© 2020 All rights reserved
Over the weeks since launching this website, the team at Vienna BioCenter was been working hard to help users implementing LAMP tests, improving the methods and looking out for more scientific opportunities. The commitment we have for developing rapid and affordable testing goes beyond publications in periodicals. As we are fully aware of the speed with which everyone in the world has to adapt to the changing situation regarding the still accelerating covid-19 pandemic, we aim to bring you the most accurate and robust protocols as soon as we develop them. Please, read these updated carefully and make sure you are using the most current version of the protocols on our page. For more information or to get in contact, please write to us at info@rtlamp.org.
Using hydroxynaphtol blue indicator dye in LAMP reactions has the immense advantage of enabling a wide range of input material to be used in the reactions, from swabs in VTM all the way to gargle and saliva. But the color change can be tricky, and we have been getting reports of it not being totally clear in some people’s hands. We heard you and we now have an approach that should fix this issue.
We are now recommending the use of extra WarmStart Bst2.0 DNA polymerase to commercial RT-LAMP reactions in order to increase the reaction yield, speed and clarify the colorimetric result. In our hands, this has proven to be a safe and efficient way to improve the color readout without compromising on issues such as false positive rate and while maintaining the low price per reaction. For a stronger color readout, add 0.32 units of WarmStart Bst2.0 DNA polymerase per microliter of final reaction volume, which is 3.2 units of extra polymerase per 10 µl reaction. Read more on the commercial RT-LAMP protocol page.
Assembling each master mix for every testing session from scratch is not necessary, however freezing prepared master mixes increases the risk of running into sensitivity or specificity issues. We’ve tested the possible combinations and now we can tell you what you can and can’t premix for easier reaction assembly without compromising on the quality of the tests. The premixing instructions for commercial RT-LAMP reactions and open-access RT-LAMP reactions are now to be found in their respective protocol pages, as well as here.
| Reagent | Per 100 reactions (10 μl size) |
|---|---|
| WarmStart LAMP Kit (2x) | 500 |
| dUTP (100 mM) | 7 |
| Magnesium sulfate (100 mM) | 7 |
| Thermolabile UDG (optional) | 20 |
| WarmStart Bst2.0 DNA polymerase (120 000 U/ml) | 2.6 |
| total | 535 |
Add 5.35 µl of this mix per 1 reaction.
Note: A concentrated version of WarmStart Bst2.0 polymerase (NEB cat. nr. M0538M) is used to minimise the volume of the premix.
Store at -20 °C.
| Reagent | Per 100 reactions (10 μl size) |
|---|---|
| 10x LAMP primer mix | 100 |
| nuclease-free water | 100 |
| total | 200 |
Add 2 µl of this mix per 1 reaction.
Note: You may use the As1, E1 or ACTB primers to make a LAMP primer premix. Make sure to label the mix adequately.
Store at -20 °C.
| Reagent | Per 100 reactions (10 μl size) |
|---|---|
| HNB Dye solution (3 mM) | 40 |
| nuclease-free water | 25 |
| total | 65 |
Add 0.65 µl of this mix per 1 reaction.
Store at 4 °C.
The premixes for commercial RT-LAMP enable you to have three tubes in your freezer and fridge and to assemble reactions easily using 5.35 µl of LAMP reaction buffer premix, 2 µl of LAMP primer premix and 0.65 µl of LAMP HNB premix per 10 µl reaction. We have observed these premixes to be stable for a month at the specified storage conditions, but they are most likely good even for longer.
The premixes for open-access RT-LAMP enable you to have two tubes in your -20 °C freezer and to assemble reactions easily using 7.5 µl of LAMP reaction buffer premix and 0.5 µl of LAMP enzyme premix per 10 µl reaction. We’ve observed their stability to be unchanged after 1 week’s storage at -20 °C, but they can most likely last for longer. It is crucial to work quickly with the enzyme mix and to work with it on ice or on the cold.
| Reagent | Stock concentration | To add per 100 reactions (10µl size) | |
|---|---|---|---|
| Isothermal amplification buffer | 10X | 100 μl | |
| dNTP mix | 25 mM | 56 μl | |
| dUTP | 100 mM | 7 μl | |
| Magnesium sulfate | 100 mM | 60 μl | |
| As1 primer mix | 10X | 100 μl | |
| HNB dye | 20 mM | 6 μl | |
| Syto9 fluorescent dye (optional) | 100 μM | 20 μl | |
| Betaine | 5 M | 80 μl | |
| nuclease-free water | 321 μl | ||
| total | 750 μl |
Add 7.5 µl of this mix per 1 reaction.
Store at -20 °C.
| Reagent | Stock concentration | Amount per reaction | To add per 100 reactions (10µl size) |
|---|---|---|---|
| HIV-RT enzyme | variable | 75 ng | variable |
| BstLF enzyme | variable | 200 ng | variable |
| Thermolabile UDG enzyme (NEB) | 1 U/μl | 0.2 U | 20 μl |
| storage buffer | to a total of 50 µl | ||
| total | 50 µl |
Add 0.5 µl of this mix per 1 reaction.
The volumes for enzymes are listed as “variable” since each enzyme purification yields a different concentration of enzyme. We recommend measuring the yield you get from your purifications and make the calculations yourself, so as to reach 75 ng of reverse transcriptase and 200 ng of DNA polymerase, respectively. Then multiply the volumes for 75 ng and 200 ng of enzyme, respectively, by 100 to fill in the table. Add storage, as described in the enzyme expression and purification protocol on the open access protocol pages, to a total of 50 µl.
Store at -20 °C.
The premixes for open-access RT-LAMP enable you to have two tubes in your -20 °C freezer and to assemble reactions easily using 7.5 µl of LAMP reaction buffer premix and 0.5 µl of LAMP enzyme premix per 10 µl reaction. We’ve observed their stability to be unchanged after 1 week’s storage at -20 °C, but they can most likely last for longer. It is crucial to work quickly with the enzyme mix and to work with it on ice or on the cold.
We would like to discourage the use of NEB N-gene-A and DETECTR N-gene, primer set, as listed in our preprint and as described in the publications of Zhang, Odiwuor, et al., 2020 and Broughton et al., 2020. In our hands, they have proven to be liable to non-specific amplification.
On the other hand, we would like to endorse RT-LAMP users to employ the As1, As1e, E1 and ACTB primer sets for their excellent sensitivity and very high specificity. These primer sequences were published by Rabe & Cepko, 2020 for the As1 and As1e primers targeting Orf1ab region of the genome, and by Zhang, Ren, et al., 2020 for the E1 region of the SARS-CoV-2 genome and the ACTB human transcript.
© 2020 All rights reserved