RES E AR C H Open Access
Detection of SARS-CoV-2 RNA using RT-
LAMP and molecular beacons
Scott Sherrill-Mix
1,2
, Young Hwang
1
, Aoife M. Roche
1
, Abigail Glascock
1
, Susan R. Weiss
1
, Yize Li
1
, Leila Haddad
3
,
Peter Deraska
3
, Caitlin Monahan
3
, Andrew Kromer
3
, Jevon Graham-Wooten
2
, Louis J. Taylor
1
, Benjamin S. Abella
4
,
Arupa Ganguly
3
, Ronald G. Collman
2
, Gregory D. Van Duyne
5*
and Frederic D. Bushman
1*
* Correspondence: vanduyne@
pennmedicine.upenn.edu;
bushman@pennmedicine.upenn.
edu
5
Department of Biochemistry and
Biophysics, Perelman School of
Medicine, University of
Pennsylvania, Philadelphia, PA
19104, USA
1
Department of Microbiology,
Perelman School of Medicine,
University of Pennsylvania,
Philadelphia, PA 19104, USA
Full list of author information is
available at the end of the article
Abstract
Background: Rapid spread of SARS-CoV-2 has led to a global pandemic, resulting in
the need for rapid assays to allow diagnosis and prevention of transmission. Reverse
transcription-polymerase chain reaction (RT-PCR) provides a gold standard assay for
SARS-CoV-2 RNA, but instrument costs are high and supply chains are potentially
fragile, motivating interest in additional assay methods. Reverse transcription and
loop-mediated isothermal amplification (RT-LAMP) provides an alternative that uses
orthogonal and often less expensive reagents without the need for thermocyclers.
The presence of SARS-CoV-2 RNA is typically detected using dyes to report bulk
amplification of DNA; however, a common artifact is nonspecific DNA amplification,
which complicates detection.
Results: Here we describe the design and testing of molecular beacons, which allow
sequence-specific detection of SARS-CoV-2 genomes with improved discrimination in
simple reaction mixtures. To optimize beacons for RT-LAMP, multiple locked nucleic
acid monomers were incorporated to elevate melting temperatures. We also show
how beacons with different fluorescent labels can allow convenient multiplex
detection of several amplicons in “single pot” reactions, including incorporation of a
human RNA LAMP-BEAC assay to confirm sample integrity. Comparison of LAMP-
BEAC and RT-qPCR on clinical saliva samples showed good concordance between
assays. To facilitate implementation, we developed custom polymerases for LAMP-
BEAC and inexpensive purification procedures, which also facilitates increasing
sensitivity by increasing reaction volumes.
Conclusions: LAMP-BEAC thus provides an affordable and simple SARS-CoV-2 RNA
assay suitable for population screening; implementation of the assay has allowed
robust screening of thousands of saliva samples per week.
Keywords: COVID-19, SARS-CoV-2, Coronavirus, Loop-mediated isothermal
amplification, Molecular beacon, LAMP-BEAC, RT-LAMP
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Sherrill-Mix et al. Genome Biology (2021) 22:169
https://doi.org/10.1186/s13059-021-02387-y
Background
Since its first detection in December 2019, the beta-coronavirus SARS-CoV-2 has
spread around the world, at this writing infecting over 150 million people and causing
over 3 million deaths. Frequen t asymptomatic spread of this virus means that frequent,
rapid, and affordable screening and surveillance testing are essential to controlling this
pandemic [1–3].
Numerous methods have been developed to detect SARS-CoV-2 infection. The most
common method is RT-qPCR to detect SARS-CoV-2 RNA [4]. RT-qPCR has the ad-
vantage of providing accurate and sensitive detection, but supply chain issues have at
times limited testing, motivating the development of additional methods using orthog-
onal materials. RT-LAM P has been widely studied as an alternative [5–9]. LAMP assays
use a “rolling hairpin” mechanism to allow amplification at a single temperature using
polymerase enzymes different from those used for PCR, helping avoid supp ly chain bot-
tle necks. In addition, RT-LAMP can be implemented on neat saliva, or on RNA puri-
fied using simple reagents available in bulk [9], again helping bypass supply chain
issues and adding robustness to assays.
RT-LAMP assays are typically not as sensitive as RT-qPCR assays [1], but the import-
ance of this varies with the application. Clinical diagnostic tests typically require high
sensitivity; however, studies suggest that infected individuals are far more infectious
during periods of peak viral loads, so methods for screening asymptomatic populations
can be adequate even with lesser sensitivities [1, 2]. A recent study emphasized tha t fre-
quency of testing and speed of reporting results are much more important than assay
sensitivity for reducing transmission, emphasizing the value of assays like RT-LAMP
that may be implemented efficiently and inexpensively [1 ].
