Clinical Chemistry 63:3
714–722 (2017)
Point-of-Care Testing
Two-Stage Isothermal Enzymatic Amplification for
Concurrent Multiplex Molecular Detection
Jinzhao Song,1 Changchun Liu,1 Michael G. Mauk,1 Shelley C. Rankin,2 James B. Lok,2
Robert M. Greenberg,2 and Haim H. Bau1*
BACKGROUND: The wide array of pathogens responsible
for infectious diseases makes it difficult to identify causative pathogens with single-plex tests. Although multiplex PCR detects multiple targets, it is restricted to centralized laboratories, which delays test results or makes
multiplexing unavailable, depriving healthcare providers
of critical, real-time information.
METHODS: To address the need for point-of-care (POC)
highly multiplexed tests, we propose the 2-stage, nestedlike, rapid (⬍40 min) isothermal amplification assay,
dubbed rapid amplification (RAMP). RAMP’s first-stage
uses outer loop-mediated isothermal amplification (LAMP)
primers to amplify all targets with recombinase polymerase amplification (RPA). First-stage amplicons are aliquoted to second stage reactors, each specialized for a
specific target, to undergo LAMP. The assay is implemented in a microfluidic chip. LAMP amplicons are detected in situ with colorimetric dye or with a fluorescent
dye and a smartphone.
RESULTS: In experiments on a benchtop and in a microfluidic format, RAMP demonstrated high level of multiplexing (ⱖ16); high sensitivity (i.e., 1 plaque-forming
unit of Zika virus) and specificity (no false positives or
negatives); speed (⬍40 min); ease of use; and ability to
cope with minimally processed samples.
CONCLUSIONS: RAMP is a hybrid, 2-stage, rapid, and
highly sensitive and specific assay with extensive multiplexing capabilities, combining the advantages of RPA
and LAMP, while circumventing their respective shortcomings. RAMP can be used in the lab, but one of its
distinct advantages is amenability to simple implementation in a microfluidic format for use at the POC, providing healthcare personnel with an inexpensive, highly sen-
1
Department of Mechanical Engineering and Applied Mechanics, School of Engineering and
Applied Sciences, University of Pennsylvania, Philadelphia, PA; 2 Department of Pathobiology, School of Veterinary Medicine, University of Pennsylvania, Philadelphia, PA.
* Address correspondence to this author at: Department of Mechanical Engineering and
Applied Mechanics, University of Pennsylvania, 233 Towne Bldg., Philadelphia, PA
19104. Fax +215-573-6334; e-mail [email protected].
Received July 13, 2016; accepted October 31, 2016.
Previously published online at DOI: 10.1373/clinchem.2016.263665
714
sitive tool to detect multiple pathogens in a single sample,
on site.
© 2016 American Association for Clinical Chemistry
The ability to concurrently detect multiple pathogens
infecting a host is crucial for accurate diagnosis of infectious diseases, identification of coinfections, assessment
of disease state and treatment efficacy, assessment of drug
resistance, and profiling of microbial flora. Pathogens
that cause infectious diseases are often coendemic, making it imperative to distinguish single infections from
coinfections, which can alter host responses, disease prognosis, transmission dynamics, and treatment strategies
and outcomes (1– 6 ). Syndromic panels for infectious
diseases can assist healthcare personnel to determine effective treatments and the potential need for additional
ancillary diagnostic testing. Standard, culture-based microbiological tests suffer from long turnaround times,
ranging from hours to days, and require skill and laboratory facilities. Moreover, culture conditions for many organisms of interest are not known (7 ). Parasitological
and serological methods for detecting infections can be
inaccurate, labor-intensive, and unreliable (8, 9 ).
Molecular methods, particularly PCR, have expanded the range of pathogens that can be identified and
greatly shortened time to detection. Existing diagnostic
assays are, however, either limited in scope or highly
complex (10 ). Although multiplex PCR (mPCR)3 has
the potential to amplify many nucleic acid (NA) targets
in a single reaction (11, 12 ) and, in principle, is suitable
for multipathogen detection, it has several limitations.
