA5 Omicron but a positive on XBB 1.5. And to test this, we didn't have HBV 1.5 RNA available, so we made the invitro transcription cassette ourselves. But before I go into that, I just wanted to tell you what is the composition of our latest Master Mix night. So it screens for 7 mutations in one reaction. 4 of them are in this main assay, which will only be positive if it takes BV 1.5. I'd like to highlight right there. And then we have updated our SC2 assay internal to incorporate S483R. So that will be positive in all of these situations. Now we're only looking at the current Omicron because these are the variants of concern. And right now, it's only XBB and beyond in the U.S. So what does the data look like? ECT now below 37% is considered a positive based on the revised CDC guidelines if you're running 40 cycles. So I don't have time to show you that. I'm only about 9 minutes left. So in order to go quickly, I would just summarize the key mutations we made as our assay world for Master Mix 1 to Master Mix 9. So if it is not highlighted in red, the mutation is conserved in the next assay, Master Mix 2, we added C67V, we added the human RSP internal control. In Master Mix 5, we upgraded our K474 primer, we incorporated S477N. And in Master Mix 6 onwards, we incorporated S483R in our internal control C2. And we added the 486P mutation, which was in the BQ 0.1 variants of Omicron, that got changed to 486P when we went to as Master Mix 9 and XBB started dominating and displacing the BQ variants. And that's what the current mix is. So if you're interested in validating any samples going forward because this virus is not going away, it will be the XBB lineages and derivatives of that, that will be happening in the future. So if you want to detect XBB or just coming into your country, you want to test at the airport, I would recommend using Master Mix 9. And I think besides [indiscernible], is the only one that has the capability to detect it right now, but it's an open system. So let's look at the data. I'll just quickly summarize. So in order to test it, I had to make the RNA. So all our assays were stitched together under the T7 promoter, and this J-Block asset in size, T7-npolymerase in vitro transcription was done, the RNA was purified and was tested in RT-PCR as a control with Master Mix 9. They were linearized with BA5. My negative control was the Omicron BA5. So here's what the data looks like. I would like you to focus on just the top assay here. So let's talk about this one, which will only light up if the XBB is positive in red and in blue was Omicron negative. It was negative across because we were looking for 486P mutation in this one besides the other 3. So that gave us a lot of confidence that this assay is working positive and it's working pretty well up to 50 copies. I think it will be more sensitive because this is in vitro purified RNA. If I had pure RNA or a clinical sample, this will be even more better to test. And it clearly disseminates that it doesn't light up for the 69/70 deletion. So which is a hallmark of Omicron BA5. And then, of course, if you use internal control, it tells you that have equivalent amount of RNA added for both of them as shown in this asset. All right. So that tells you that we have an XBB detecting assay available. And now we'd like to see what you would like to use this in the future. So we have the final poll question for you. Over to you.

So future multiplex PCR assays. Respiratory viruses, SARS COV 2 and flu AB plus RSV. That's a powerful combination. Virus panels, STIs, very important parasites, oncology, lung cancer panels, diagnostics, I like that. Breast and prostate cancer panels, because which one do you pay?

Yes. I think the top one is definitely an obvious offering. We already have the designs for that. We'll test it and hopefully, we should be able to show the data in the future. And the other thing I'd also like to make a plug for is we have another product coming along with the MAC PrEP cell-free DNA purification kit, which is magnetic-bead based and MAC PrEP viral RNA purification kit. And those could be very helpful for people and sample perhaps, both in COVID detection as well as in liquid biopsy. So we'll have a white paper coming out very soon on showing the GD-PCR data for lung cancer targets, EGFR mutations, PIK3CA, LH5ta, T790M. And our assay, the MAC PrEP kit should be launched pretty soon next year.

Okay. So a quick question for you then. So you've seen partial acute PCR assays, I am partial collateral flow test. Which do you see best off in the -- in developing regions?

I think both exists. Lateral flow is nice because you can get an asset cheaper, but then we have to develop the assets and may not have as much sensitivity as the qRT-PCR or qPCR assays have. But I agree, both of them for low-income countries and economy of scale and taking it to the field, lateral flow has an advantage over qPCR because you need a cycler for that, and you need a flosses reader for that. So I think both exists and both can mutually exist, and they have different applications and fit of use cases.

Awesome. All right. Well, let's see what the audience thinks.