However, a complication is that RT-LAMP reactions often result in non- specific
amplification in the absence of target, particularly at longer reaction times , limiting sen-
sitivity. This off-target amplification is especially problematic because LAMP reactions
are commonly quantified using colorimetric or fluorescent dyes reporting only bulk
DNA synthesis. To address these problems, improvements based on sequence-specific
detection have been proposed such as incorporating DNA sequencing (LAMP-seq) [10]
or CAS enzymes (DETECTR) [11]. These methods are promising, but as presently de-
signed they typically require opening of RT-LAMP tubes and secondary manipulation
of reaction products, which has the potential to result in contamination of subsequent
reactions with amplification products from previous assays. In another detection
method, a quencher-fluorophore duplex can be created by adding a short oligonucleo-
tide complementary to a standard LAMP primer which is then displaced upon amplifi-
cation [12–15]. These methods are more specific than bulk reporters but are still
potentially vulnerable to false positives from spurious amplification.
Previous research has shown the potential for molecular bea cons [16] to allow
sequence-specific detection of LAMP products in “single-pot” assays [17, 18]. Here, we
adapt molecular beacons to detect SARS-CoV-2 sequences, a method we have named
LAMP-BEAC (Fig. 1a). Molecular beacon s are target-specific oligonucleotides labeled
with a fluorophore on one end and a quencher on the other. The beacons are designed
to incorporate complementary sequences on their 5′ and 3′ ends such that at low tem-
peratures the ends anne al to form a hairpin, bringing the quencher and fluorophore
into close proximity and quenching fluorescence. When the target of interest is present,
Sherrill-Mix et al. Genome Biology (2021) 22:169 Page 2 of 17
the complementary target-specific beacon sequence anneals to its target, separating the
fluorophore from the quencher and greatly increasing the fluorescent signal. The bind-
ing sites for beacons can be targeted to amplicon sequences not present in oligonucleo-
tides used for priming, thereby enhancing specificity. The increase in fluorescence
resulting from annealing of the beacon probe can be detected without manipulation of
the product or opening the reaction tube. Here we describe (1) development of molecu-
lar beacons for detection of SARS-CoV-2 RNA in LAMP-BEAC react ions, (2) develop-
ment of a LAMP-BEAC method to detect human RNA to validate sample integrity, (3)
combinations of LAMP-BEAC assays for single-pot multiplex detection, (4) develop-
ment of custom polymerases allowing inexpensive expression and purification of re-
quired enzymes, (5) use of LAMP-BEAC to screen infected subjects for viral RNA in
saliva, and (6) increased sensitivity accessible using the high specificity of molecular
beacons.
Results
Designing molecular beacons for SARS-CoV-2 RT-LAMP
Several beacons were tested for detection of SARS-CoV-2 RNA in RT-LAMP reactions
(Additional file 2: Table S1). Optimization required identifying sequence designs that
performed properly under the conditions of the RT-LAMP reaction, which is typically
run at temperatures around 65°C. Function of the beacon requires that the hairpin re-
main mostly folded in the hairpin stru cture at this temperature, while still opening suf-
ficiently often to allow annealing to the target RT-LAMP cDNA product. The annealed
beacon-target cDNA duplex must then be sufficiently stable at 65°C to result in
unquenching and an increase in fluorescence. To increase beacon affinity for use at
Fig. 1 LAMP-BEAC: RT-LAMP assayed using molecular beacons. a Product from a LAMP reaction is depicted
emphasizing its loop region which forms single-stranded loops during amplification along with an example
molecular beacon in its annealed hairpin form, which is quenched. Binding of the beacon to the target
complementary sequence LAMP amplicon separates the fluorescent group and the quencher, allowing
detection of fluorescence. The red loops on the beacon indicate locked nucleic acids used to increase
binding affinity. b A genome map of SARS-CoV-2 showing the locations of primer binding sites for the
LAMP primer sets used in this study. c Example of visual detection of LAMP-BEAC fluorescence using an
orange filter with blue illumination. Multiplexed SARS-CoV-2-targeted Penn and human control STATH
primer sets were used to amplify samples consisting of water or inactivated saliva with or without synthetic
SARS-CoV-2 RNA (10,000 copies per reaction). Molecular beacons Penn_LF_S1 conjugated to a FAM
fluorophore fluorescing green and Stath_LB_S2 conjugated to Cy3 fluorescing yellow were included in the
reaction. The image was captured using the “Night Sight” mode of a Google Pixel 2 cell phone
Sherrill-Mix et al. Genome Biology (2021) 22:169 Page 3 of 17
higher temperatures, we substituted multiple dNTP positions within the target se-
quence of each beacon with locked nucleic acids [17]. Locked nucleic acids reduce the
conformational flexibility of dNTPs and make the free energy of nucleic acid annealing
more favorable [19]. We tested the performance of 28 molecular beacons using five
previously reported SARS-CoV-2 (Fig. 1b) and three human control RT-LAMP ampli-
cons (Additional file 1: Fig. S1, Additional file 2: Table S1).
