Vol. 53 | No. 2 | 2012
Sequence-specific isothermal nucleic acid
amplification techniques represent a growing
sector of molecular diagnostics, offering
rapid, sensitive detection without the need
for thermal cycling equipment as required
for the PCR. Furthermore, isothermal
techniques typically provide comparable or
better detection limits compared with PCR
but in a fraction of the reaction time (1,2).
These methods have particular interest to field
or point-of-care molecular diagnostics due to
advantages in efficiency, cost, and instrumen-
tation (3). In contrast to PCR, which denatures
double-stranded DNA (dsDNA) with heat,
isothermal amplification techniques typically
use enzymatic activity to provide strand
separation of dsDNA. Sequence specificity
is provided by oligonucleotide primers that
anneal to the target sequence and are extended
by a strand displacing DNA polymerase.
Several techniques require multiple enzymes
to work in concert [e.g., strand displacement
amplification (SDA), helicase dependent
amplification (HDA), and isothermal and
chimeric primer-initiated amplification of
nucleic acids (ICAN)], but loop-mediated
isothermal amplification (LAMP) provides
sequence-specific amplification using only
a strand-displacing DNA polymerase
(techniques reviewed in References 1 and
2). In addition to the DNA polymerase,
LAMP uses four core primers (FIP, BIP, F3,
and B3) recognizing six distinct sequence
regions on the target (Figure 1), with two
primers containing complementary sequence
in order to create loop structures that facil-
itate exponential amplification (4). The use
of multiple target sequence regions confers a
high degree of specificity to the reaction. Two
additional primers, termed loop primers, can
be added to increase reaction speed, resulting
in six total primers used per target sequence
(5). The LAMP reaction rapidly (completed
in as little as 5 min) generates amplification
products as multimers of the target region in
various sizes and is substantial in total DNA
synthesis (>10 µg, >50× PCR yield) (4–6).
Measurement of the LAMP product
is typically performed by fluorescence
detection of dsDNA with an intercalating
or magnesium-sensitive fluorophore (4,7),
bioluminescence through pyrophosphate
conversion (8), turbidity detection of precip-
itated magnesium pyrophosphate (9,10), or
even visual examination through precipi-
tated Mg2P2O7 or fluorescence (11,12). These
methods are robust and familiar, and visual
methods are ideal for use in field diagnostics,
but detect total DNA amplification in a
reaction and are thus limited to detection
of a single target. As isothermal techniques
are further adopted as diagnostic tools, the
ability to detect multiple targets in a single
sample will be important. Currently, quanti-
tative PCR (qPCR) enables probe-specific
multiplex detection and the ability to perform
tests with an internal standard for definitive
negative results. However, qPCR probes
require extensive design and optimization for
use and may not effectively translate to the
LAMP reaction (13–16). Previous studies have
demonstrated methods of multiplex LAMP
detection, although they have been limited in
capability. A variety of methods have used end
point analysis, through agarose gel (17,18) or
pyrosequencing (19) methods, but these do not
allow real-time detection and require further
processing and instrumentation. In addition,
the sensitivity of LAMP reactions to carryover
contamination is so great that manufacturer
recommendations (Eiken Chemical, Tokyo,
Japan) suggest not opening LAMP reaction
vessels, or doing so in separate facilities with
separate equipment, further decreasing the
desirability of post-LAMP manipulation.
Previous real-time methods use nonspecific
quenching, either through loss-of-signal
Simultaneous multiple target detection in real-time
loop-mediated isothermal amplification
Nathan A. Tanner, Yinhua Zhang, and Thomas C. Evans, Jr.