Yes. There you go. The RSV panel yes, is bang on. So I'm glad to see that. And I'm also glad to see that the #2 is the oncology lung cancer panel. And actually, people are more interested in sorry, is the breast and prostate cancer panel, so thanks for that direction. Look out for that coming forward in the future. And in the last few minutes, I'd like to just tell you what can we do beyond now that all these QPCR machines are available, people are using -- waiting to use them for good applications. So Loon for our RSV containing influenza AB and SARS-CoV2 multiplex mix coming out soon. And then, of course, if needed, we can always design assays for doing coronavirus, MERS and SARS because those are pretty much history right now. And then you can make similar assays to detect. So I would like to make a plug for our custom design facility, which is led by Ramu. And if any of you are interested in any of these other viruses, I've just listed a few, like herpes, moms and adeno, whereas UTI infections are also caused the very popular and parasites and sexually transmitted infections. So if you want assays developed to detect these, please let us know which ones you're interested in. We could custom make these for you besides purifying them without any contamination, et cetera. In terms of plexes, we can also help you with the design part and particularly if you want to combine these and make a multiplexing panel on demand. And then going on to liquid biopsy applications in oncology, that is a fascinating area where lung and prostate cancers are dominating. And we've got very good assays already designed for the ones that have listed and the risk can be added as you address it in -- so remember, we are a research use only. We are not to the IVD business. We can help you and make these high quality and purity without contamination that will help you make your own LDDs and apply for clear or for IVD and [indiscernible] certification. In summary, I'd like to conclude saying that we've developed a nice multiplex RT-PCR assay mix, which is primers and probes to detect as SARS-CoV-2 variants of concern. It's only for research use, and it's optimized blend so that you don't have to worry about primers and probes and we purposely added less of the ASC 2 because of the sub genomic RNA that accumulates in infected samples towards the end of the virus genome. So we kept that in mind. We can detect very sensitive. VR assay is very sensitive. It can detect 10 copies of RNA or less. And it has been clinically validated for Alpha, Beta, Delta and Omicron variants as you saw the data. We upgraded the assets subsequently to detect the 46p, which is a characteristic mutation of XBB 1.5 recombinant variant and its lineages. The various assay of concern is now Master Mix 9 that can detect a total of 7 mutations in 1 reaction. And we've also developed an accompany multiplex RT-PCR buffer mix using our own enzymes and our own DTP cocktail, and this can be ordered on custom. The multiplexing assay and the use of various applications, like you are all interested in, in the respiratory virus file for flu A/B, RSV and SARS-CoV-2. And of course, for liquid biopsy applications in lung cancer panels, I couldn't have done all this work by myself. So there's a huge team that I need to thank. They are all listed here. I quickly call them out. Ramu for the design. And David, as an intern right here, Gavin, Josh, Nole, Pierre, Carol and Brian for their advice, Tom Russell. Thank you for your support. And Tracy and Aaron, thanks for your support and our marketing team, Mark, Dan, Niraj, Alex, Devin, Samantha, Patricia and Costa in South Africa. Adam, Adrian and Keith for a clinical validation in USI in the U.S. and Kathleen and Florence for the validation in South Africa. I didn't have time to show you the bioinformatics team who did a nice job. Thanks, J.J. and Raja for their analysis versus 300,000 virus sequences from SID genome to make sure we are not missing any SNFs in our prime approves and they have to be relevant for across the stream. And thanks to Matt and Darla and their team for the product training. Thank you for your guidance, help support. So to be part of this great team and take any questions you might have. I'd like to mention if you have any further follow-up contacts, for custom assets and R&D inquiries, feel free to reach out to me and here is my e-mail vikas.palanmilliposigma.com. And for customer orders, as far as oligos, primes, probes go, reach out to Tom Russell, he's our Product Manager for custom. And to Scott Weber. He's a senior scientist and my team. His e-mail is right there. For any custom multiplex, one-step RT-PCR mix with UTP1. We can also make a PCR mix without the RT for you guys. Thank you so much. So that's perfect. I look forward to taking any questions you might have.

Yes. Vikas, thank you for sharing your expertise with us today. We're waiting for questions to come in. I know one that we're going -- definitely going to get, I mean we can head it off right now is regarding intellectual property, right, as far as people wanting to develop custom multiplex panels. Can you shed some light on to that for us?

