Molecular diagnostics in a teacup: Non-Instrumented Nucleic Acid Amplification (NINA) for rapid, low cost detection of Salmonella enterica

Abstract

We report on the use of a novel non-instrumented platform to enable a Loop Mediated isothermal Amplification (LAMP) based assay for Salmonella enterica. Heat energy is provided by addition of a small amount (<150 g) of boiling water, and the reaction temperature is regulated by storing latent energy at the melting temperature of a lipid-based engineered phase change material. Endpoint classification of the reaction is achieved without opening the reaction tube by observing the fluorescence of sequence-specific FRET-based assimilating probes with a simple handheld fluorometer. At or above 22°C ambient temperature the non-instrumented devices could maintain reactions above a threshold temperature of 61°C for over 90 min-significantly longer than the 60 min reaction time. Using the simple format, detection limits were less than 20 genome copies for reactions run at ambient temperatures ranging from 8 to 36°C. When used with a pre-enrichment step and non-instrumented DNA extraction device, trace contaminations of Salmonella in milk close to 1 CFU/mL could be reliably detected. These findings illustrate that the non- instrumented amplification approach is a simple, viable, low-cost alternative for field-based food and agricultural diagnostics or clinical applications in developing countries.

Keywords: DNA; LAMP; assimilating probe; biosensor; food safety; molecular diagnostics.

Figure 1

Hardware for non-instrumented nucleic acid amplification. (a) 3-D perspective cross sectional view of NINA cartridge; (b) photograph of mobile pathogen detection lab including clockwise from top left electric kettle, NINA cartridge, thermos with lid, and custom handheld fluorometer with smart phone interface.

Figure 2

Average temperatures observed in reaction wells of NINA devices run at ambient temperatures of: 8°C (- - -); 22°C (— — —), and; 36°C (– – –). Boiling water is added to NINA devices at time=0. Horizontal lines show design temperature (—) with upper and lower bounds (···).

Figure 3

Relative fluorescence values read by custom handheld device compared to commercial fluorometer (●; —; R²=0.982; y=84.15x+14190).

Figure 4

Endpoint fluorescence values for DNA standards subjected to Non-Instrumented Nucleic Acid Amplification with Assimilating Probe. Reactions were run at ambient temperatures of 8°C (blue circles), 22°C (green triangles), and 36°C (red squares), with standards classified as positive designated by filled symbols those classified as negative by open symbols. Pooled data fit a logistic curve (a=41400 RFU; b=−1.69; x₀=122 fg; y₀=14390 RFU; R²=0.92, RMSE=5314 RFU) resulting in a detection limit of 92 fg DNA (vertical solid line, equivalent to 18 copies of the S. enterica genome). A single genome copy equivalent (5.1 fg) is represented by the vertical dashed line.

Figure 5

NINA assay results for DNA extracted directly from Salmonella culture (●; ———; detection limit 13000 CFU/mL), compared to results from contaminated milk samples without enrichment (○; — — —; detection limit ~28000 CFU/mL) and pre-enriched by incubating at 35°C for 8 h (Δ; ······; detection limit ~790 CFU/mL) or 24 h (□ - - -; detection limit 1.4 CFU/mL).