New England Biolabs, Ipswich, MA, USA
BioTechniques 53:81-89 (August 2012) doi 10.2144/0000113902
Keywords: LAMP; isothermal DNA amplification; multiplex; real-time; detection
Supplementary material for this article is available at www.BioTechniques.com/article/113902
Loop-mediated isothermal amplification (LAMP) is a rapid and reliable sequence-specific isothermal nucleic acid
amplification technique. To date, all reported real-time detection methods for LAMP have been restricted to sin-
gle targets, limiting the utility of this technique. Here, we adapted standard LAMP primers to contain a quench-
er-fluorophore duplex region that upon strand separation results in a gain of fluorescent signal. This approach
permitted the real-time detection of 1–4 target sequences in a single LAMP reaction tube utilizing a standard real-
time fluorimeter. The methodology was highly reproducible and sensitive, detecting below 100 copies of human
genomic DNA. It was also robust, with a 7-order of magnitude dynamic range of detectable targets. Furthermore,
using a new strand-displacing DNA polymerase or its warm-start version, Bst 2.0 or Bst 2.0 WarmStart DNA poly-
merases, resulted in 50% faster amplification signals than wild-type Bst DNA polymerase, large fragment in this
new multiplex LAMP procedure. The coupling of this new multiplex technique with next generation isothermal
DNA polymerases should increase the utility of the LAMP method for molecular diagnostics.
Vol. 53 | No. 2 | 2012
guanine quenching (20) or gain-of-signal
fluorescence using labeled primers and an
intercalating dye (21). These methods can be
less sensitive, and nonspecific quenching limits
the selection of fluorophores available for
multiplexing. Our approach was to develop a
gain-of-signal, target-specific LAMP detection
that is easily implemented and not limited in
design or sensitivity.
The LAMP forward and back interior
primers (FIP and BIP) contain 5′ flaps (Figure
1; F1c sequence) that, upon synthesis and
displacement, will anneal to a complementary
downstream region (F1). We chose this region
for development of detection probes, as it is
inherent to LAMP and contains sequence
that is specific to each target, precluding
any need for probe sequence optimization.
LAMP also requires a strand displacing DNA
polymerase (typically Bst DNA polymerase,
large fragment), a component we utilize for
detection through strand displacement. Using
previously designed LAMP primers as a basis,
we synthesized the FIP modified at the 5′ end
with either a dark quencher or fluorophore.
For probe creation, we annealed oligonu-
cleotides complementary to the flap region
(F1c) with a 3′ dark quencher or fluorophore
spectrally overlapping with the fluorophore
or dark quencher of the FIP (Figure 1A).
This duplex primer maintains its function as
a LAMP primer, but upon synthesis from the
reverse direction the flap duplex is separated,
resulting in detection of amplification by
release of quenching (Figure 1B). A similar
method using a short nested probe rather than
amplification primer was applied to qPCR
(22). Our method, which we termed detection
of amplification by release of quenching or
DARQ, is adaptable for any LAMP reaction
and requires no additional probe design or
testing, merely synthesis of a 5′-modified
FIP and a 3′-modified complementary probe
(termed Fd). Similar quencher:fluorophore
duplex primers have been previously used in
isothermal amplification, including LAMP
with suggested use for mulitplexing, but to
date no demonstration of real-time, multiplex
LAMP has been achieved in this fashion
(23–25). Here, we demonstrate the use of
this method for LAMP detection in single
and multiplex reactions, detecting up to four
distinct LAMP targets in a single reaction.
Materials and methods
LAMP primers were designed either manually
or using PrimerExplorer V4 (Eiken Chemical).
Sequences can be found in Supplementary
Table S1, and all synthetic oligonucleotide
primers, Q-FIP, and Fd were synthesized by
Integrated DNA Technologies (Coralville,
IA, USA). The dark quencher was either
Iowa Black FQ or RQ, and fluorophores used
were 6-FAM, HEX, ROX, Cy5, and Cy5.5,
each corresponding to one of five channels
in a CFX96 Real Time System (Bio-Rad
Laboratories, Hercules, CA, USA), used for
performing LAMP reactions (Supplementary
Q-FIP:Fd duplexes were annealed by
heating 50 µM Q-FIP and 50 µM Fd to
98°C and slowly cooling the mixture to room
temperature. LAMP reactions with Bst 2.0
DNA polymerase or Bst 2.0 WarmStart
DNA polymerase (New England Biolabs,
