Bst-XT: An improved Bst DNA polymerase to support loop mediated isothermal amplification

Burcu Minsky:

Good afternoon, and thank you for joining us today. I am Burcu Minsky, and I am accompanied by my colleague, Kristine Werling. She and I are development scientists at NEB in the DNA amplification group. We are part of a team that develop a new Bst enzyme, Bst-XT, to support lube mediated isothermal amplification.

Today we will discuss the properties of this new Bst enzyme, highlight its unique features, compare Bst-XT to our current Bst enzymes, and provide recommendations for a few specific applications.

Before delving into the specifics of Bst-XT polymerase, I would like to explain how NEB supports molecular assay development. NEB has over 60 years of experience in molecular biology and enzymology, and has the expertise to support development of molecular assay research and development by providing high quality reagents to the life science community. Molecular assays are typically developed to robustly and accurately identify and quantitate specific DNA or RNA sequences within sample of interest, and nucleic acid amplification is needed to detect the presence of these targets.

Nucleic acid amplification and detection is commonly performed by either PCR or isothermal methods. Real-time PCR is the gold standard in molecular diagnostic assays. It's a well-established technology that provides specific and sensitive detection of RNA and DNA targets, and it relies on terminal cycling for amplification. On the other hand, isothermal amplification has become alternative to PCR in molecular testing. Isothermal amplification methods provide detection of a nucleic asset target sequence in a streamlined exponential manner, and they are not limited by the constraint of thermal cycling.

There are many ways to perform isothermal amplifications. These methods have simpler implementation due to single temperature incubation requirements, and they enabled development of point of care and field deployable devices. Among these isothermal amplification methods, loop-mediated isothermal amplification, also known as LAMP, has shown increased utility in rapid molecular testing. The COVID-19 pandemic notably brought attention to the need for fast, sensitive, and cost-effective pathogen detection strategies. LAMP could easily meet these requirements, as it enables sensitive detection of nucleic acids, and the reactions can typically be performed in under 30 minutes. Additionally, progress in robust and high performance reagents, knowledge to support essay design, and improve primary design tools, have established LAMP as a widely recognized and validated application strategy for molecular diagnostics. On this note, several at home lab-based point of care diagnostics were granted emergency use authorization to support soft testing for COVID-19.

This figure further highlights some of the key differences between the isothermal and PCR-based amplification. PCR is carried out using two primers, and it is driven by thermal cycling. Following denaturation of the DNA strands with high heat, the reaction temperature is lowered to enable primer annealing and extension by a thermostable DNA polymerase. This process creates discrete amplification products. The detection can be accomplished in one to two hours, and it can be quantitative when the reaction is monitored in real time. PCR-based thermal cycling requires significant power source and precise temperature control to heat and cool the reactions. Therefore, a real-time PCR instrument is needed to perform the reaction.

In contrast to PCR, LAMP requires only single incubation temperature and reactions are fast, typically performed in under 30 minutes. LAMP requires four to six primers and a strand displacing DNA polymerase to generate products that have characteristic thermal structures. These structures have multiple priming sites, which allows for the rapid generation of highly branched concatemerics. LAMP offers flexibility in detection methods and amplification can be detected in variety of ways such as using fluorescence, turbidity, lateral flow, or even a colorimetric visual readout.

However, the results are semi-quantitative at best, even though LAMP reactions can also be monitored in real time. Additionally, the single temperature requirement makes it easier to implement LAMP, as it doesn't need a complex instrumentation like PCR. The amplification can be achieved using a simple heat block or water bath. Designing primers for LAMP assays is generally more challenging than designing PCR primers. And as a result, we build the NEB LAMP primer design tool to streamline and simplify LAMP primer design. This tool is readily available online at NEB.com, and it only requires input of the target sequence to design all four to six primers required in LAMP.

The unique primer design and amplification chemistry are the core features of LAMP. So I would like to describe these in more detail to highlight LAMP's exceptional power to amplify DNA. LAMP uses four core primers, which are essential for amplification, and two loop primers can be added to accelerate the reaction. These primers recognize six to eight distinct regions of target DNA. Amplification is initiated from strand invasion by one of the inner primers, and these inner primers are specially designed to facilitate formational self-hybridizing dumbbell loop structures. The resulting dumbbell structures have multiple priming sites which enable amplification to proceed in an exponential fashion. Loop primers bind and initiate synthesis on the single standard DNA loop regions in the dumbbell structure to accelerate the amplification. As a result of this distinctive amplification chemistry, LAMP produces DNA in large quantities.