I think as far as sequences for detecting things that are present in nature for viruses, there is no IP on the primer and probe design because it is used in nature. That's why unless you made some customer modification to it. So I think most of the IP would reside on modifications you made to your enzyme to make it work better, be enabled tolerant or something like that, not so much of the primes and probes. Yes, sorry, go ahead.

Yes. So I was thinking more towards our custom design service in which we'll help you design the primers and probes but those belong to you. We don't keep those designs -- we don't share them...

Yes. Yes. So there is a silo between each customer and us, your enzymes and your targets are unique to the customer and we do not use them. That's for sure.

So all right, first question here. Why don't you -- or why aren't you detecting the human RNase P as a signal in some of your runs?

Okay. That's because intentionally, we have put that primary and probe at a very low concentration because we don't want that assay to dominate and start preventing -- use up all the resources in terms of the dNTPs and the magnesium and the enzymes so that you can't detect the viral targets. That's the reason why.

Okay. Next question here. Can you cherry-pick and customize your variant SNPs mutation detection assay and order the -- and can you order them in different floors and country combinations?

Yes. All that exists. So because we try on the custom business. So whatever combinations you want, once you see them on our website on manuscript, feel free to let us know from a white paper, what combination you're interested in. And we have been supplying this to South Africa to Slovenia, to another places. Yes.

Great. Looks like we've got one more question left here. We certainly have time for a couple more if they come in. If a future COVID variant does not have any of the mutations that are present in your latest master mix, would miss detecting SARS-CoV-2 and give it a false negative result?

No, not really because we've got on internal control assay in a highly conserved region, which we think can speculate will not change, which is the SC2 and we have upgraded it to include all the variants that have come right now, where we saw the ASOs in the SC2 assay. So the engine is very conserved and highly unlikely. But we will be on top of it. That's why I recommend even for CDC, those watching. If you are following the CDC multiplex assay for influenza and SCI, you can get better results following the AC2 probe as data has shown to you today.

That's great. So before we end today, I again want to remind our audience of the extra resources that we've attached to this webinar, lots of good information there. And before you log off today, please take a few moments to complete our brief survey and download your certificate of participation. And with that, Vikas, I would once again like to thank you for sharing your expertise and insights with us today. Thank our audience for their participation. And if you have any additional questions, please feel free to reach out directly to myself or to Vikas. And with that, I'd like to thank you for your time, and have a wonderful rest of your day.

Matt, just one thing. We have 6 minutes left. If there's a chance I could show the appendix for the single-plex assay that I couldn't cover.

Yes, market stuff out.

Sure. So I'd just like to remind the audience that this appendix slides will be available when you download it. I'll let you just mention quickly that there are several of these assays that are available as a single plex for those of you who want to do epidemiology studies to see how the variants evolve using the inventory of your samples and the freezers. So S477N plusT478k, which was observed in [indiscernible] of Omicron, BA1, BA2, BA3, the earlier microns. And then E484K, there was characteristic of Beta, E484Q, that was characteristic of Capa. And then the AL452R, which was characteristic of Delta as well as the Omicron BA4 and 5 lineages. So we have data to show that these guys are very specific and they work. And you can choose whatever combination you want to put this in. You will have to optimize the combination. But if it exists in one of my master mixes that we've used in now study, those could be used in the same combination. So this is S477N and T478K. Omicron in red and blue showing that it doesn't detect. It's very specific for macro, and E484Q that detects beta and not gamma. So that's a very nice job done by Gavin showing its specificity, We also crossed check on Gama, Alpha, Delta to show Alpha and Delta are not positive. And if you care for Beta only, then you have E484Q. So that is, again, Gavin's work here to show that it's very specific, and it's only for Delta at this point. And then here's the L452 assay that was tested personally to show this one works at 57 degrees. And that's why we've kept all our assets to work at 60 degrees, but this one, because of the nature of the sequence, it only worked efficiently at 57, where it doesn't pick on wild-type only on Delta. So we paid a lot of due diligence and testing our assets. And particularly, when we are optimizing them, we had made several mixes. And this is to tell you that a mix that we've selected is very specific and clearly outperforms the benchmark that mix. And for this assay, which is in an 8-year rich neighborhood. All right. Thank you for your time. That's all I wanted to share. And then no primary dimes, of course.

Awesome. Great. Thank you. Once again, Vikas, wonderful work, super impressive. And thanks again to our audience for taking the time to be with us today. Thanks, and enjoy the rest of your day.

Bye guys. Thank you very much.