Ipswich, MA, USA) were performed in
1× Isothermal Amplification Buffer (New
England Biolabs): 20 mM Tris-HCl (pH 8.8,
25°C), 10 mM (NH4)2SO4, 50 mM KCl, 2
mM MgSO4, 0.1% Tween-20 supplemented
to 8 mM MgSO4, and 1.4 mM each of dATP,
dCTP, dGTP, and dTTP. LAMP reactions
with standard Bst DNA polymerase, LF (New
England Biolabs) used a similar buffer based
on ThermoPol DF (New England Biolabs),
identical in composition as above but with
10 mM KCl in place of 50 mM KCl. LAMP
reactions contained: 1.6 µM FIP (or 0.8 µM
FIP and 0.8 µM Q-FIP:Fd), 1.6 µM BIP, 0.2
µM F3 and B3, 0.4 µM LoopF and LoopB,
and 0.64 U/µL Bst DNA polymerase, LF, Bst
Figure 1. DARQ in LAMP. (A) Schematic depiction of a DARQ probe, with a 5′-quencher FIP (F1c + F2
sequence) annealed to a 3′-fluorophore Fd. The quencher and fluorophore moieties are represented
by Q and F, respectively. (B) Outline of DARQ LAMP reactions, with core LAMP primers FIP (F1c +
F2), BIP (B1c + B2), F3 and B3, and the DARQ oligonucleotide, Fd (Q, black; F, red). For clarity,
LoopF and LoopB primers are not shown. (1) LAMP initiates at the F2c sequence of the target,
with the Fd probe quenched through annealing to Q-FIP. This new strand is displaced by upstream
synthesis from the F3 primer. (2) The BIP primer anneals to the B2c site in the newly synthesized
strand. (3) Synthesis from the primer annealed to the B2c sequence displaces the Fd probe. This
releases the quenching resulting in a gain of signal. The newly synthesized strand is displaced by
extension from the B3 primer. (4) The resulting structure undergoes exponential amplification in
the LAMP reaction. Subsequent initiations at FIP give rise to additional release of Fd, resulting in
exponential signal detection.
Vol. 53 | No. 2 | 2012
2.0 DNA polymerase, or Bst 2.0 WarmStart
DNA polymerase. For multiplex reactions,
total primer concentrations were kept to those
described for the standard LAMP reaction,
but with each set representing 1/n of the total,
where n is the number of targets. Bacterio-
phage λ genomic DNA (5 ng/reaction) and
HeLa genomic DNA (100 ng/reaction) were
from New England Biolabs, Escherichia coli
genomic DNA (5 ng/reaction) was from
Affymetrix (Santa Clara, CA, USA), and
Caenorhabditis elegans genomic DNA (82.5
ng/reaction) was purified using standard
procedures. Reactions were performed at
65°C in triplicate, and all presented Ct values
represent an average ± standard deviation.
Results and discussion
In DARQ, the standard FIP primer is
modified with a 5′ quencher and annealed to
an F1c-complementary detection probe, Fd
(Figure 1). To test this method for LAMP
detection, we designed four sets of LAMP
primers with Q-FIP and accompanying Fd
probes, each with a different fluorophore
and quencher pair. These targeted genes from
different organisms and genome complexities:
E. coli dnaE (Iowa Black RQ/Cy5); C. elegans
lec-10 (RQ/ROX); human cystic fibrosis trans-
membrane conductance regulator (CFTR;
FQ/6-FAM); and human BRCA1 (RQ/
Cy5.5). Additionally, we adapted a set of
LAMP primers for bacteriophage λ DNA (5)
with the quencher and fluorophore positions
reversed (5′-HEX FIP/3′-FQ) to examine
any effect of quencher/fluorophore location.
Q-FIP:Fd duplexes were made for each primer
set, and LAMP reactions performed using
duplex FIP primers.
Figure 2 shows LAMP amplification
of dnaE from E. coli genomic DNA with
DARQ detection using Bst DNA polymerase,
large fragment or standard and WarmStart
versions of Bst 2.0 DNA polymerase. Release
of quenching can be seen as an increase in Cy5
signal, and use of all three polymerases resulted
in robust amplification and signal (Figure 2A).
The reaction was found to proceed faster when
using either the standard or WarmStart versions
of Bst 2.0 DNA polymerase as compared with
wild-type Bst DNA polymerase (Figure 2 and
Supplementary Figure S1). Therefore, Bst
2.0 DNA polymerase was used in all subse-
quent experimentation. Use of 5′ modified
FIP primer alone had no effect on LAMP,
but we observed some relative inhibition of
amplification when Q-FIP:Fd duplex primer
completely replaced the standard FIP primer
(Supplementary Figure S1). This inhibition
was significantly reduced through the use of
equimolar standard FIP primer and Q-FIP:Fd
duplex (Supplementary Figure S2). This effect
was likely due to faster target generation with
FIP and easier incorporation of duplex FIP
during exponential amplification, and using
equimolar amounts maintains rapid threshold
detection with high fluorescence signal
amplitude, important for detecting amplifi-
cation in multiplex reactions.