Designing six primers for LAMP may pose a challenge. However, I would like to reemphasize using NEB's primer design tool to streamline the primer design and mitigate this inherent complexity. In addition to the unit primer design, using a strand displacing polymerase is essential for driving LAMP reactions. And Bst DNA polymerase, which is known for its robust strand displacement activity, is the enzyme of choice for LAMP.

NEB currently offers Bst 2.0 WarmStart and Bst 3.0 as primary Bst enzymes to support LAMP reactions. Bst 2.0 and Bst 3.0 are engineered variants of Bst. And Bst 2.0 is fused to a novel nucleic acid binding domain for improved amplification speed. Bst 2.0 WarmStart contains a diversible bound aptamer, which inhibits the polymerase activity below 45 degrees, and therefore enhances amplification specificity. I'll describe this WarmStart feature in more detail later in the webinar.

The table shows the comparison between Bst 2.0 WarmStart and Bst 3.0 regarding key features of amplification speed and specificity. And here we use the term specificity to indicate how prone the enzyme is to generate off-target amplification. Bst 2.0 WarmStart shows strong specificity with limited off-target amplification. However, its amplification speed is slower than Bst 3.0. On the other hand, Bst 3.0 displays faster amplification, but it tends to produce non-specific amplification. Considering the outline advantages and disadvantages of these two enzymes, we aim to re-engineer BSD enzymes to enable these two key features in a single enzyme.

While engineering the new enzyme, we follow a multistep and collaborative approach, including multiple teams from engineering to manufacturing. At NEB, we have accumulated decades of knowledge and experience in enzymology and enzyme engineering, and we are dedicated to continuously improve the available tools. We test candidate enzymes in challenging assays to verify that the selection process is robust. We achieve this by developing diverse assays, utilizing high throughput screening approaches to increase the statistical power of the selection and perform extensive functional testing until the desired selection criteria are satisfied. Once the enzyme selection process is completed, the manufacturing team establishes a scalable workflow for enzyme manufacturing. The implemented procedure is designed to ensure reproducibility while achieving desired quality metrics.

In addition to enzyme engineering and manufacturing, we also employ innovative methods to control enzyme activity. Our approach to control enzymatic activity prior to thermal incubation is using diversibly bound aptamers. Aptamers are highly modified oligonucleotides that have been engineered to shut down enzymatic activity at room temperature by binding non-covalently to the protein. Initiation of the reaction using heat causes the aptamers to quickly release from the enzyme so it can perform its function. And the release can be attuned to a desired temperature. It is also fully diversible, meaning the aptamer will rebind its target upon cooling the reaction to room temperature. Using aptamers to control enzymatic activity enhances the robustness of molecular assays by eliminating off-target amplification and allowing room temperature reaction setup. Traditional methods for controlling enzymatic activity in amplification reactions, such as antibodies, generally require long activation times and very high temperature incubations that are incompatible with essential enzymes used in isothermal methods or reverse transcription.

Aptamers can also control these enzymatic activities, and we refer to such enzymes as WarmStart. As LAMP requires four to six primers incubated together at a single reaction temperature, it may be prone to off-target amplification depending on the primer design. To help reduce these sporadic off-target amplification events, we utilize the WarmStart feature for our several enzyme offerings for LAMP, including our new Bst-XT enzyme.

Another cutting edge manufacturing capability that we have at NEB is to support lyophilization workflows. LAMP is particularly well suited to incorporate into portable point of care devices because of its speed and simplicity. And reagents stored at room temperature are essential to support these assays. Lyophilization is a powerful technique to enable manufacturing MBS temperature stable reagents. In addition to room temperature storage, lyophilized reagents provide additional advantages such as longer shelf life, more efficient assay setup, minimize assay contamination, and elimination of cold chain shipping and storage.

NEB supports our customers' lyophilization workflows in multiple ways. We offer ready to use lyophilize amplification reagents for RTQPCR and LAMP. We are particularly well-equipped to support requests for lyophilized products, because New England BioLabs Lyophilization Sciences brings expertise in design, development and manufacturing of the lyophilize reagents. In addition to the ready to use lyophilize products, we also offer lyo-compatible reagents and glycerol-free enzymes for customers interested in lyophilization. NEB provides select glycerol-free enzymes through our catalog while variety of glycerol-free and compatible formulations can be accessed through our customized solutions team.

