Electricity—Toward Instrument-Free Molecular Diagnostics in Low-Resource
Settings
Introduction
Clinical diagnostic assays targeted to nucleic acid (NA) markers are becoming an
increasingly important part of the clinician's toolbox. Many disease states are
difficult to diagnose due to the lack of specific and well-characterized biomarkers
in an accessible specimen. These generalizations apply in particular to infectious
disease diagnostics. The clinical signs of infection are often non-specific (e.g.,
inflammation or fever) and may originate from many possible sources, yet the
treatments are more often specific and require an accurate diagnosis to be
effective. There are many infectious diseases endemic in LRS where the lack of
simple, instrument-free, NA diagnostic tests is a critical barrier to effective
treatment, in part because of co-morbidities that confound a differential diagnosis.
These diseases include malaria, human immunodeficiency virus (HIV-1), tuberculosis
(TB), influenza, and many others.[1] Millions of lives are lost and a huge morbidity burden
incurred through inadequate diagnosis and treatment of these diseases.[1] In many cases
the need for rapid diagnostics appropriate for these LRS is so severe that mediocre
performance tests such as RDT are preferred to less accessible but better performing
NA tests.[2]
Clearly, any technology that can increase the practicality and availability of NA
assays in LRS could have a significant impact on global public health.
Nucleic acid detection, to date, has mainly been confined to wealthy, developed
countries or to the large centralized facilities in the developing world that can
marshal the resources required to perform these techniques. Like many molecular
diagnostic assays, nucleic acid amplification techniques (NAATs) typically require a
significant investment in equipment, training, and infrastructure. Economic and
infrastructural realities dictate that diagnostics for the developing world need to
be foremost inexpensive; but also, accurate, reliable, rugged, and suited to the
contexts of these low-resource settings (LRS).[3]–[5] Recent guidelines published by
the World Health Organization recommend that diagnostic devices for developing
countries should be ASSURED: Affordable, Sensitive, Specific, User-friendly, Rapid
and robust, Equipment-free, and Deliverable to end users.[6] In some diagnostic contexts in
LRS, rapid diagnostic tests (RDT) based on the immunochromatography strip (ICS) fit
the ASSURED model, albeit with limited sensitivity and specificity.[7]–[9] NAAT assays that
use polymerase chain reaction (PCR) amplification are capable of providing excellent
sensitivity and specificity but generally fail to meet the ASSURED guidelines for
affordability, rapidity and robustness, equipment-free operation, and
deliverability.[10], Appropriate, low-cost, equipment-free, pathogen-specific
NA marker assays that characterize medical care in much of the developing world
remain unavailable in LRS.
One of the primary barriers to the practicality and availability of NA assays in LRS
has been the complexity of PCR amplification. PCR is inherently impractical in LRS
where reliable electrical power, complex equipment, training, reagent storage,
quality programs and clean water, are intermittent or absent., Recently, there have been
significant developments in a class of NAATs that do not require temperature
cycling.[13]–[16] A comprehensive review of these techniques, and their
application in LRS has recently been published. These isothermal amplification
techniques vary in amplification temperature and duration, as well as complexity of
reagents required—and many are proprietary—but all have the potential to
be simpler and require less complex equipment than PCR-based assays. These methods
use a variety of reaction principles to specifically amplify NA targets through
isothermal melting, exponential amplification and intermediate target generation;
and which, in several cases, can be detected directly without the need for an
instrument.[17]–[20] Nevertheless, almost all investigators and manufacturers
currently use some type of electrically powered equipment to achieve and maintain
the temperature required for amplification, although this equipment can be much
simpler than the typical PCR thermocycler. This inherent simplicity makes isothermal
amplification more appropriate for diagnostics in LRS.
One of the letters in the acronym ASSURED — the guideline for providing
diagnostics to LRS — represents “equipment free.” We are currently
developing a non-instrumented nucleic acid (NINA) platform that requires no
detection instrument, no electrical power, no batteries, and no external reagents.