A convenient feature of LAMP F1c regions
is that they are typically 20–25 bases long and
are selected by Primer Explorer to feature Tm
greater than 60°C. Thus by the nature of the
LAMP primer design, the F1c:Fd duplex
remains stably annealed in our reactions at
Figure 2. DARQ detection in real-time LAMP reactions. LAMP primers were specific for the dnaE gene
of E. coli (5 ng genomic DNA per reaction) as described in the Materials and methods section. The
FIP primer was labeled with Iowa Black RQ and the DARQ probe had a 3′ Cy5. (A) Three Bst DNA
polymerases provided robust signal, with faster amplification seen for Bst 2.0 and Bst 2.0 WarmStart
DNA polymerases. (B) Duplicate reactions to (A) were performed after 2 h preincubation at room
temperature, with substantial negative effects on amplification time and yield for non-WarmStart
polymerases, but identical performance was observed with WarmStart Bst 2.0. The reactions were
performed as described in the Materials and methods section.
Figure 3. The detection of two targets simultaneously in real-time LAMP. DARQ detection of two distinct
LAMP amplifications in the same reaction, either with similar (A) or disparate (B) amplification times.
Data are graphed as normalized fluorescence units to account for differences in fluorophore quantum
yield. (C) Maintenance of amplification for an internal standard (C. elegans lec-10/ROX; 82.5 ng C.
elegans genomic DNA) with varying amounts of test target DNA (10 pg–100 ng HeLa; ~3–30,000
copies). The reactions were performed as described in the Materials and methods section.
Vol. 53 | No. 2 | 2012
63°–65°C, and no signal is observed in the
absence of strand-displacing DNA polymerase.
However, if nonstandard primer sequences
are required, DARQ reactions can simply be
performed at lower temperatures to accom-
modate less stable duplexes. The F1c regions
described here range in Tm from 61°–74°C
(Supplementary Table S1), and all perform
DARQ LAMP reactions at 60°–65°C,
indicating that use of F1c:Fd duplexes does not
limit primer design considerations. As described
above, we also tested a primer pair with fluoro-
phore and quencher positions switched on FIP
and Fd. Use of this reverse orientation primer
set (λ) resulted in no difference in amplifi-
cation detection efficiency (Figure 3A and
Figure 4) or duplex primer inhibition levels
(data not shown), thus we can conclude that
DARQ primers can be synthesized with either
a 5′ quencher or fluorophore FIP if necessary
to accommodate limited modified oligonucle-
otide synthesis chemistry.
A common problem in nucleic acid ampli-
fication reactions is undesired activity from
DNA polymerases during room temperature
reaction setup (26,27). This activity may not
be a problem when performing a limited
number of samples set up by hand, but it can
result in irreproducibility for medium- or high-
throughput or automated workflows. To test
whether LAMP and DARQ detection may
be affected by preincubation at room temper-
ature, identical LAMP reactions were set up
and performed with or without a 2-h preincu-
bation at 25°C. Both Bst DNA polymerase large
fragment and Bst 2.0 DNA polymerase were
negatively impacted by the 2-h preincubation,
likely due to low levels of DNA polymerase
activity at 25°C (Figure 2). In PCRs, this
problem is typically overcome through the
use of “hot start” DNA polymerases. Bst 2.0
WarmStart DNA polymerase provides this
functionality for isothermal amplification
techniques through an engineered aptamer that
inhibits polymerase activity below 50°C (28),
and its efficacy in DARQ is shown in Figure 2.
Bst 2.0 WarmStart maintains identical ampli-
fication profiles whether LAMP is performed
immediately after set up (Figure 2A) or after
2 h preincubation at 25°C (Figure 2B). This
benefit accommodates room temperature
reaction setup, but otherwise does not affect
Bst 2.0 performance, so all subsequent data
are from standard Bst 2.0.