As a result of our engineering, manufacturing and development efforts, we are excited to announce our newest Bst offering Bst-XT DNA polymerase, which is an engineered variant of Bst and it is used to a novel nucleic acid binding domain. Bst-XT combines the best properties of Bst 2.0 and Bst 3.0, enabling specific and fast nucleic acid amplification. It contains WarmStart feature enabled by a reversibly bound aptamer that allows room temperature set up and greatly reduces off-target amplification to enhance specificity. In addition to the glycerol-containing Bst-XT WarmStart, we will also offer Bst-XT WarmStart in the glycerol-free format in the catalog.

We will be providing Bst-XT WarmStart and Bst-XT WarmStart glycerol-free in a two-vial format, which includes the enzyme stored in its diluent, accompanied with its optimized 10X Bst-XT amplification buffer and 100 millimolar magnesium sulfate. Bst-XT isothermal amplification buffer is optimized specifically for Bst-XT. It is also lyo-compatible, so we have a single buffer to support both glycerol-containing and glycerol-free Bst-XT products. This is different than Bst 2.0 products that glycerol-containing angle-cell-free Bst 2.0 WarmStart have their own unique reaction buffers. Additionally, Bst-XT amplification buffer contains 2 millimolar magnesium at 1X concentration, allowing optimization of the final magnesium concentration in LAMP assays.

We are launching Bst-XT WarmStart product number in 9204 at 8,000 units per milliliter, and Bst-XT WarmStart glycerol-free product number in 9205 at 120,000 units per milliliter. Both enzymes will be available in the NEB catalog in April 2025. Based on reaction optimization, we are offering guidelines for setting up LAMP and RT-LAMP reaction using Bst-XT WarmStart. We recommend including 1X Bst-XT isothermal amplification buffer, which contains 2 millimolar magnesium at 1X, and supplementing the reaction with additional formula molar magnesium to increase the final magnesium concentration to 6 millimolar, and adding eight units of Bst-XT WarmStart in a typical LAMP reaction.

In addition to the Bst-XT WarmStart enzyme buffer and magnesium, LAMP reactions require dNTPs and Primer Mix. dNTP mix can be purchased from NEB. And as I mentioned before, we recommend utilizing NEB primer design tool to design the primer sets for a successful DNA or RNA target detection. In order to detect RNA targets robustly, including WarmStart RTX, reverse transcriptase is highly encouraged, and it can be edited at 7.5 units total per RT-LAMP reaction. After all components are edited, including the RNA or DNA sample, the reaction can be incubated at 65 degrees for 20 minutes. You may need to increase the incubation time up to 40 minutes for low copy or challenging targets. Additionally, we strongly recommend running a no template control to ensure amplification specificity. In specific cases, deviation from the standard protocol may be required. So please refer to the protocol on the product page for additional recommendations once the product is available.

I would like to highlight our recommendation for the final magnesium concentration for Bst-XT, as it is different than Bst 2.0 and Bst 3.0. For most assays, the optimal concentration for magnesium is 6 millimolar, and this concentration will ensure fast and specific length RT-LAMP amplifications while using Bst-XT. Certain assays and specific applications may require further optimization of the magnesium concentration, and we will elaborate on these applications later in the webinar.

As described previously, LAMP produces large quantities of DNA, and therefore it's compatible with a variety of detection modalities. The amplification can be detected using a DNA intercalating dye to monitor the fluorescent change in real time. And NEB LAMP fluorescent dye which has a product number B1700 can be included in the reaction at 0.1 to 1X concentration to enable detection in the cyber fem channel of a real-time PCR instrument.

Following the amplification, the CQ values detected by the qPCR instrument can be used to determine the time to detection for a given input. Alternatively, LAMP amplification can be detected via endpoint colorimetric detection, lateral flow, turbidity, or agarose gel. For endpoint non-pH-based colorimetric detection, metal indicator dyes, including hydroxynaphtol blue or calcein with manganese are common detection components, and they offer a simplified visual readout for the amplification reaction. Lateral flow readout is often preferred in point-of-care assays, and they generally require the LAMP primers to be labeled with a fluorophore and biotin.

For turbidity detection or analyses by agarose gel electrophoresis, no detection components are necessary. If LAMP reactions will be analyzed by agarose gel electrophoresis or other methods that require opening the reaction tubes post-amplification, we highly recommend to have a separate post-amplification analysis area to avoid potential contamination. Bst-XT is compatible with all these detection modalities, and we will be showcasing the performance of the Bst-XT WarmStart DNA polymerase in fluorescent LAMP assays in the upcoming slides. And later in the talk, we will also highlight the endpoint colorimetric detection compatibility of Bst-XT WarmStarts.