We believe this can be achieved by combining isothermal amplification with a novel
method for generating the required temperature profile without electrical power in a
simple disposable that contains the lyophilized assay reagents. Our first prototype
of this platform uses loop-mediated amplification (LAMP) as the model for an isothermal
amplification technique and malaria as a model diagnostic target. The amplification
protocol requires incubating the reaction mixture at ∼65°C for at least 60
minutes. This temperature requirement is sufficiently flexible that small excursions
(+/−1.5°C) around this target are tolerable.–[26] LAMP (and several other
isothermal techniques) have been shown to far less sensitive to inhibitors than PCR,
to the point where direct assay of whole blood and other unpurified specimens is
feasible.[18],
[19] In those
cases, no power or instruments are required for NA purification, as is the case with
PCR. In addition, recent advances in protein stabilization make it likely that the
reagents can be dried-down in the reaction tubes with sufficient stability to avoid
the need for a cold-chain during delivery and storage. Thus, another power consuming
“instrument” is eliminated. We have not yet attempted to package all of
these features and advances into a single prototype device; however, the successful
demonstration of electricity-free temperature-controlled heating in a disposable
format reported here is an important first step toward the long-term goal.
The prototype NINA platform exploits exothermic chemical heating, as used in
“ready-to-eat” meals and camping hand warmers. Table S1
summarizes the prior history of prototype development. Hatano and coworkers recently
described a crude heater that was able to perform a qualitative LAMP assay for
anthrax using off-the shelf pocket hand warmers and a Styrofoam box. Dominguez et
al. used a similar container with an unspecified phase change material to maintain a
stable incubation temperature for a commercial interferon gamma release assay at 37C
(although the heat source was conventional). While these interesting
approaches are compelling in their simplicity, the bulky apparatus displayed slow
warm-up (>30 min.); and for LAMP, significant temperature variation within
incubation time, and a lack of run-to-run repeatability was observed. To meet the
performance goals implied in the ASSURED guidelines, an optimized heating unit
should be engineered to eliminate or minimize all sources of variation. When
combined with the temperature-moderating characteristics of engineered phase change
materials (EPCM), we demonstrate that an engineered exothermal chemical heating unit
can produce a consistent constant-temperature incubator for isothermal NA
amplification suitable for a variety of isothermal techniques.
Heat Production and Temperature in the NINA Heater
Ten replicate runs of the optimized prototype displayed minimal variation in
temperature from run to run within the reaction tubes (Figure 1). The heater reached the optimal
incubation temperature in 15 minutes, and maintained the target temperature with
minimal drift over 60 minutes. (Drift from minimum to maximum temperature within
run, mean over all runs = 2°C.) Comparison of the
temperature plots for the CaO, EPCM, and reaction tubes in Figure 1 to Figure 1B in Hatano et al.[27] illustrates
the beneficial effect of having the EPCM component in the heater. The CaO
temperature traces show rapid and poorly controlled heat generation, with
maximum temperatures exceeding 100°C. The traces of the EPCM at the
interface with the CaO have a pattern similar to the CaO, but the initial
temperature excursions are reduced in magnitude, and the plots are far more
repeatable. Finally, the reaction tubes display only a uniform ramping to the
target temperature followed by a prolonged stable isothermal phase. The
temperature in the NINA reaction appears more uniform than that shown by Hatano
et al.[27] for
their hand-warmer device.
These results evince the potential of EPCM in an optimized design for controlling
exothermic reactions in a simple NINA. This level of temperature control is
important to enable conformance to the “sensitive”,
“specific”, and “robust” aspects of the ASSURED
guidelines. Once the abundant heat from the CaO reaction begins to melt the
EPCM, the additional heat produced by the exothermic reaction is converted into
the latent heat of fusion of the EPCM. Thus, the temperature in the EPCM remains
constant at the selected melting temperature until the solid to liquid
transition is complete (provided heat transfer within the EPCM is rapid). Once
the CaO reaction has reached equilibrium, the energy stored as latent heat keeps
the two-phase EPCM at the target temperature until complete solidification.
In our optimization work we observed that the purity of the CaO need not be high,
although it should be consistent to yield consistent heat profiles (data not
shown). The ability to use less pure CaO is important for minimizing the cost
per amplification, addressing the “affordable” aspect of the ASSURED
guidelines. Other key physicochemical parameters of raw CaO (particle size,
particle porosity, presence of unoxidized calcium carbonate “grit”,
etc.) that result from variation in kiln calcination of limestone[29] (the
industrial manufacturing process) also must be kept consistent for consistent
heat profiles. However, we were able to produce precise heat profiles in our
prototypes with commodity grades of CaO. This makes the only disposable
materials (CaO, water, and PCR tubes) in the device very inexpensive. The
reaction of CaO and water can be tuned somewhat to control the steepness of the
temperature ramp and the maximum temperature for a given reaction chamber,
although flexibility and precision is greatly improved by including the
EPCM.