With DARQ detection validated, we next
sought to extend the method to multiple targets
detected in a single reaction. Figure 3 presents
fluorescence curves from LAMP reactions
containing two distinct, complete primer
sets and their corresponding genomic DNA
targets. For multiple target amplification, we
maintained the total amount of primer in a
standard LAMP reaction; using each set at full
concentration resulted in suboptimal perfor-
mance as concentration increased (i.e., total
primer concentration was kept to 4.4 µM
regardless of the number of templates, with each
primer set adjusted by 1/n, where n is number
of targets in the reaction). As shown in Figure
3, DARQ robustly detects distinct targets
in a single LAMP reaction. Curves shown
are normalized to maximum fluorescence
signal in that channel to account for differ-
ences in the quantum yield of various fluoro-
phores. The detection provided robust signal
for each target regardless of the speed of their
independent amplification, which will vary
based on the nature of the primers, templates,
and target copy number. Despite the quicker
amplification reaching exponential phase
sooner, the slower amplification was observed
to proceed unaffected, obviating the need for
consideration of amplification speed when
multiplex targets are selected (Figure 3B). This
independent nature of DARQ reactions allows
maintenance of sensitivity when performed
with multiple targets, as shown in Figure 3C,
where FAM-CFTR is detected to ~2.9 copies
of HeLa genomic DNA (10 pg) in the same
reaction as robust LAMP for ROX-lec-10
(82.5 ng C. elegans DNA, ~7.6 × 105 copies).
Thus a robust LAMP standard curve can
be generated across a copy number range of
Target 1 (here, CFTR) while Target 2 (lec-10)
is detected simultaneously. Amplification of
the constant target remains unchanged (all 5
ROX Ct values 11.8 ± 0.03 min) across the copy
number range of the variable target providing
Figure 4. Multiplex real-time LAMP using DARQ. Detection of three (A) and four (B) LAMP targets in the
same reaction using distinct sets of DARQ primers. A slight increase in detection time accompanies
the decrease in specific primer concentration due to added target sequences. Data are graphed as
normalized fluorescence units to account for differences in fluorophore quantum yield. The reactions
were performed as described in the Materials and methods section.
Figure 5. Nontemplate, negative control signals in DARQ LAMP reactions. Panel A shows a primer set
with a high nontemplate signal, E. coli dnaE primers, and a primer set with no nontemplate signal, C.
elegans lec-10 primers. In the absence of DNA template, the E. coli primer set gave an amplification
signal (red dotted line), whereas the C. elegans primer set gave no signal (blue dotted line). In
the presence of appropriate DNA template, each primer set gave a robust positive signal that was
unchanged whether performed as singleplex (dashed lines) or duplex (solid lines) reaction. Panel B
shows real-time DARQ reactions for bacteriophage λ, C. elegans, E. coli, and HeLa (BRCA1) DNA. The
primers were used in singleplex (dashed lines) or quadruplex (solid lines) reactions. The nontemplate
control reactions (dotted lines) contained the three unrecognized genomic DNAs appropriate for each
primer set. Nontemplate signals were high for E. coli and bacteriophage λ primer sets but negative
for C. elegans and HeLa primer sets. Note that the positive signals for each primer set were the same
in the singleplex and quadruplex reactions, indicating that there was no spurious amplification due to
the presence of three nonspecific genomic DNAs in each quadruplex reaction.
Vol. 53 | No. 2 | 2012
a reliable positive control (Figure 3C). This property allows LAMP to
be performed with an internal standard, an important consideration
for diagnostic applications. Figure 3C demonstrates DARQ perfor-
mance at low copy numbers, but high copy numbers are also reliably
detected, as seen in Figure 2 (5 ng E. coli genomic DNA, ~106 copies)
and Figure 3A (5 ng λ DNA, ~108 copies). Use of DARQ detection
thus imposes no limitation to the sensitivity of the LAMP reaction.
Similarly, the dynamic range of LAMP is unaffected by duplex DARQ,
which maintains robust detection from 10–108 copies.
DARQ can easily be extended to three and four target reactions
(Figure 4), again with total primer concentration constant and each
set adjusted for number of targets. Reducing primer concentration 3-
or 4-fold does accordingly increase time to reach threshold. This drop
in time was consistent, making template quantification reliable, and
the reaction times were still rapid with Bst 2.0 DNA polymerase. The
multiplexed reactions display robust amplification of three (Figure 4A)
or four (Figure 4B) targets, with loss of signal amplitude accompa-
nying decreased concentration of the fluorophore-containing primer.