In this part of the talk, I will discuss the comparison between Bst-XT WarmStart and other NEB Bst enzymes, including Bst 2.0 WarmStart and Bst 3.0, first in LAMP, and then RT-LAMP assays. I will specifically describe the properties in detection speed and specificity. For this comparison, LAMP experiments were performed with Bst 2.0 WarmStart, Bst 3.0, and Bst-XT WarmStart, in the respective isothermal amplification buffers, and recommended final magnesium concentration. The reactions were set up in triplicate over three logs of total human gDNA at 10 milligrams down to 0.1 nanogram in 96 cell plate format and at 25 microliter reaction volume. All reactions contained 1X LAMP primer and 0.5X nonfluorescent dye. Reactions were incubated at 65 C° for 30 minutes, and fluorescence was monitored in the cyber fam channel of a real-time thermal cycler.

The graphic on the left describes how we determine the time to detection. As the amplification reaction proceeds, the fluorescent signal crosses the instrument defined threshold and we report this as time to detection. And therefore, earlier time points reflect faster amplification. The time to detection are presented in the graph on the right for each enzyme. And the highlighted column represents Bst-XT WarmStart amplification data. Please note the dots that represents time to detection frequent to overlap for the replicates, and all replicates were detected successfully unless otherwise is indicated on the graph. For this particular DNA target, Bst 2.0 WarmStart exhibits lower detection across all template input as seen with the blue dots in the graph. Detection gets faster when using Bst 3.0, indicated with the gold dots, and Bst-XT WarmStarts shown in red dots. And both enzymes demonstrate very similar detection speed for all template inputs.

As noted earlier, LAMP assays may be prone to non-template amplification based on primer design. Therefore, we also evaluated control reaction without template, which is indicated as NTC in the graph on the right column to assess the specificity of the amplification. Bst 2.0 showed fast amplification for no template controls within 30 minutes of incubation, which is indicated with the gold dots in the graph. On the other hand, Bst 2.0 WarmStart and Bst-XT WarmStart show no off-target amplification. As you can appreciate, the NTC columns are clear for both enzymes.

We tested additional human DNA targets and observed the same trend. Bst 2.0 and Bst-XT WarmStart DNA polymerase showed fast amplification and better performance at low template input compared to Bst 2.0 WarmStart. Only Bst 3.0 exhibited undesired amplification in the no-template controls, while Bst-XT WarmStart maintained its specificity within 30 minutes of incubation. We also compared Bst 2.0 WarmStart, Bst 3.0 and Bst-XT WarmStart in RT- LAMP assay in detecting three different human RNA targets at 10 milligrams down to 0.1 nanogram total template input, including no template controls. As previously mentioned, we highly recommend including WarmStart RTx reverse transcriptase to improve speed and sensitivity for RNA detection. Therefore, all testing was done in the presence of NEB WarmStart RTX.

I also want to highlight that we included WarmStart RTX for all RNA detection data that we will be presenting later in the webinar. Similar to the testing we performed to detect DNA targets, we use respective isothermal amplification buffers and follow the recommendations for the final magnesium concentrations for each enzyme. RNA targets exhibited same pattern as DNA target detection. Bst 2.0 and Bst-XT WarmStart DNA polymerase showed fast amplification and better performance at low template inputs compared to Bst 2.0 WarmStart. However, Bst 3.0 exhibited amplification in the no-template control as can be seen with the gold dots in the NTC column, while Bst 2.0 WarmStart and Bst-XT did not show any NTC amplification.

Overall, both DNA and RNA detection data highlights that Bst-XT WarmStart DNA polymerase combines the specificity of Bst 2.0 WarmStart DNA polymerase and the amplification speed of Bst 3.0 DNA polymerase.

Now, I will hand off the presentation to Kristine, and she will continue with explaining additional characteristics of Bst-XT WarmStart DNA polymerase.