Nonspecific products generated through primer–primer
interactions may interfere with the amplification of targets, decreasing sensitivity and selectivity. Moreover, detection of multiple amplicons requires bead arrays, mi-
© 2016 American Association for Clinical Chemistry
Nonstandard abbreviations: mPCR, multiplex PCR; NA, nucleic acid; nmPCR, nested
mPCR; LAMP, loop-mediated isothermal amplification; isoPCR, 2-stage amplification
combining PCR and LAMP; POC, point-of-care; RPA, recombinase polymerase amplification; mRPA, multiplex RPA; RAMP, rapid amplification; ZIKV, Zika virus; HPV, human
papilloma virus; PFU, plaque-forming units; T1/2, the threshold time (the time required
for the signal to reach half its saturation value); LCV, Leuco crystal violet; gDNA, genomic
DNA; dsDNA, double-stranded DNA.
3
New Method for Multiplex Molecular Detection
croarrays, or probes with different color fluorophores,
which complicates the assay and increases cost (13–15 ).
Recently, nested mPCR (nmPCR) (16 –18 ) and
2-stage amplification combining PCR and loopmediated isothermal amplification (LAMP) (isoPCR)
(19, 20 ) have emerged as powerful methods to concurrently detect multiple pathogens. Both involve 2 successive steps of NA amplification, with the first step comprising mPCR and the second step either target-specific
PCR or LAMP (21 ). Amplicons of the first step serve as
templates in the second step. Both nmPCR and isoPCR
significantly enhance limits of detection and specificity
over single-stage, mPCR. However, both methods require at least one thermal cycling (PCR) process. Thermal cycling complicates instrumentation and is not
compatible with point-of-care (POC) applications.
Moreover, the need to transfer first-stage amplicons to
the second-stage exposes the NA-rich first-stage tube to
the environment, potentially contaminating the work
space. A closed system for 2-stage amplification is, therefore, preferable.
In recent years, there has been a growing interest in
isothermal amplification methods (22 ) such as recombinase polymerase amplification (RPA) (23 ) and LAMP
(21 ) for POC diagnostics. A few groups have developed
multiplexed RPA and multiplexed LAMP assays (24 –
28 ) for codetection of a small number (i.e., ⱕ4) of targets
(24 –28 ).
To improve the sensitivity of the amplification process, enable concurrent detection of multiple NA targets,
and benefit from the advantages of isothermal amplification (such as simple instrumentation and low power consumption), we propose a 2-stage, isothermal–isothermal,
enzymatic amplification method, dubbed rapid amplification (RAMP), that consists of a first-stage RPA and
second-stage LAMP. To demonstrate RAMP’s capabilities, we first carried out benchtop experiments to optimize the assay and compare its sensitivity to that of
LAMP alone and RPA alone. Second, we examined multiplex assays to detect pathogens that are prevalent in
low-resource settings, and, in some cases, coendemic. To
eliminate the need for pipetting, to carry out the entire
RAMP assay in a closed system, and to enable testing
by minimally trained personnel, we implemented the
RAMP assay in a microfluidic format.
Methods
RAMP consists of 2 successive isothermal enzymatic amplification stages. The first stage is a RPA isothermal amplification at approximately 37 °C for 10 –20 min, with
primers for all the targets. First-stage amplicons are then
distributed among individual LAMP reactors, each specific to a target, and serve as templates in second-stage
LAMP reactions at 60 – 65 °C, typically for 30 min.
LAMP is monitored in real time, with either a nonspecific intercalating fluorescent dye or a colorimetric dye.
The first-stage RPA uses a mixture of all F3 and B3
primers of the second LAMP stage concurrently amplifying all the targets present in the sample (Fig. 1). In
contrast, each of the second-stage reactors operates with a
set of 6 LAMP primers and amplifies a specific target.
Typical amplicons’ length range from 180 –330 bp.
RAMP was implemented either on benchtop (Fig. 1A) or
in a microfluidic format with spontaneous distribution of
first-stage products to second-stage reactors (Fig. 1C).
In our experiments, we used different types of samples: (a) purified DNA and RNA, (b) serum from Schistosoma mansoni-infected mice, (c) spiked human serum
and whole blood, and (d) spiked urine samples. See Supplemental Materials and Methods in the Data Supplement that accompanies the online version of this article at
http://www.clinchem.org/content/vol63/issue3 for additional details of the experimental procedures.
Results
We first describe our benchtop experiments with the
RAMP assay, which, among other things, serve to optimize the assay. Then, we describe the implementation of
the assay in a microfluidic format.