This fluorescence decrease emphasizes the need for bright fluorophores,
with high quantum yield and appropriate spectral matching with the
fluorescence detection channels. In DARQ reactions, ROX gave the
best results (>20,000 background-subtracted fluorescence counts in
single-plex reactions), likely due to a 15 nm wider detection channel.
Cy5, Cy5.5, and HEX gave similarly high signal (10,000–15,000), but
6-FAM (<5,000) gave lower fluorescence signal that became difficult
to distinguish from background fluorescence when diluted 3- or 4-fold
for multiplex reactions. Thus DARQ is accommodating of any fluoro-
phore that can be quenched and detected, but for detecting more than
three targets simultaneously brighter dyes are preferred.
An essential consideration for amplification techniques or any
diagnostic methodology is proper identification of positive reactions
from false positive or negative reactions, either from nontemplate (ampli-
fication of primers without template DNA) or nonspecific amplification
(amplification of a nontarget DNA present in the sample), although the
latter is unlikely in LAMP due to the use of six sequence-specific primers
per target. LAMP and other techniques can result in nontemplate ampli-
fication due to several factors, including the nature of LAMP primers
and the reaction conditions, which include high concentrations of primer
(4.4 µM total) and magnesium (8 mM). The degree of this nontemplate
amplification is determined by the specific primer sequences (27), and
we demonstrate this in DARQ in Figure 5, which shows a high degree
of nontemplate amplification for one primer set (E. coli dnaE), an inter-
mediate level for another (bacteriophage λ), and none for two sets (C.
elegans lec-10 and human BRCA1). Importantly for diagnostic testing
and multiplexing, the reactions containing many different templates
maintained their level of specificity, i.e., the positive and nontemplate
(nonspecific) signal was identical for a primer set whether no template
or several nonspecific genomic DNAs were present. Figure 5B shows
this specificity, with each primer set amplifying target identically in the
presence of either a single genomic DNA (dashed lines) or four genomic
DNAs (solid lines). Each negative control reaction (dotted lines) was
carried out with three nonspecific genomic DNAs, and no effect was
seen on nontemplate amplification, e.g., C. elegans lec-10 primers gave
no signal without any genomic DNA (Figure 5A) or in the presence
of bacteriophage λ, human, and E. coli DNA (Figure 5B). This allows
accurate detection and quantification of a target (e.g., human BRCA1) in
the presence of high levels of other genomic DNAs (e.g., 100 ng bacterio-
phage λ, 82.5 ng C. elegans, and 100 ng E. coli). Although we did not test
clinical samples with DARQ, LAMP has been widely used for specimen
diagnostics, afforded by an increased tolerance to typical contaminants
and inhibitors (29). There is no obvious reason that DARQ detection
would not effectively translate into diagnostics using clinical samples,
however this needs to be experimentally verified.
DARQ detection provides a method for multiplex detection of
LAMP amplification with no need for additional primer optimi-
zation or probe design, requiring only the use of a 5′ modified LAMP
primer (FIP) with complementary detection oligonucleotide (Fd). This
detection methodology may be extendable to other nucleic acid ampli-
fication techniques by the addition of a duplex “tail” to a primer in the
reaction. Upon extension from the opposite direction, the duplex would
be displaced with detection upon release of quenching. Our detection
method is a simple extension of LAMP to accommodate robust, target-
specific, and multiplex detection. As molecular diagnostics become more
prominent and accepted in healthcare, the ability to detect multiple
targets and use of internal controls will further the utility and flexi-
bility of LAMP.
The authors thank Dr. Gregory Lohman for helpful discussion, Drs.
Andrew Gardner, Gregory Lohman, and Jennifer Ong for critical
reading of the manuscript, and New England Biolabs, Inc. for financial
and material support.
The authors are employed by New England Biolabs, Inc., manufacturer
of the described enzymes.
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Phone: 1-800-617-3363 • http://www.biospec.com
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Received 27 April 2012; accepted 9 July 2012.
Address correspondence to Thomas C. Evans, Jr.,
New England Biolabs, Inc., 240 County Rd., Ipswich,
MA, USA. e-mail: email@example.com
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