Kristine Werling:

Thanks, Burcu. Now I will move on to discuss some data that highlights the advantages of the WarmStart feature for Bst-XT. So here we're showing data for Bst-XT WarmStart, where two different reaction setups were run in parallel. One reaction was set up on ice, as usual, and is labeled as Ice on the graph shown. And the other reaction was set up on the bench top simply at room temperature. And this is indicated as Room Temp on the graph. We tested one DNA target and one RNA target, and the resulting time to detection is shown for 10 nanograms of input in orange, or one nanogram of input in purple. So by comparing the Ice versus Room Temp column, you can see that the time to detection is really equivalent between these two conditions. And if you now look over to the right-hand column in the NTC reactions, you can see that no non-specific amplification was observed within 30 minutes for either of these conditions, really demonstrating that the specificity of Bst-XT WarmStart is maintained even when reactions are set up at room temperature.

Besides simple room temperature setup, the WarmStart feature of Bst-XT may also be particularly useful for applications where the complete reaction mix will be incubated together for more extended periods of time, such as when adopting for lyophilization or automated workflows. Additionally, the WarmStart feature could also be beneficial in cases where you're testing a complex sample with high amounts of background nucleic acid, such as testing a human biological sample for the presence of a specific pathogen. WarmStart helps ensure that the reaction does not initiate until elevated temperatures are reached, and this helps limit off-target amplification. As Burcu mentioned previously, Bst-XT WarmStart will be available in both a glycerol-containing and glycerol-free format through the NEB catalog. So here we are showing side-by-side data for those two formats of Bst-XD WarmStart, with glycerol-containing version shown in blue, and glycerol-free version shown in gold.

So looking across three DNA targets on the left and three RNA targets on the right, either at 10, 1, or 0.1 nanograms input, as well as an NTC condition, we see that these enzymes perform equivalently in terms of both amplification speed and specificity. Therefore, you can really be confident in selecting the formulation format that best suits your needs. And furthermore, just to reiterate, the same isothermal amplification buffer is provided with both enzymes and it is biocompatible.

Next, we will look at the temperature range at which Bst-XT WarmStart can be used to perform LAMP reactions. As outlined earlier, in a standard LAMP reaction, we recommend heating to 65 °C for 20 minutes to achieve optimal performance of Bst-XT WarmStart for most targets. But depending on your particular application, it may be desirable to have more flexibility with reaction temperature, perhaps going a little bit higher or lower than the standard 65 °C.

So here we tested the performance of Bst-XT WarmStart compared to Bst 2.0 WarmStart and Bst 3.0 in LAMP reactions run across a range of temperatures. So looking at this graph, you can see that the reaction temperatures are displayed along the X-axis. And on the Y-axis, we plotted the time to detection for a DNA target with one nanogram of input. Bst-XT data is shown in red, Bst 2.0 in blue, and Bst 3.0 in gold. And once again, faster time to detection is better. So we are assessing whether the target was amplified within 30 minutes in this assay. And as you can see, Bst-XT WarmStart performs the best across a broader temperature range than either Bst 2.0 WarmStart or Bst 3.0. And more specifically, we see that from 50 °C up to 70 °C, Bst-XT WarmStart is able to detect target within 30 minutes.

Instead, for Bst 2.0 and Bst 3.0, they tolerate a narrower range of reaction temperatures before that time to detection starts to get delayed beyond 30 minutes. And notably, Bst-XT WarmStart yields the fastest amplification when reaction temperatures are run below the standard 65 °C. So if you're interested in running LAMP reactions at slightly lower temperatures, Bst-XT WarmStart would be the enzyme of choice.

So now we have demonstrated the key features and capabilities of Bst-XT WarmStart. And in summary, it enables fast amplification, high specificity, and room temperature startup. It will also be available in a glycerol-free format and has a broader optimal temperature range for LAMP reactions outperforming Bst 2.0 and Bst 3.0 at lower temperatures.

Now that you are familiar with the core characteristics of Bst-XT WarmStart, we will move on to discuss data showing how Bst-XT performs in the presence of a few different additives that may be carried over into your LAMP reaction from either your sample type or your sample preparation method. Specifically, we will look at the performance in the presence of Viral Transport Medium, ethanol, and monovalent salt.

First, we tested the performance of Bst-XT WarmStart compared to Bst 2.0 WarmStart and Bst 3.0 in the presence of increasing amounts of Viral Transport Medium, or VTM for short. We tested from 0% up to 20% VTM in a 25 microliter reaction. And looking at this graph, you can see the percent VTM tested labeled across the top. And the data on the left shows time to detection for a DNA target. And the data on the right shows the results of the NTC reactions up to 40 minutes.

