(soft gentle music)
- Welcome to the COVID-19 Researcher Spotlight Series.
Today, we're speaking with Greg Patton,
a development scientist here at New England Biolabs.
Greg was one of the developers
of the SARS-CoV-2 Rapid Colorimetric LAMP Assay Kit,
which was just released by NEB.
Greg is gonna tell us about how the kit works,
what type of samples are compatible with it,
and he'll explain the kit's intended uses.
Hi, Greg, thanks so much for taking time out
of your schedule to join us today.
- Thanks for having me back on the podcast.
Although the circumstances are a little different this time.
- Oh, they certainly are.
So thanks for joining me remotely.
As one of the developers of NEB's new
SARS-CoV-2 Rapid Colorimetric LAMP Assay Kit,
could you tell our listeners what the kit does?
- Yeah.
So, the kit is a set of reagents designed
to determine the presence of SARS-CoV-2 RNA
in a sample within 30 minutes,
using a simple visual color change.
The kit is a research use only kit,
and it relies on loop mediated isothermal amplification
or LAMP.
LAMP is an isothermal technique that involves
four to six primer sets that target a specific nucleic acid
and allows amplification of that nucleic acid
at one uniform temperature.
So, here, the kit uses 65 degrees Celsius.
The kit relies on colorimetric detection,
and to be able to do that, we take advantage
of the chemistry of amplification.
So, when a DNA polymerase incorporates a nucleotide,
a proton is released in the process.
LAMP makes a lot of DNA, which means there are
a lot of nucleotides that get incorporated and thus,
there are a lot of protons that are released.
And so, what we do is we couple the reaction
with a pH indicator.
So, if there is amplification, you get a generation
of a lot of protons.
Those protons then basically change the color
of the pH indicator.
So, if you don't have amplification,
the reaction stays pink, but if you have amplification,
the reaction changes color to yellow.
And so, it allows a very simple way
to determine basically the presence of SARS-CoV-2 RNA.
- And how many samples can be tested per kit?
- Yeah, so the kit is essentially able
to test 24 different samples.
It's a 96 reaction kit, but we envision four reactions
being set up per sample that you want to interrogate.
So there's a no template control,
a positive control, an internal control.
And that internal control just makes sure
that your sample input is compatible with the Assay,
and also makes sure that you actually have sample there.
And then the final reaction is your SARS-CoV-2 test
to determine the presence or absence of SARS-CoV-2 RNA.
- What kind of samples are compatible with the kit?
- We recommended isolated total nucleic acid going
into the kit, but there are a lot of people who are trying
to go directly from saliva, for example.
And we have tested some of those workflows,
and it certainly may work with different sample input types,
but there may be some optimization that's required
to do some different sample types or go direct.
- Understood.
And what does the kit contain?
- Yeah, so the kit is essentially a six component kit.
The first main component is the WarmStart® Colorimetric LAMP
Master Mix with Antarctic Thermolabile UDG and dUTP.
- And what do those kit components do?
- Yeah, so thermolabile UDG and dUTP
enable carryover prevention,
and carryover prevention is sort of an old technique
that prevents previous amplicons
from contaminating future reactions,
and prevents false positives from occurring.
So, basically, amplicons that are made
with the carryover prevention mix,
so the thermolabile UDG and dUTP end up containing uracil,
and then uracil containing amplicons are a substrate
for thermolabile UDG.
So, any previous amplicons that get carried over
into a future reaction will be destroyed
by thermolabile UDG during setup.
And then thermolabile UDG is quickly inactivated
upon basically incubating your reactions
at 65 degrees Celsius, which is the isothermal
amplification step that drives amplification,
so that you can essentially amplify your target nucleic acid
without the thermolabile UDG actually destroying
what is being amplified.
- Understood.
What are the other components of the kit?
- Yeah, so the second component is a positive control.
It is essentially the N gene of a SARS-CoV-2 RNA genome
in a plasmid.
And it just makes sure that basically
you're getting detection of the control
as you set up the kit.
The next two,
basically, reagents are the primers.
So there's an internal control primer set
that targets the Actin RNA to ensure again
that your human sample RNA is present,
and make sure that your sample is compatible with the kit.
And then the other primer set
is the SARS-CoV-2 RNA primer set,
which is a mixture of primers that target the N gene
and E genes of SARS-CoV-2 RNA.
- And how did we land on these primers?
- Yeah, so there was a lot of development work
that was done on the best primer combo.
Really the N and E combined together
give us the best sensitivity, and so we settled upon using
a dual primer set for the kit.
The last two components of the kit are basically water,
nuclease-free water to set up the reactions,
and then guanidine hydrochloride.
- Why guanidine hydrochloride?
- We showed in a Biotechniques paper
that guanidine hydrochloride actually
is an enhancer of LAMP.
Particularly for our N2 and E1 primer sets
that are part of the SARS-CoV-2 primer set,
basically 40 to 60 millimolar of guanidine hydrochloride
enhances detection.
It basically improves sensitivity and gives
them more robust yellow color after 30 minutes.
