Electricity—Toward Instrument-Free Molecular Diagnostics in Low-Resource

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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.