So as you can see, Bst-XT WarmStart shown in red, maintains fast amplification even in reactions with up to 20% BTM. And furthermore, comparing to 2.0 in blue or 3.0 in gold, you can see that in the presence of VTM, Bst-XT WarmStart yields the fastest time to detection for the target. Also looking at the NTC results on the right, we can see that only Bst 3.0 produces non-specific amplification within 40 minutes. And we don't see any NTC signal for reactions with Bst-XT across the range of BTM concentrations tested, demonstrating that it also maintains its specificity.

Next, we tested the tolerance of NEB's Bst enzymes to the presence of ethanol in a LAMP reaction. Ethanol could be carried over into your reaction from an upstream nucleic acid purification step. In here, we tested zero to 10% ethanol in a 25 microliter reaction. Once again, on the left, we are showing the time to result data for a DNA target. And on the right, we have the results for the NTC reactions.

So first, looking at the data for Bst 2.0 shown in blue, we see that Bst 2.0 is only able to tolerate up to 5% ethanol. Instead, Bst 3.0 in gold and Bst-XT in red can both tolerate 7.5% ethanol. Now, looking specifically in the column for 7.5% ethanol, we see that Bst-XT WarmStart is faster than Bst 3.0, and better able to tolerate this carryover component. And now, if we look over at the NTC data on the right, we once again see that Bst-XT also remains specific.

Finally, we also tested the tolerance of Bst-XT WarmStart to the presence of increasing concentrations of monovalent salts, which could be present in certain biological sample types and get carried over into the LAMP reaction. So to test this, reactions were run with Bst-XT WarmStart in its provided isothermal amplification buffer and with the standard recommended 6 millimolar final concentration of magnesium sulfate. Then on top of that, additional monovalent salt was spiked into these standard reaction conditions. The amount of additional salt that was added is indicated on the figure in + mM amounts. And here, the time to detection for one nanogram of DNA target is shown in red. As you can see, when 90 millimolar of additional monovalent salt is spiked into the standard reaction conditions, Bst-XT maintains nearly identical time to result as control conditions, which are shown in the first column. This demonstrates that for this assay, Bst-XT can tolerate up to 90 millimolar of additional salt without a negative impact on performance.

Moving across the graph in the condition with 115 millimolar of additional salt, the time to detection here is still quite fast coming in under 15 minutes, but amplification has started to slow compared to the control. And at 140 millimolar of additional salt, Bst-XT shows more significant slowing with a time to result around 25 minutes. And then finally on the far right, you can see that, for this assay, Bst-XT could not tolerate an additional 165 millimolar of monovalent salt, as amplification has completely dropped off here. So all in all, based on our findings from this assay, if you are working with a high salt sample type, it's likely that up to 90 millimolar of additional salt will be well tolerated in a LAMP reaction with Bst-XT. Higher than that, you might start to expect to see some delay in amplification. And based on these data, we do not currently recommend more than 140 millimolar of added salt.

Next, we will move on to discussing a few recommendations that we have for using Bst-XT in specific reaction conditions. For instance, when using UDG and dUTP for carryover prevention, as well as when using metal sensing dyes for endpoint color metric detection.

So first, what is carryover contamination? Well, as previously mentioned, during the course of a LAMP reaction, a high amount of amplified double-stranded DNA is produced. And while this is beneficial for a variety of different detection strategies, it can also create a challenge if you are repeatedly running LAMP reactions in the same lab space, as it increases the chance that amplicon produced in an earlier reaction will contaminate a subsequent reaction. And this can lead to false positive results that can be hard to eliminate. To address this challenge, NEB recommends use of a carryover contamination prevention system. And this system requires two components: dUTP and antarctic thermolabile UDG.

When dUTP is included in your LAMP reactions, the Bst DNA polymerase will incorporate uracils into the newly synthesized amplicons. If the uracil-containing amplicon accidentally gets into a future reaction, antarctic thermolabile UDG will be active simply while you are setting up your reaction, and it will remove the uracils from the contaminating amplicon and leave abase excites. And critically, this prevents re-amplification of the carryover products. UDG is then deactivated at elevated temperatures and does not interfere with amplification. Therefore, including both UDG and dUTP in your LAMP reactions greatly reduces the chance of false positive results.