SINGLE-PLEX RAMP
We first investigated RAMP’s performance with purified
samples with single targets on the benchtop (see Supplemental Results 1 in the online Data Supplement). RAMP
was consistently more sensitive (typically, ⫻10) than
LAMP alone and more specific and efficient than RPA
alone. Any spurious first-stage RPA amplicons did not
amplify in the specific second-stage LAMP and did not
produce any detectable signals. In contrast to RPA (23 ),
RAMP’s amplicons can be detected in situ with nonspecific dyes without a need to discharge the amplicons to a
hybridization array.
Next, we examined the effects of first-stage (RPA)
reaction time and primer combinations on overall RAMP
performance (see Supplemental Results 2 in the online
Data Supplement). A 20-min RPA, followed with a
15–20 min LAMP yielded detectable signals even in the
presence of low-abundance targets. Specifically, the
single-plex RAMP detected successfully 0.5 fg S. mansoni
DNA. We also examined various primer combinations.
RAMP assay with F3-B3 primers in stage 1 and the 4
primers FIP, BIP, Loop F, and Loop B in stage 2 provided highly specific results and were used throughout
this report. The sequences of the various primers used in
this work are listed in Supplemental Table 1A in the
online Data Supplement.
Clinical Chemistry 63:3 (2017) 715
Fig. 1. The working principle of the RAMP assay.
(A), RAMP procedure on the benchtop. (B), The primer sets used in RAMP. (C), A microfluidic chip for RAMP’s amplification in a closed system,
featuring a central multiplex RPA (mRPA) reactor (green) and 16 branching LAMP reactors (blue) for specific targets and controls.
MULTIPLEX RAMP ASSAY
Although RAMP has higher sensitivity than LAMP and
is useful for this reason alone, RAMP’s main benefit is its
capacity to detect multiple targets. We first carried out a
4-plex RAMP assay for the detection of HIV, S. mansoni,
Plasmodium falciparum, and Schistosoma hematobium,
and found it highly sensitive and specific (see Supplemental Results 3 in the online Data Supplement). Encouraged by this successful performance, we increased
the level of multiplexing to 16 targets. We designed an
assay to detect S. mansoni, HIV-1 clade B, S. hematobium, P. falciparum, Schistosoma japonicum, Brugia malayi, Strongyloides stercoralis, drug-resistant Salmonella,
Zika virus (ZIKV)-America strain (mex 2– 81, Mexico),
ZIKV-Africa strain (MR 766, Uganda), human papilloma virus (HPV)-58, HPV-52, HPV-35, HPV-45,
HPV-18, and HPV-16. The primers’ sequences are listed
in Supplemental Table 1A in the online Data Supplement. The above targets were selected for proof of concept, taking advantage of reagents and targets available in
our lab, and not for clinical reasons (though many of the
selected pathogens are coendemic). The targets are both
716
Clinical Chemistry 63:3 (2017)
DNA and RNA (HIV-1 and ZIKV), and range from
viruses to multicellular metazoans.
Fig. 2 depicts amplification curves of samples containing the following according to the panels listed here:
(A) HPV-16 (100 copies) and ZIKV [50 plaque-forming
units (PFU), American strain]; (B) HPV-18 (100 copies)
and ZIKV (50 PFU, African strain); (C) HIV-1 clade B
(100 copies), P. falciparum (300 fg DNA), the schistosome S. japonicum (1 pg DNA), the filarial nematode B.
malayi (13 pg DNA), the soil-transmitted nematode S.
stercoralis (1 pg DNA), and drug-resistant Salmonella
(100 copies); and (D) no targets (negative control). Once
again, RAMP proved to be highly sensitive and specific,
with no false positives or negatives. At the tested concentrations, the assay successfully discriminated among various strains of HPV and between American and African
strains of ZIKV.
To examine assay sensitivity and the dependence of
the threshold time on target concentration, we repeated
our experiments by using a dilution series. In the interest
of brevity, we show results only for the American strain of
the ZIKV. Fig. 2E depicts the amplification curves ob-
New Method for Multiplex Molecular Detection
Fig. 2. A 16-plex, 2-stage RAMP assay designed to detect (1) S. mansoni, (2) HIV-1 clade B, (3) S. haematobium, (4) P. falciparum, (5)
S. japonicum, (6) B. malayi, (7) S. stercoralis, (8) drug-resistant Salmonella, (9) ZIKV-America strain (mex 2– 81, Mexico), (10)
ZIKV-Africa strain (MR 766, Uganda), (11) HPV-58, (12) HPV-52, (13) HPV-35, (14) HPV-45, (15) HPV-18, and (16) HPV-16.