Testing LAMP-BEAC
An example of a successful bea con design is Penn_LFMB_S1 (Additional file 2: Table
S1). The RT-LAMP amplicon targets the orf1ab coding region and was first reported
by El-Tholoth and coworkers at the University of Pennsylvania (named “Penn”)[7].
The favored beacon was designed to target sequences within the forward DNA loop
generated during LAMP; thus, the beacon is designated Penn loop forward beacon,
contracted to Penn_LFMB_S1. Detection can be accomplished with laboratory plate
readers or PCR machines (below), and even visually with a simple blue light and orange
filter (Fig. 1c).
Figure 2 shows use of the Penn_LFMB_S1 system to detect synthetic SARS-CoV-2
RNA. Tests were carried out with commercial LAMP polymerase and reverse tran-
scriptase prep arations. In addition, to avoid possible supply chain problems and allow
potential production of reagents in resource limited settings, we produced and purified
novel DNA polymerase and reverse transcriptase enzymes, which were assayed in paral-
lel with commercial preparations for some tests (described below).
To compare standard LAMP amplification with LAMP-BEAC, reactions were pre-
pared containing both fluorescent dye (Fig. 2a), which detects bulk DNA by inter-
calation, and the molecular beacon Penn_LFMB_S1 (Fig. 2b). Re action pr odu cts
were detected at two wavelengths, allowing separate quantification of the bulk dye
and the molecular beac on in single reactions. The non-specific intercalating dye re-
ported bulk DNA production in positive samples earlier than the water controls,
but the negative controls did amplify shortly after. This spurious late amplification
is commonly seen with RT-LAMP, though the mechanism is unclear. The primers
may interact with each other t o form products and launch amplification, or per-
haps the reaction results from a mplification of adventitious environmental DNA. In
separate tests, synthesis of DNA products was shown to depend on addition of
LAMP primers (data not show n).
Molecular beacon Penn_LFMB_S1 in the same reactions showed more clear-cut dis-
crimination (Fig. 2b). The positive samples showed positive signal, but no signal was
detected for the negative water controls. Lack of amplification in negative controls has
been reproducible over multiple independent reactions (examples below).
The nature of the produ cts c ould be assessed using th ermal denaturation (Fig. 2c
and d). Reactions were first cooled to allow full anne aling of complementary DNA
strands, then slowly hea ted while recording fluorescence intensity. The fluoresc ent
signal of the intercalating dye started high but dropped with increasing
temperature in all samples (Fig. 2c), consistent with denaturation of the duplex
and release of the intercalating dye into solution. In contrast, the beacon’s fluores-
cent signal in the water controls started at low fluorescence (Fig. 2d), consistent
Sherrill-Mix et al. Genome Biology (2021) 22:169 Page 4 of 17
with annealing of the beacon DNA termini to form the hairpin structure (Fig. 1a).
At temperatures above 70°C, the fluorescence modestly increased, consistent with
opening of the hairpin and reptation of the beacon as a random coil in solution.
For reactions containing the RT-LAMP product and Pe nn_LFMB_S1 beacon, fluor-
escence values were high at lower temperatures, consistent with formation of the
annealed duplex, then at temperature sufficient for denaturation, the fluorescence
values fell to match those of the random coil ( Fig. 2d). Th us, the LAMP-BEAC
assay genera tes strong fluorescence signals during LAMP amplification in the pres-
ence of target RNA but not in negative controls, and the thermal melting proper-
ties are consistent with formation of the expected products.
Fig. 2 Reaction progression curves comparing RT-LAMP using the Penn primer set assayed using an
intercalating dye and the Penn_LFMB_S1 molecular beacon in the same reactions. a Conventional RT-LAMP
assay using non-specific dye to detect amplification of synthetic SARS-CoV-2 RNA diluted in water. Time
after reaction initiation (x-axis) is compared to the relative fluorescence intensity (y-axis). The copy numbers
of SARS-CoV-2 RNA in the reaction mixtures are shown in the key at the bottom. b Detection of the
amplification of SARS-CoV-2 RNA using a LAMP-BEAC molecular beacon in the same reactions shown in A.
Lines are colored as in A. c, d Thermal melting curves to characterize amplification products. The results
shown are for the same reactions as in a and b. Reaction products were cooled to room temperature, then
slowly heated for the melt curve analysis. c Characterization of the fluorescence intensity produced by non-
specific intercalating dye (y-axis) with RT-LAMP end products over varying temperatures (x-axis). Lines are
colored as in a. d Characterization of the fluorescence intensity produced by a LAMP-BEAC molecular
beacon with RT-LAMP end products over varying temperatures. Markings as in c
Sherrill-Mix et al. Genome Biology (2021) 22:169 Page 5 of 17