Bst-XT WarmStart is able to incorporate dUTP into synthesized DNA. So UDG-based carryover contamination prevention can be used with this enzyme. However, as shown here for a DNA target on the left and an RNA target on the right, when UDG and dUTP are included in a standard Bst-XT reaction with 6 millimolar final magnesium, we saw that there can be a delay in time to detection compared to reactions without UDG and dUTP. But we found that time to detection can largely be recovered if the final magnesium concentration is simply increased to 7 millimolar, shown here in the far right column for both the DNA and RNA targets. Therefore, when using UDG and dUTP for carryover contamination prevention with Bst-XT, we recommend deviating from the standard protocol in using 7 millimolar final magnesium to better maintain fast amplification.

Now, we will move on to briefly discuss the compatibility of Bst-XT WarmStart with another LAMP detection strategy. Throughout this talk, we have presented data using NEB's intercalating LAMP fluorescent dye and a real-time PCR instrument. But as previously discussed, there are many different ways that LAMP reactions can be detected. And one alternative detection strategy that is particularly common is endpoint colorimetric detection. One class of dyes that can be used for a non-pH-based colorimetric detection in LAMP is metal sensing dyes. These dyes change color as the concentration of free metal ions shifts during an amplification reaction. Endpoint colorimetric detection is particularly valuable because it doesn't require real time monitoring with a PCR instrument, and can simply be assessed by eye after the reaction is complete.

One common metal sensing dye is calcium. Calcium was pre-incubated with manganese chloride and then tested with Bst-XT WarmStart by spiking it into standard LAMP reactions. Reactions were then heated at 65 °C for 25 minutes and then imaged with either a scanner or camera phone. And those images are shown here. Overall, calcium worked as expected without additional optimization. Under normal light, the NTC or negative reactions were a light orange color, while the positive reactions with one nanogram of template DNA appeared a bright yellow. This color change from orange to yellow is reasonably visible under normal light, but the difference between positive and negative reactions is even more obvious when viewed under UV light. And here we use just a simple handheld UV flashlight. And as you can see in the second row of data here, the positive reactions produce a strong glow that is easily distinguishable from the dark NTC tubes. Therefore, calcium can be used with Bst-XT in standard conditions for endpoint colorimetric detection.

In addition to calcium, another common metal sensing dye used in LAMP is hydroxynaphtol blue, or HNB. HNB is expected to appear a dark purple and negative reactions and then changed to a bright blue and positive reactions. We first tested HNB with Bst-XT WarmStart using standard LAMP reaction conditions, including having a final magnesium concentration of 6 millimolar. We incubated the reaction at 65 °C for 25 minutes and then captured images. But as you can see in the top row for the HNB data, when the standard 6 millimolar of final magnesium was used, the color contrast between the positive and negative reactions was very poor, and they all appeared a similar shade of blue. Since HNB senses free magnesium ions, we tried increasing the final magnesium concentration up to 8 millimolar. And as you can see, when eight mM final magnesium was used, shown in the bottom row of HNB data. The color contrast between positive and negative reactions was greatly improved. And now we see the expected dark purple color for the negative reactions, and the bright blue color for the positive reactions.

Therefore, Bst-XT is compatible with HNB for endpoint colorimetric detection, but you may need to increase final magnesium up to 8 millimolar to achieve optimal color contrast between positive and negative reactions.

So overall, today we've demonstrated the exciting properties of Bst-XT WarmStart and highlighted its key features, as well as how it's unique from Bst 2.0 WarmStart and Bst 3.0. We also described the performance of Bst-XT in a few more specific scenarios, and shared recommendations for optimal use.

Briefly in summary, Bst-XT WarmStart combines the high specificity of Bst 2.0 WarmStart with the fast amplification speed of Bst 3.0. It also has a WarmStart feature that enables LAMP reactions to be set up at room temperature. And it will be available in glycerol-containing and glycerol-free formats with the same lyo-compatible buffer and magnesium for optimization. Furthermore, Bst-XT can be used to perform LAMP reactions across a broader temperature range, and can be used with dUTP and UDG for carryover prevention, as well as with metal sensing dyes for endpoint color metric detection.

So now that you're familiar with Bst-XT, we will leave you with a brief summary of the upcoming products. As mentioned, Bst-XT WarmStart will be available in the NEB catalog in both a glycerol-containing and glycerol-free format. But we also wanted to note that the non-WarmStart versions of these enzymes will be available through our customized solutions channel. So if you're interested in those offerings, please get in touch with us at neb.com/customized solutions.

Thank you so much for tuning in, and we will now welcome your questions.

Burcu Minsky:

Hi, we have a question about applications of glycerol-free Bst-XT. "So using Bst-XT in the glycerol format will be very useful to support lyophilization and automation workflows, because in both of these processes glycerol may interfere with the process."