The first amplification stage (RPA) operates with a mixture of all the F3 and B3 primers of the targets. Each second-stage LAMP operates with
the 4 primers. Amplification curves when the sample contains (A), HPV-16 (100 copies) and ZIKV (American strain, 50 PFU); (B), HPV-18 (100
copies) and ZIKV (African strain, 50 PFU); (C), HIV-1 clade B (100 copies), P. falciparum (300 fg), S. japonicum (1 pg), B. malayi (13 pg), S.
stercoralis (1 pg), and drug-resistant Salmonella (100 copies); and (D), no targets are present (negative control). (E), Samples with various
numbers of ZIKV: 1, 5, 50, and 500 PFU. Note the high sensitivity of the assay, detecting 1 PFU. When the number of templates is ≥5 PFU, the
data are highly reproducible. (F), T1/2 as a function of the number of target viruses (PFU).
tained with the 16-plex RAMP assay in the presence of 0,
1, 5, 50, and 500 PFU of the American ZIKV. Note that
1 PFU of ZIKV is detected. When the number of Zika
templates was equal to or larger than 5 PFU, the thresh-
old time [the time required for the signal to reach half its
saturation value (T1/2)] was a linear function of the number of target ZIKV (PFU) (Fig. 2F) and data were highly
reproducible (with a relative standard deviation in
Clinical Chemistry 63:3 (2017) 717
Fig. 3. Microfluidic chip-mediated 4-plex RAMP.
(A), Design of the microfluidic chip. (B), Fluorescence image of RAMP reaction in the microfluidic chip for multiplex detection of HIV RNA (1000
copies), drug-resistant Salmonella gDNA (approximately 100 copies), and S. mansoni gDNA (0 fg). The fluorescence image was taken at t = 50
min. (C), Colorimetric detection of amplicons from 100 copies HIV RNA in tube with LCV dye that changes from colorless to violet in the
presence of dsDNA. (D), Colorimetric multiplex detection of HIV, S. mansoni, P. falciparum, S. haematobium on chip, by using LCV dye. The
simulated samples contained (i) no targets, (ii) P. falciparum DNA (300 fg), (iii) P. falciparum DNA (300 fg), and HIV RNA (1000 copies). S. m.,
S. mansoni; P. f., P. falciparum; S. h., S. hematobium.
threshold time of 2%). If we were to process a 100-␮L
sample, the RAMP’s detection limit for ZIKV would be
better than 50 PFU/mL. This is orders of magnitude
lower than the ZIKV concentrations, ranging from 103–
106 PFU/mL, in symptomatic Zika-infected patients
(29 ). Moreover, our data suggest that multiplex RAMP
can genotype HPV strains and, at the target concentrations tested, differentiate the American ZIKV from the
African strain.
To reduce test complexity, it is occasionally desirable to minimize, or even eliminate, sample preparation.
To assess RAMP compatibility with minimally processed
samples, we used RAMP to detect pathogens in urine,
serum, and whole blood samples without NA extraction
(see Supplemental Results 4 in the online Data Supplement). RAMP demonstrated robustness and was refractory to inhibitors. Specifically, the16-plex RAMP of this
section successfully and reproducibly detected 5 PFU
ZIKV when 5 ␮L urine was added directly to the firststage reaction volume.
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Clinical Chemistry 63:3 (2017)
IMPLEMENTATION OF RAMP IN A MICROFLUIDIC FORMAT
To enable the use of RAMP at the POC by minimally
trained personnel, avoid the tedium of pipetting firststage products into multiple second-stage reactors, and
minimize risk of contamination, it is desirable to automate the process of distributing first-stage products into
second-stage reactors and implement it in a closed system.
Fortunately, these objectives can readily be achieved.