The EPCM used here is tunable for many of its important characteristics (melting
temperature range, specific heat capacity, thermal conductivity, etc.) making
this device a flexible incubation platform potentially applicable to a number of
isothermal amplification techniques. When evaluated by differential scanning
calorimetry, the EPCM melts over a range of temperatures around the target
(±2°C), and displays some hysteresis in the phase change, presumably
due to polydispersity in polymer chain length and supercooling of the EPCM
(personal communication from Renewable Alternatives). It is unclear at this time
how this behavior contributes to variation seen in the results of the LAMP
assay; however, the manufacturer of the EPCM is confident that further
development of the EPCM for this application will mitigate this behavior. The
EPCM is a fully hydrogenated fat product, so it is resistant to environmental
oxidation and should be very stable. While the EPCM is not currently as readily
available as CaO, and is not a commodity product like CaO, similar materials
have been used in consumer products in the US. These EPCMs are made mainly from
bio-based fats — namely beef tallow, palm oil, coconut oil and soybean oil
— so local, low-cost production of the EPCM in the developing world should
be feasible.
Portable energy for heat production could, of course, be supplied with
conventional batteries, so a comparison seems appropriate. A cost analysis
indicates that on a per calorie per test basis, using CaO as a thermal battery
is several times less expensive than mass-produced, disposable, dry-cell
batteries. Costs are scaled by the projected number of analytical runs possible
and include both energy source and control hardware. CaO disposables are single
use, while dry cells are expected to last five runs based on their energy
density (four D-cells would be required). Two grades of CaO (reagent grade and
soap grade), with an EPCM are compared to three possible dry-cell
implementations (with an EPCM, with microprocessor closed-loop control, with
thermostat closed-loop control). With a projected cost per run of
US$0.56, the soap-grade CaO/EPCM combination is clearly the
least-expensive alternative (compared to $1.40, $1.17,
$1.21, and $1.16 for reagent-grade CaO/EPCM, D-cell/EPCM,
D-cell/microprocessor, and D-cell/thermostat, respectively.) Costs were
estimated from MSRP. Increased value of CaO over the alternatives could be
realized at increased production volumes. Any special disposal or recycling
required does not seem any more onerous than what is required for common
batteries.
The data shown here were not gathered under any stringent external environmental
control; therefore, given that testing was performed in an air-conditioned
laboratory, it is likely that the system was not challenged in the same way as
it would be at its intended point of use. The wide external temperature ranges
found in LRS could significantly change the ramp time and/or duration at the
desired temperature of the heater, possibly significantly, but the
characteristics of the EPCM will ensure that the desired temperature is held for
some period of time, regardless – without calibration to the
ambient conditions or closed-loop control. First principles of heat
transfer dictate that the effects of ambient on ramp time and/or duration should
be greatest when the desired temperature is furthest from ambient. Thus, the
problem should be appropriately non-dimensionalized to identify states of
similitude. We plan to explore these phenomena and to evaluate their effects
once we have improved our understanding of the intrinsic variation in the assay
chemistry sufficiently to evaluate those effects. This evaluation will include
trials under actual field conditions.
LAMP Assay Demonstration and Comparison to a Reference Heater
Representative images of the qualitative results (Figure 2) shows 1) the NINA heater is capable
of supporting LAMP, 2) that samples incubated in the NINA heater give results
that are virtually identical to those incubated in parallel in the GeneAmp®
9600. For both incubators the turbidimetric readout method (Figure 2A) is difficult to interpret, but
turbidity due to accumulating LAMP product is observed (relative to the
no-template control, or NTC). The fluorescence of the Calcein reagent when
illuminated with a UV lamp (Figure
2B) is more easily seen as an increase in intensity (relative to NTC)
for the dilutions that are >1 pg/µL. Note that there is some background
fluorescence visible in the NTCs with both heaters. These
observations conform to those noted by the operator at the actual time of the
analysis, so no artifacts have been introduced by the photographic process.
