A simple, inexpensive device for nucleic acid amplification without electricity-toward instrument-free molecular diagnostics in low-resource settings

Abstract

Background: Molecular assays targeted to nucleic acid (NA) markers are becoming increasingly important to medical diagnostics. However, these are typically confined to wealthy, developed countries; or, to the national reference laboratories of developing-world countries. There are many infectious diseases that are endemic in low-resource settings (LRS) where the lack of simple, instrument-free, NA diagnostic tests is a critical barrier to timely treatment. One of the primary barriers to the practicality and availability of NA assays in LRS has been the complexity and power requirements of polymerase chain reaction (PCR) instrumentation (another is sample preparation).

Methodology/principal findings: In this article, we investigate the hypothesis that an electricity-free heater based on exothermic chemical reactions and engineered phase change materials can successfully incubate isothermal NA amplification assays. We assess the heater's equivalence to commercially available PCR instruments through the characterization of the temperature profiles produced, and a minimal method comparison. Versions of the prototype for several different isothermal techniques are presented.

Conclusions/significance: We demonstrate that an electricity-free heater based on exothermic chemical reactions and engineered phase change materials can successfully incubate isothermal NA amplification assays, and that the results of those assays are not significantly different from ones incubated in parallel in commercially available PCR instruments. These results clearly suggest the potential of the non-instrumented nucleic acid amplification (NINA) heater for molecular diagnostics in LRS. 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 should enable electricity-free NA testing for many important analytes.

Figure 1. Temperature monitoring of the prototype designed for ∼65°C LAMP assays.

Note the repeatability of results at three different locations over 10 replicate runs. (---)  =  target temperature (63°C). Red  =  Temperature of the CaO, Green  =  Temperature at the CaO/EPCM interface, Blue  =  Temperature of the amplification reaction. Sampling frequency  = 1 Hz.

Figure 2. LAMP with qualitative visual readouts performed in both the NINA heater and a reference instrument.

LAMP assays were performed for a dilution series of P. falciparum genomic DNA (see figure for concentrations) with qualitative visual readouts and with amplification performed in both the NINA heater and a reference instrument (Perkin Elmer GeneAmp Thermocycler 9600, set at 63°C). NTC =  no template control. A) A turbidimetric readout based on the scattering of accumulated magnesium pyrophosphate precipitate (a by-product of the amplification reaction). B) A fluorescence readout based on the Loopamp Calcein reagent. Pyrophosphate (a by-product of the amplification reaction) competitively displaces the Calcein fluorophore from manganous ions (Mn++), relieving the quenching effect of the Mn++. Further fluorescent enhancement results from the binding of magnesium ions (Mg++) by Calcein at the site vacated by Mn++.

Figure 3. Quantitative method comparison of LAMP performed in both the NINA heater and a reference instrument.

LAMP assays were performed for a dilution series of P. falciparum genomic DNA (see figure for concentrations), with amplification performed in both the NINA heater and a reference instrument (ESE-Quant Tube Scanner, set at 63°C) for the same amount of time. Fluorescence intensity of the Calcein dye was then read on the SpectraMax M2 plate reader with λ_ex = 485 nm and λ_em = 515 nm. A) Linear regression analysis of the method comparison. The error bars represent ±2 s using the best unbiased estimate for replicate noise available from the data set. B) Bland-Altman analysis of the same data.

Figure 4. Temperature monitoring of prototypes designed for other contexts.

A) A representative plot for a CaO prototype with a temperature set point of 55°C, suitable for EXPAR assays. B) Ten replicate plots of the NaAc prototype with a temperature set point of 38°C, suitable for a variety of uses. As yet, this prototype has not been exhaustively optimized for precision as have the CaO/EPCM based units. Red  =  Temperature of the CaO, Blue  =  Temperature of the amplification reaction. ( --- )  =  target temperature. Sampling frequency  = 1 Hz.

Figure 5. Fifth-generation prototype design made from a reusable $4 insulated soup thermos container.

(A) assembled incubator, (B) incubator lid with built-in spring timer, (C) CaO chamber (w/ CaO added), (D) assay tubes, and (E) thermocouple wires (only required in temperature monitoring experiments).