We designed, fabricated, and tested a prototype of a
microfluidic chip for multiplex detection of DNA and
RNA targets with RAMP. Fig. 3 and Fig. 1C show, respectively, 4-plex and 16-plex chips. Fig. 3A depicts schematically the structure of the 4-plex plastic chip, which
includes a main chamber for the first-stage RPA reaction
and 4 branching chambers for second-stage LAMP amplifications (the number of these branching chambers can
be adjusted according to the number of desired targets as
shown in Fig. 1C). In the embodiment shown in Fig. 3A,
the first-stage 25-␮L (RPA) amplification chamber is in
direct communication with the second-stage chambers,
New Method for Multiplex Molecular Detection
each 15 ␮L in volume. In another embodiment (with
which we have experimented, but do not discuss here),
we sealed the connectors between the first-stage chamber
and the second-stage chambers with paraffin that melts
once the chip’s temperature exceeds 60 °C. During the
first stage (37 °C), the paraffin remains solid and blocks
the passages between the first-stage RPA chamber and the
LAMP chambers. When the chip’s temperature increases
to 65 °C for the second-stage LAMP, the paraffin melts,
opening the passages and allowing the RPA products diffuse into the second-stage LAMP chambers to serve as
templates in the second-stage amplification.
Our hypothesis is that the first-stage amplicons are
uniformly distributed in the first-stage chamber. This
hypothesis is borne out by the reproducibility of our data,
but requires further study. The rate of diffusion of the
amplicons from the first stage to the second stage is controllable by the size of the passages leading from the firststage chamber to second-stage chambers.
In the experiments described here, the targets and
RPA cocktail were introduced into the first-stage chamber and incubated at 37 °C for a predetermined time (15
min). The first-stage amplicons diffused into the secondstage chambers. After 15 min, the chip was heated to
63 °C to carry out LAMP amplifications. The prestored
LAMP reagents (currently, in liquid form) included either an intercalating fluorescent dye such as EvaGreen or
a colorimetric dye such as Leuco crystal violet (LCV)
(30 ). The fluorescence emission and the change of color
in the second-stage chambers were, respectively, monitored during the amplification process with a smartphone
camera (31 ) and by eye. We did not observe any crosstalk
among the second-stage chambers. Nevertheless, in future implementations, we will encapsulate the lyophilized LAMP reagents in paraffin for refrigeration-free,
long shelf life and just-in-time release (32 ).
As a proof of concept, we prestored in the secondstage chambers LAMP primers targeting HIV clade B
(chambers 1 and 3), drug-resistant Salmonella (chamber
2), and S. mansoni DNA (chamber 4). We then added
1000 copies of HIV RNA and 100 copies of drugresistant Salmonella genomic DNA (gDNA) (and no S.
mansoni gDNA) in RPA reaction buffer and inserted the
reaction buffer in the first-stage chamber. We monitored
the emissions from the LAMP reactors as functions of
time (see the Supplemental Video in the online Data
Supplement). Fig. 3B depicts the fluorescence emission
from the second-stage reactors 50 min after the start of
the assay. The second-stage reactors specific for HIV (2
reactors) and Salmonella emit strong fluorescence signals.
The S. mansoni reactor that serves here as a negative (no
target) control emits no signal, as expected. This experiment indicates that the RAMP assay is amenable to simple microfluidic implementation that eliminates the need
for manual transfer of first-stage amplicons to the second
stage. Moreover, the transfer from the first to the second
stage takes place in a closed system, eliminating the risk of
contaminating the environment with NAs or picking up
contaminants from the environment.
Although we can excite fluorescence with a cell
phone flash and detect emission with the cell camera
(31 ), this requires the use of filters to separate between
the excitation and emission spectra, which may add
slightly to the device’s cost. To further simplify the assay,
we replaced fluorescence-based detection with a colorimetric LCV (30 ) dye, allowing us to read the RAMP
results by naked eye, without any detector, or monitor
them with a cell phone camera.
Fig. 3C shows colorimetric detection of 100 copies
of HIV RNA targets in PCR vials. Notice the welldefined color contrast between the colorless negative control and the violet positive test. The dye-based detection
can be readily implemented into our microfluidic format.
Fig. 3D depicts examples of microfluidic RAMP assays
with different samples, by using dye-based detection.