A quantitative comparison of Calcein fluorescence corroborates the qualitative
study. A statistical method comparison by the two most common techniques
indicates substantial quantitative agreement between samples incubated in the
NINA heater to those incubated in parallel in the ESE-Quant Tube Scanner. Linear
regression of the fluorescence intensity units (FIU) observed for samples
incubated in the NINA heater as a dependant variable of the FIU observed for
samples incubated in the ESE-Quant (Figure 3A) results in a slope of 0.98 and a y-intercept of 37.5 FIU,
with a coefficient of determination of 0.87. Bland-Altman analysis (Figure 3B) reveals a mean
difference (ESE – NINA) of −26 FIU, no dependence of difference on
mean, and all differences lie within the ±2s interval that indicates the
differences are random, not systematic. Although these experiments were intended
to quickly assess the agreement between heater types and were not designed to
rigorously define the dose response relationship of a nascent assay, closer
inspection of the FIU for each concentration (Figure 3A) reveals a general increase in
response with increasing dose, within the experimental noise limits of this
admittedly small sample set. As with the qualitative assay demonstration,
considerable background fluorescence was observed in NTC reactions in
both heaters (Figure 3A and 3B).
These results clearly show that the NINA heater can incubate isothermal reactions
predictably and precisely with no electricity and without any form of
closed-loop control. We also demonstrate that it can be used for LAMP assays,
with no discernable difference when compared to two reference heaters, the
GeneAmp® 9600 and the ESE-Quant Tube Scanner. There is a bias between the
NINA heater and the ESE-Quant (NINA higher), but this is not a significant
finding considering we are comparing FIU without any assay calibration. This
bias would be easily removed by applying a standard curve. Although we did not
intend to rigorously qualify the LAMP assay for malaria here, these results
suggest that a quantitative assay with a clinically significant lower limit of
detection and three decade dynamic range might be possible with further
development of the protocol. Planned work will comprehensively compare
incubation of several isothermal assays with the NINA heater to incubation with
conventional, electrically-powered instruments by many metrics –
sensitivity, specificity, accuracy, precision, and other standard figures of
merit must all be assessed before equivalence can be rigorously inferred.
However, these prelimary results are very encouraging.
Other Isothermal Techniques
We have also explored heaters with temperature profiles suitable for other
isothermal amplification techniques requiring different incubation temperatures,
e.g., the Exponential Amplification Reaction (EXPAR), Nicking Enzyme
Amplification Reaction (NEAR), or Recombinase Polymerase Amplification (RPA),
could be integrated with this method. These prototypes are not significantly
different in form, but use different EPCMs, and in one case a different
exotherm. A CaO heater with a different EPCM formulation has been shown to yield
a temperature profile suitable for EXPAR with a nominal temperature of 55°C
(Figure 4A). Evaluation
with EXPAR reactions are in process. We have also explored a similar heater
approach with sodium acetate (NaAc, Figure 4B). Hand warmers based on the crystallization reaction of
NaAc are common. In a purified form, at typical ambient temperatures, liquid
NaAc is thermodynamically unstable but kinetically stable due to the absence of
nucleating sites for crystal formation. The application of a mechanical shock
initiates the exothermic crystallization, and when mixed as a 25% aqueous
solution the phase change occurs repeatably at ∼37°C. In this system,
NaAc acts as both the exothermic reactant and the EPCM. This system has the
advantage of being regenerable (immersion of the NaAc in boiling water is
sufficient). For isothermal amplification methods operating at temperatures
below 45°C as well as for other diagnostic applications requiring heating
(e.g., smart-polymer-based analyte pre-concentration[30]), NaAc is the preferred
exothermic/phase change system. These results establish that the heater is a
flexible platform for a number of isothermal detection techniques.
Assay Specific Limitations of This Investigation
We have shown results for an instrument-free LAMP assay with a simple qualitative
visual readout. As operated here, LAMP is an exponential rate assay being
assessed with an endpoint measurement. Thus, the timing of reaction
interrogation and/or a reliable “stop” reaction are required for
quantitative precision. If quantitative results are required, improvements to
the entire assay system to facilitate precise timing will be necessary. This
could include, for example, a different heater-lid or incubation-vessels to
facilitate access, or a “reading window” in the heater to enable
visual interrogation while the vessel is still in the heater. An elevated
temperature “stop” (>80°C) is generally used for LAMP. In our
experimental work here, we used an electrical heat block for this purpose;
however, this could be accomplished with the electricity-free heater by the
inclusion of a parallel heating unit at a higher temperature (essentially, by
including a second incubation chamber that uses a different EPCM, or no EPCM at
all). Alternatively, a chemical “stop” could be developed, or a
boiling water bath could be kept at hand. Other assays perform best with a
pre-amplification, high-temperature denaturation step (“hot start”).