And we decided to include that as a separate component,
rather than putting it in the master mix itself
to give users flexibility.
There's a lot of work being done
on, basically, upfront sample prep workflows.
Some of those workflows may include guanidine,
and so if a user has a total nucleic acid
or RNA direct sample with guanidine present in the solution,
they're going to be carrying that into the reaction.
And so, we're gonna ask users to basically
omit the guanidine, so that you don't
have too much guanidine within the reaction,
because too much guanidine actually starts
to basically
cause a loss of performance.
- What makes the kit unique?
- I think one of the things that makes the kit unique is
essentially the minimum amount of equipment you need
to basically set up and detect SARS-CoV-2 RNA.
Isothermal amplification methods benefit
from traditional methods such as RT-qPCR,
in that they really just need a heat block.
And a set of pipettes to assemble the reactions.
And this will really allow this kit
to be used in low resource settings.
Something like RT-qPCR, you need instrumentation,
a real-time instrument to be able
to detect the SARS-CoV-2 RNA,
and basically, real time instruments
a PCR requires a dedicated thermocycler,
which draws a lot of power because it has to cycle
between temperatures in order to drive amplification,
which is in direct contrast to an isothermal method,
which you could use a standard heat block or water bath
to do the reactions.
So, I see the real advantage of the kit being used
in low resource settings.
- Understood.
So, what are the intended uses of the kit?
You mentioned that it's research use only,
but obviously, there will be interested in using this kit
to test patient samples.
So, what are the intended use cases for this kit?
- Yeah, as you mentioned, it's a research use only kit,
but certainly someone could take that kit
and basically put it in their diagnostic workflow
if they were interested in doing so.
They would need to collect the relevant clinical data
and submit to the FDA in order
to get the emergency use authorization
on using their workflow containing this kit
in diagnosing of patients.
- I see.
Were there any particular challenges that arose
when you were developing the kit?
- Yeah.
There were a number of challenges during the development
of this kit.
I think one of them was the rapid timeline
in which we needed to develop this product
to meet the humanitarian need.
I just wanna take a moment to thank all
of the NEB scientists and our collaborators
who have helped get this kit to market.
It's really been a collaborative effort
over the last few months to make it happen.
But I think one of the biggest challenges we faced
in the last couple of months
was really improving the sensitivity of the kit.
So, when we started this project back in March,
we had an original primer set,
which we called the NA primer set,
that we used in a paper that was published in collaboration
with the Wuhan Institute of Virology,
to basically show proof of principle,
that the colorimetric LAMP could be used
to use on patient samples and show detection
of SARS-CoV-2 RNA.
The problem with that original primer set really was,
it wasn't giving us the best sensitivity.
The LOD (limit of detection) of that primer set
was probably a couple 100 copies,
and that wasn't good enough.
The gold standard is RT-qPCR and in those workflows
you can generally detect five to 10 copies.
So, we spent a lot of time optimizing
around the primer sets, and we tested a number
of different primer sets.
The N2 and E1 primer sets that are now present in the kit,
individually perform really well.
And when we put them together,
we really got that synergistic effect,
and it allows us to detect down to 50 copies
with 95% confidence.
So we're claiming the LOD is 50 copies on synthetic RNA.
- That's amazing.
So, you mentioned RT-qPCR as being the gold standard
of SARS-CoV-2 diagnostics currently.
How does this test compare to RT-qPCR
in terms of sensitivity?
- Yeah.
So there are limitations of, certainly, colorimetric LAMP.
Again, 50 copies is a reasonable LOD.
RT-qPCR can go lower, five to 10 copies.
But again, you sort of require that dedicated thermocycler
to be able to enable amplification and you need some way
to determine amplification with RT-qPCR.
Usually it's quantitative real-time PCR that's being used,
so you need a fluorimeter
to monitor the amplification reaction.
Here, with colorimetric, the colorimetric LAMP,
you essentially get a nice visual readout,
no fancy equipment required other than your eyes.
And so, it's a quick way to basically get to whether
or not SARS-CoV-2 RNA is present.
- Well, I'm really appreciative of all the hard work
that you and your team at NEB have put into it,
all of the scientists at NEB, as well as the collaborators
around the world who have been helping validate the kit
and testing the validity of the results.
And I think it's really promising.
I know a lot of our customers are really excited to see it
on the market.
So, thank you so much.
- Yeah. Thank you.
- [Lydia] Thanks joining us for this episode
of the COVID-19 Researcher Spotlight Series.
If you'd like to learn more
about NEB SARS-CoV-2 rapid colorimetric LAMP Assay kit,
you can visit the product page at neb.com/e2019.
Hope you can join us
for the next COVID-19 Researcher Spotlight,
because it's a great one.
I'll be interviewing Chris Mason of Weill Cornell Medicine.
Chris is a voracious collaborator and has built a consortium
of researchers devoting themselves to understanding
and detecting SARS-CoV-2.
He's also working to bring rapid testing
to communities across the United States,
as well as to analyze the evolution of SARS-CoV-2
through genome sequencing.