The second-stage reactors were specialized to detect
HIV-1 clade B, S. mansoni, P. falciparum, and S. hematobium. Fig. 3D(i) shows results obtained with a negative
(no targets) sample. None of the second-stage reactors
turned violet, consistent with negative tests. Fig. 3D(ii)
shows results for a sample containing 300 fg P. falciparum
DNA and no other targets. Note that only the reactor
specific for P. falciparum turned violet, while the 3 other
chambers remained colorless, consistent with a positive
test for P. falciparum and negative tests for the other 3
targets. Fig. 3D(iii) shows results for a sample containing
300 fg P. falciparum DNA, 1000 copies of HIV RNA,
and no other targets. Only the reactors specialized for P.
falciparum and HIV turned violet, consistent with a positive test for P. falciparum and HIV and negative for the
other 2 targets included in the assay. The multiplexed
assay operated well, producing no false positives and no
false negatives. To eliminate subjectivity, we found we
could delegate the test reading to a smartphone that imaged the signal, compared it to a control, and applied
preprogrammed thresholds to discriminate between positive and negative signals.
Discussion
Different infections can cause similar overt symptoms,
but require diverse disease management strategies (i.e.,
the current ZIKV epidemic) (1, 6 ). Patients may be subject to coinfections that alter symptoms, immune responses, and therapies (2–5 ). These are just 2 examples in
which multiplexed platforms would provide timely, costeffective diagnosis. Although PCR-based multiplexed
platforms are available, for the most part, they require
expensive equipment and highly skilled personnel, which
precludes their use in resource-poor settings and causes a
Clinical Chemistry 63:3 (2017) 719
Table 1. Qualitative comparison of a few NA amplification methods for multiplex detection.
Method
RAMP
Multiplex
capability
Sensitivity
Specificity
Tolerance to
inhibitors
16
Platform
Amplification
time
++++
++++
++++
Isothermal
≤40 min
RPA, Kersting et al. (27 )
4
+++
+
++++
Isothermal
≤30 min
LAMP, Liang et al. (28 )
4
+++
++++
++
Isothermal
≤40 min
mPCR, Reddington et al. (12 )
15
++
+++
+
Thermal cycler
⬵5 h
nmPCR, Sotlar et al. (16 ), Poritz et al. (17 ),
Popowitch et al. (18 )
18
++++
++++
+
Thermal cycler
⬵1–2 h
isoPCR, Søe and Warthoe (20 )
24
++++
++++
+
Thermal cycler
⬵1 h
significant delay between sample collection and test
results. There is a great need in both developed and
developing countries to enable healthcare providers
and epidemiologists to make timely, informed decisions regarding treatment options and efficacy. Economic pressures also motivate efforts to develop multiplexed diagnostic procedures that can be carried out at
the patient’s side.
To address this need, we propose a 2-stage, hybrid
isothermal enzymatic amplification method, dubbed
RAMP, comprising a first-stage RPA reaction, in which
all the targets in the sample are amplified concurrently,
combined with a second, highly specific LAMP. RAMP
merges the advantages of RPA and LAMP, while circumventing their shortcomings. RAMP benefits from RPA’s
high tolerance to inhibitors while overcoming RPA’s tendency to produce spurious amplicons. RAMP can concurrently detect multiple DNA and RNA targets without
undue demands on sample volume, operating with volumes similar to those used in single-plex detections.
Specifically, RAMP’s first stage uses outer LAMP
primers F3 and B3 of all targets while the second stage
uses the other 4 RAMP primers. This nested-like principle provides high sensitivity, specificity, and robust, extensive multiplexing capabilities. RAMP’s sensitivity is,
respectively, about 10- and 100-fold better than LAMP
when operating with purified and crude samples. RAMP
is also more efficient and specific than RPA. Although
spurious amplicons are produced in RAMP’s first stage,
they do not undergo further amplification in the second
stage and their concentration is sufficiently low as not to
produce false signal, permitting the use of inexpensive
nonspecific dyes to identify the presence of doublestranded DNA (dsDNA).
We demonstrated 2 detection modalities. In a few of
our experiments, we used an intercalating fluorescent dye
that can be excited with a smartphone flash and detected
with a phone camera (31 ). In other experiments, we
adapted the colorimetric dye LCV, which does not require excitation. The color change induced by the presence of amplicons can be visualized by eye or detected
720
Clinical Chemistry 63:3 (2017)
with a cell phone camera. Both labeling methods are
compatible with POC applications. In contrast, detection of RPA products requires specific molecular beacons
and/or hybridization arrays.