A second incubator chamber could facilitate this feature even more readily.
These data were gathered on contrived samples diluted in buffer. It has already
been demonstrated that LAMP assays can be performed on clinically relevant
specimens without NA extraction/purification and without a pre-amplification,
high-temperature denaturation step.[31]–[35] Recent results of an HIV
assay on the NINA platform with clinical samples from HIV-positive infants will
be reported elsewhere.
Furthermore, neither turbidity nor Calcein reactions are sequence-specific
signals—as a result non-specific amplification will also produce a strong
signal—a possible cause of the NTC background fluorescence noted above.
Greater analyte specificity should be possible by incorporating a fluorescent
molecular beacon probe specific to an internal region of the target amplicon,
thus minimizing non-specific signal. Alternatively, the
amplified product could be the input to a lateral flow strip test with a visual
readout.–[39]
Any of these potential improvements should be approached with a secondary aim of
minimizing the potential for amplicon contamination from previous tests.
Wherever possible, opening of the amplicon container after amplification should
be avoided. This may be challenging if molecular beacon quenchers need to be
added, or aliquots for ICS testing need to be removed. Regardless of how the
system and assays are improved, we have clearly demonstrated that the NINA
heater is an effective device that can facilitate the electricity-free
amplification of NA using an isothermal technique.
Future Directions
There are several applications of this technology that could have an impact on
diagnostics for LRS. One application is as a modular amplification unit where a
sample and the required reagents would be introduced to the heater and amplified
product withdrawn for subsequent analysis by any simple detector. In this
embodiment a standard PCR tube can be the reaction chamber and could be used
later as a cuvette for fluorometric analysis to resolve the presence of
amplicons. This would free the user from the high power requirements of
electrical heating but would still require some sort of detection instrument or
device with its attendant requirements. One could also imagine how a properly
tuned, stand-alone heater unit could be applicable to any field analytical or
preparative method that requires a constant heat source; e.g., cell lysis or
temperature-responsive polymer mediated concentration. More compelling is the
potential of the NINA heater as the core component of a stand-alone assay kit,
capable of providing a result without external electrical power, a reader
instrument, or any complex ancillaries. Such a device might include the NINA
heater, reaction chambers containing lyophilized reagents, sample metering
devices, a readout chamber or lateral flow strip for visible interrogation, and
an LED “penlight” for fluorescence excitation (if required). We
envisage versions of the kit that are fully disposable (for high-value
applications in developed countries such as for home testing and for
first-responder biothreat detection) and partially reusable (primarily for LRS
use). Most of the components necessary to create such a NINA kit already exist.
We are currently working to combine them into a field-ready, instrument- and
electricity-free, sample-to-result, molecular diagnostic test system (Figure
S1).
Conclusion
We have demonstrated the ability of an optimized NINA heater prototype, based on
exothermic chemical reactions and EPCM, to support isothermal NA amplification
assays and established its equivalence to commercially available PCR
instruments. The disposable heater described is a component of an
instrument-free point of care molecular diagnostics system under development.
When combined with other innovations in development that eliminate power
requirements for sample preparation, cold reagent storage, and readout, the NINA
heater will comprise part of a kit that enables electricity-free NA testing for
many important analytes. Replicate temperature profiles display minimal
variation between runs and far less variation than any similar devices,
highlighting the advantages of including an EPCM in the design. Versions of the
prototype for several isothermal techniques have been presented, clearly
evincing the potential of the NINA heater.
