False-negative rate and recovery efficiency performance of a validated sponge wipe sampling method.

Recovery of spores from environmental surfaces varies due to sampling and analysis methods, spore size and characteristics, surface materials, and environmental conditions. Tests were performed to evaluate a new, validated sponge wipe method using Bacillus atrophaeus spores. Testing evaluated the effects of spore concentration and surface material on recovery efficiency (RE), false-negative rate (FNR), limit of detection (LOD), and their uncertainties. Ceramic tile and stainless steel had the highest mean RE values (48.9 and 48.1%, respectively). Faux leather, vinyl tile, and painted wood had mean RE values of 30.3, 25.6, and 25.5, respectively, while plastic had the lowest mean RE (9.8%). Results show roughly linear dependences of RE and FNR on surface roughness, with smoother surfaces resulting in higher mean REs and lower FNRs. REs were not influenced by the low spore concentrations tested (3.10 × 10(-3) to 1.86 CFU/cm(2)). Stainless steel had the lowest mean FNR (0.123), and plastic had the highest mean FNR (0.479). The LOD(90) (≥1 CFU detected 90% of the time) varied with surface material, from 0.015 CFU/cm(2) on stainless steel up to 0.039 on plastic. It may be possible to improve sampling results by considering surface roughness in selecting sampling locations and interpreting spore recovery data. Further, FNR values (calculated as a function of concentration and surface material) can be used presampling to calculate the numbers of samples for statistical sampling plans with desired performance and postsampling to calculate the confidence in characterization and clearance decisions.

Rapid microchip-based electrophoretic immunoassays for the detection of swine influenza virus.

Towards developing rapid and portable diagnostics for detecting zoonotic diseases, we have developed microchip-based electrophoretic immunoassays for sensitive and rapid detection of viruses. Two types of microchip-based electrophoretic immunoassays were developed. The initial assay used open channel electrophoresis and laser-induced fluorescence detection with a labeled antibody to detect influenza virus. However, this assay did not have adequate sensitivity to detect viruses at relevant concentrations for diagnostic applications. Hence, a novel assay was developed that allows simultaneous concentration and detection of viruses using a microfluidic chip with an integrated nanoporous membrane. The size-exclusion properties of the in situ polymerized polyacrylamide membrane are exploited to simultaneously concentrate viral particles and separate the virus/fluorescent antibody complex from the unbound antibody. The assay is performed in two simple steps--addition of fluorescently labeled antibodies to the sample, followed by concentration of antibody-virus complexes on a porous membrane. Excess antibodies are removed by electrophoresis through the membrane and the complex is then detected downstream of the membrane. This new assay detected inactivated swine influenza virus at a concentration four times lower than that of the open-channel electrophoresis assay. The total assay time, including device regeneration, is six minutes and requires <50 microl of sample. The filtration effect of the polymer membrane eliminates the need for washing, commonly required with surface-based immunoassays, increasing the speed of the assay. This assay is intended to form the core of a portable device for the diagnosis of high-consequence animal pathogens such as foot-and-mouth disease. The electrophoretic immunoassay format is rapid and simple while providing the necessary sensitivity for diagnosis of the illness state. This would allow the development of a portable, cost-effective, on-site diagnostic system for rapid screening of large populations of livestock, including sheep, pigs, cattle, and potentially birds.