Although we applied a 16-plex RAMP to samples
that included similar targets, such as different strains of
HPV and the ZIKV, the assay did not produce any false
positives or false negatives and at the concentration tested
was able to discriminate among the various strains. Although more extensive testing is needed, the initial results
are highly promising. We demonstrated here a successful
16-plex RAMP. It is likely that concurrent detection of
more than 16 targets is feasible.
RAMP has many advantages over conventional multiplexing strategies such as mPCR, nmPCR, and isoPCR.
RAMP is faster and more inhibitor-tolerant than mPCR,
nmPCR, and isoPCR. RAMP takes ⬍40 min, even in
the presence of low-abundance samples. In contrast,
amplification times of nmPCR (16, 19, 33, 34 ) and
isoPCR (19 ) exceed 2 h and 1 h, respectively. Since in
our RAMP experiments, we did not optimize the primers, we allocated 15–20 min to the first-stage RPA and 20
min to the second-stage LAMP. With optimized primers,
such as elongated F1-B1 primers, it may be possible to
further reduce the duration of the RPA (23 ) and thereby,
of the entire RAMP.
RAMP inherits RPA’s tolerance to impurities. In the
presence of high-abundance targets, RAMP can detect
targets in minimally purified samples, which is not possible with PCR (35, 36 ). By equipping the first-stage
reaction chamber with an NA isolation membrane, as we
have previously described (37 ), we decouple the sample
volume from the reaction volume, purify the NAs in situ,
and achieve sensitivities on par with state-of-the-art
laboratory-based equipment. Finally, since RAMP is
based on 2 isothermal processes and does not require
thermal cycling, it can operate with much simpler instrumentation than PCR. Table 1 provides a qualitative comparison of RAMP with other multiplexing strategies.
One of RAMP’s major advantages is its easy implementation in a microfluidic format, wherein first-stage
New Method for Multiplex Molecular Detection
amplicons diffuse into the second-stage reactors without
a need for mechanical aliquoting. The entire RAMP process can be carried out in a closed system without exposing the NA-rich, first-stage products to the environment,
minimizing contamination risk. The ability to implement RAMP in a simple microfluidic format and its minimal demands on instrumentation, make it an ideal candidate for POC applications: at the sample collection site,
next to the patient, at home, in the clinic, or in the field.
For POC applications, it is necessary, however, to prestore all reagents in the diagnostic cassette, refrigerationfree. Although we did not discuss reagent storage in this
report, we describe a possible strategy elsewhere (32 ).
In summary, RAMP is a hybrid, 2-stage, rapid, highsensitivity and -specificity assay with extensive multiplexing capabilities. RAMP can be used in the lab, but one of
its distinct advantages is its amenability to implementation at the POC, providing healthcare personnel with a
tool to detect multiple pathogens in a single sample without a need to send the sample to a centralized laboratory.
We demonstrate the feasibility of the RAMP assay, showing concurrent detection of DNA and RNA from several
pathogens, including retroviruses, flaviviruses, papillomavirus, bacteria, protozoa, and parasitic helminths. In
the future, we hope to use the RAMP concept to develop
various diagnostic panels of medical significance such as
vector-transmitted pathogens and HPV genotyping.
Author Contributions: All authors confirmed they have contributed to
the intellectual content of this paper and have met the following 3 requirements: (a) significant contributions to the conception and design, acquisition of data, or analysis and interpretation of data; (b) drafting or revising
the article for intellectual content; and (c) final approval of the published
article.
Authors’ Disclosures or Potential Conflicts of Interest: Upon manuscript submission, all authors completed the author disclosure form. Disclosures and/or potential conflicts of interest:
Employment or Leadership: None declared.
Consultant or Advisory Role: None declared.
Stock Ownership: None declared.
Honoraria: None declared.
Research Funding: J. Lok, NIH; R. Greenberg, NIH; H. Bau, NIH
grant 1R41AI104418-01A1 (to the institution).
Expert Testimony: None declared.
Patents: H. Bau, US provisional patent application no. 62/278,095.
Role of Sponsor: The funding organizations played no role in the
design of study, choice of enrolled patients, review and interpretation of
data, and final approval of manuscript.
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