Materials
In the NINA heater for LAMP, we used the exothermic reaction of calcium oxide
(CaO, or quicklime; Science Stuff, Inc., Austin, TX, USA, Cat # C1450) and water
to generate the necessary heat. To keep the isothermal device within the
temperature band required for LAMP, the reaction chambers were surrounded with
an engineered fat-based compound with a high specific heat capacity and specific
melting range centered around 65°C (Renewable Alternatives, Inc., Columbia,
MO, USA). While several prototype heater designs have been explored, the
optimized heater uses an off-the-shelf insulated food storage container (a
“thermos”) to provide an insulated housing with two chambers (Figure 5). The bottom chamber
contains the exothermic reaction, and the upper chamber contains the EPCM and
reaction wells. To facilitate directed heat transfer to the reaction wells, an
aluminum “honeycomb” material (Plascore, Inc., Zeeland, MI, USA) was
added to the upper chamber prior to introduction of the EPCM. The machined
reaction wells, sized to closely fit a standard 200-µL PCR tube, are
embedded in the EPCM. Three reaction wells were used for most prototypes (one
for a positive control, one for a negative control, and one for an unknown
specimen); however, the existing prototype could easily be modified to accept
several times this number without significant loss of performance, based on the
available space in the EPCM and first principles of heat transfer. An
inexpensive spring timer (manufacturer's suggested retail price
[MSRP] ≈10 US$) with an audible “ready”
indicator was affixed to the lid of the heater unit for added electricity-free
functionality.
Loopamp® DNA LAMP kits were purchased from Eiken Chemical Co. Ltd (Tokyo,
Japan, Code No.: LMP206). The Eiken Loopamp® Fluorescence Detection Reagent
(Code No.: LMP221), a Calcein-based reagent that indirectly indicates the
progress of DNA amplification in the LAMP reaction, was used for fluorescence
experiments. LAMP primer sequences for P. falciparum
(Integrated DNA Technologies , Coralville, IA, USA) were as described by Poon et
al.[40]
Genomic DNA from P. falciparum for preparing contrived samples
was obtained from the PATH laboratory specimen collection.
Either an ESE-Quant Tube Scanner (ESE GmbH, Stockach, Germany) or a PE
GeneAmp® Thermocycler 9600 (Applied Biosystems, Carlsbad, CA, USA ) was used
as a quantitative reference instrument for temperature incubation. The ESE-Quant
Tube Scanner also served as a reference for quantitative fluorescence
measurement. A SpectraMax M2 fluorescence plate reader (Molecular Devices,
Sunnyvale, CA, USA) was also used (as noted in individual experiments).
Physitemp IT-23, T-type thermocouples (Clifton, NJ, USA) and an Omega DaqPRO
5300 Data Recorder (Omega Engineering, Stamford, CT, USA) were used to monitor
temperature.
Methods
Thermocouples were installed in a reaction well (below its microcentrifuge tube)
at the bottom of the chamber containing the EPCM and in the exothermic reaction
chamber for experiments performed to characterize the temperature profiles of
the heaters. The temperature acquisition rate was 1 Hz. Initial experiments were
focused on refining the dimensions of the heater, optimizing the quantity and
quality of CaO and water, and testing EPCM formulations—with the goal of
minimizing initial pre-heating time and variability during the specified
incubation period. In the optimized device, 20 gm of CaO and 6.8 mL of water
were added to the bottom chamber and mixed by rotary stirring for five strokes
to initiate the heating and then the components were assembled as discussed
above.
To verify that the device could incubate a LAMP assay, the Eiken kits were used
as per package insert instructions, except where noted below. Clinically
relevant dilutions of genomic DNA were made in Eiken kit buffer to yield the DNA
concentration and approximate parasite count noted for each experiment. All
dilutions were prepared as single solutions and then aliquoted across treatment
conditions to minimize preparation variation. No template controls (NTC) were
prepared first and immediately sealed to reduce the possibility of
contamination. Mineral oil was layered on the tops of the samples to minimize
evaporation. All reactions were incubated at 63°C.
Qualitative readout experiments were performed both with and without the Calcein
reagent to determine if turbidimetric readouts were possible. To compare the
performance of the NINA heater to a reference heater, these experiments were
also performed in parallel with reactions incubated in both the test device and
in the GeneAmp® thermocycler, programmed for a constant incubation at
63°C.
Quantitative fluorescence experiments were performed in parallel with reactions
incubated in both the test device and in the ESE-Quant Tube Scanner, programmed
for a constant incubation at 63°C. For NINA incubated reactions, LAMP was
terminated at the time (∼36 min.) when the signal from the parallel
reactions on the Tube Scanner began to indicate detectable amplification to
avoid signal saturation in all dilutions. Termination was accomplished by flash
chilling and later inactivating the reaction by heating at 80°C for 5
minutes. The fluorescence signals of the NINA incubated samples were then read
on the SpectraMax M2 plate reader with
λex = 485 nm and
λem = 515 nm.