Kristine Werling:

Okay. And we have another question asking when Bst-XT will be available for purchase in the catalog and how much it will cost, as well as a few questions about getting the non-WarmStart version of Bst-XT.

So all of these products will be available for purchase next month in April 2025. And the pricing will be very similar to our current offerings for Bst 2.0 WarmStart. And then as we showed on that last slide, but just to reemphasize, the non-WarmStart version of these enzymes will be available in the customized solutions channel, also in April.

Burcu Minsky:

So we have another question about the fidelity of Bst-XT. So we performed some preliminary measurements using a single molecule sequencing assay, and its fidelity is similar to Bst 2.0. So this assay confirmed that.

Kristine Werling:

Okay. So now we also have a few questions regarding a few other inhibitors and asking how tolerant Bst-XT is to those particular inhibitors. And from the preliminary data that we shared here today, it suggests that Bst-XT is more tolerant to general inhibitors than Bst 2.0, but we haven't done extensive testing on all possible inhibitors or different sample types to know definitively how Bst-XT will perform in those different specific conditions.

Burcu Minsky:

So we have one question about the shelf life of Bst in glycerol format. So it can be stored at -20 for two years.

Kristine Werling:

Okay. So we also have a question regarding endpoint colorimetric detection, specifically whether Bst-XT is compatible with pH-based color change. And pH-based colorimetric detection does require having a weekly buffered reaction condition. So the reaction buffer that's provided with Bst-XT is fully buffered. So pH-based colorimetric detection will not be compatible with these reaction conditions.

Burcu Minsky:

So we have one question about multiplexing capacity for Bst-XT. So Bst-XT can be used to perform multiplex LAMP reactions. And we tested this for both lateral flow and fluorescent pro base assays. But we just want to emphasize that additional optimization may be required for your particular assay when you multiplex using Bst-XT.

Kristine Werling:

Okay. We also have some questions regarding the temperature range data that we shared for Bst-XT, and wondering whether those are for WarmStart or non-WarmStart Bst-XT. And all the data that we shared regarding the temperature profile was with WarmStart Bst-XT. So those reactions that we showed from 50 up to 70 °C were with the WarmStart version of that enzyme. So if you're interested in those lower temperatures, that was with the WarmStart version.

Burcu Minsky:

So we have a question about the particular detection method that works most effectively with Bst-XT DNA polymerase. As we emphasize in our presentation, we tested Bst-XT in different detection modalities, including fluorescence and endpoint colorimetric. And also we tested lateral flow and Bst-XT performed very well in all of these detection methods. Of course, LAMP is very versatile in terms of different detection methods. So there's definitely a room for using in different detection modalities. But again, we just want to emphasize we will have a very detailed protocol for all of these methods. Then the product will be available. You can see that in our product page. But if you want to use a different detection method, maybe slight modifications are needed for particular application.

Kristine Werling:

Okay. We also have a question regarding the RT activity of Bst-XT, whether it's reduced or absent. And the inherent RT activity of Bst-XT is present. So if you detect an RNA target with just a single enzyme, you are able to get detection. But as Burcu mentioned, to really enhance that detection and improve the speed of amplification, we do recommend using an RT in those reactions.

Burcu Minsky:

So we have a question about asking Bst-XT and Bst 3.0 are faster than Bst 2.0. Is this SA dependent? My answer is no, because we've been seeing this consistently across different RNA and DNA targets and with different detection methods. So Bst-XT and Bst 2.0 are generally faster. In most assays, it's faster than Bst 2.0.

Kristine Werling:

Okay. We also have some questions regarding the buffer for Bst-XT. And we just want to say in regards to that, that the Bst-XT isothermal amplification buffer provided with the enzyme has been specifically optimized for the best performance of Bst-XT. And that's for both the glycerol tree and glycerol-containing versions. So we do recommend using the guidelines that we'll provide on the product page. And once again, this buffer is also LIO compatible.

Moderator:

Looks like we are coming up on time, so we have time to take one more question.

Burcu Minsky:

So there is one question about deactivating Bst-XT. So we recommend if you need to deactivate Bst-XT, you can heat up the reaction to 85 degrees for 20 minutes.

Moderator:

Thank you so much. Thank you to Kristine and Burcu for taking the time to present today. If you guys have any other additional questions, please feel free to email us at webinars@neb.com. The webinar will be available on demand after today's live event. And we hope you all have a wonderful day. Thank you.