Ralstonia solanacearum is a pathogenic bacterium that causes wilt in over 200 plant species. Here we report a rapid and sensitive detection of R. solanacearum using an isothermal method for copying DNA known as loop-mediated amplification (LAMP). A set of four primers was designed to replicate the gene coding for the flagellar subunit, fliC, and conditions for detection were optimized to complete in 60 min at 65 degrees C. Magnesium pyrophosphate resulting from the amplification reaction could be detected optically as an increase in the solution turbidity, and the DNA products spread in a reproducible ladder-like banding pattern after electrophoresis in an agarose gel. Replication of the fliC gene was detected only from R. solanacearum. The detection limit of this LAMP assay was between 10(4) to 10(6) colony forming units/ml, and the technique may be useful for developing rapid and sensitive detection methods for the R. solanacearum pathogen in soil and water.

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Exciting discoveries in material science and molecular interactions are resulting in many promising electrochemical biosensor technologies. Compact, high-quality instrumentation is critical to adaptation of these new technologies especially for distributed applications in the agriculture and food industries. To this end, we have developed ABE-Stat, a fully open-source, battery-powered potentiostat project including a wireless Android interface. ABE-Stat is capable of conducting routine electrochemical analyses including cyclic voltammetry (CV), differential pulse voltammetry (DPV), high impedance potentiometric measurements, and can connect directly to the internet through WiFi or indirectly through the Android interface. Importantly it is the first fully opensource potentiostat capable of evaluating electrochemical impedance spectroscopy (EIS) across a wide frequency spectrum (0.1 Hz to 100 kHz) with user selectable amplitude and bias. Current noise was observed to be over an order of magnitude larger than the nominal resolution of the embedded 24-bit analog to digital converter (ADC), but were largely consistent with the actual ADC specifications. In this manuscript we share detailed documentation for ABE-Stat including hardware design and source code, and evaluation of the performance of all avaiable analyses. We also suggest design improvements that could improve the noise performance of ABE-Stat and consistency of EIS measurements across the spectrum.

Isothermal nucleic-acid amplification methods such as Loop-Mediated isothermal AMPlification (LAMP) are increasingly appealing alternatives to PCR for use in portable diagnostic system due to the low cost, weight, and power requirements of the instrumentation. As such, interest in developing new probes and other functionality based on the LAMP reaction has been intense. Here, we report on the development of duplexed LAMP assays for pathogen detection using spectrally unique Assimilating Probes. As proof of principle, we used a reaction for Salmonella enterica as a model coupled with a reaction for λ-phage DNA as an internal control, as well as a duplexed assay to sub-type specific quarantine strains of the bacterial wilt pathogen Ralstonia solanacearum. Detection limits for bacterial DNA analyzed in individual reactions was less than 100 genomic equivalents in all cases, and increased by one to two orders of magnitude when reactions were coupled in duplexed formats. Even so, due to the more robust activity of newly available strand-displacing polymerases, the duplexed assays reported here were more powerful than analogous individual reactions reported only a few years ago, and represent a significant advance for incorporation of internal controls to validate assay results in the field.