Defining cell culture conditions to improve human norovirus infectivity assays.

Significant difficulties remain for determining whether human noroviruses (hNoV) recovered from water, food, and environmental samples are infectious. Three-dimensional (3-D) tissue culture of human intestinal cells has shown promise in developing an infectivity assay, but reproducibility, even within a single laboratory, remains problematic. From the literature and our observations, we hypothesized that the common factors that lead to more reproducible hNoV infectivity in vitro requires that the cell line be (1) of human gastrointestinal origin, (2) expresses apical microvilli, and (3) be a positive secretor cell line. The C2BBe1 cell line, which is a brush-border producing clone of Caco-2, meets these three criteria. When challenged with Genogroup II viruses, we observed a 2 Log(10) increase in viral RNA titer. A passage experiment with GII viruses showed evidence of the ability to propagate hNoV by both quantitative reverse transcription polymerase chain reaction (qRT-PCR) and microscopy. In our hands, using 3-D C2BBe1 cells improves reproducibility of the infectivity assay for hNoV, but the assay can still be variable. Two sources of variability include the cells themselves (mixed phenotypes of small and large intestine) and initial titer measurements using qRT-PCR that measures all RNA vs. plaque assays that measure infectious virus.

Evaluation of the FilmArray® system for detection of Bacillus anthracis, Francisella tularensis and Yersinia pestis.

AIMS: To evaluate the sensitivity and specificity of the BioFire Diagnostics FilmArray(®) system in combination with their Biothreat Panel for the detection of Bacillus anthracis (Ba), Francisella tularensis (Ft) and Yersinia pestis (Yp) DNA, and demonstrate the detection of Ba spores. METHODS AND RESULTS: DNA samples from Ba, Ft and Yp strains and near-neighbours, and live Ba spores were analysed using the FilmArray(®) Biothreat Panel, a multiplexed PCR-based assay for 17 pathogens and toxins. Sensitivity studies with DNA indicate that the limit of detection is 250 genome equivalents (GEs) per sample or lower. Furthermore, the identification of Ft, Yp or Bacillus species was made in 63 of 72 samples tested at 25 GE or less. With samples containing 25 CFU of Ba Sterne spores, at least one of the two possible Ba markers was identified in all samples tested. We observed no cross-reactivity with near-neighbour DNAs. CONCLUSIONS: Our results indicate that the FilmArray(®) Biothreat Panel is a sensitive and selective assay for detecting the genetic signatures of Ba, Ft and Yp. SIGNIFICANCE AND IMPACT OF THE STUDY: The FilmArray(®) platform is a complete sample-to-answer system, combining sample preparation, PCR and data analysis. This system is particularly suited for biothreat testing where samples need to be analysed for multiple biothreats by operators with limited training.

Continuous spore disruption using radially focused, high-frequency ultrasound.

We report on the development of a novel, continuous-flow, radially focused ultrasonic disruptor capable of lysing Bacillus spores in the absence of added chemical denaturants, enzymes, or microparticles. Greater than 99% disruption was achieved for Bacillus globigii spores and Escherichia coli and Bacillus subtilis vegetative cells with sample residence times of 62, 12, and 12 s, respectively. Microscopic and SEM images indicated that at equivalent power levels, the incidence of cell death or loss of viability typically exceeded the efficiency of (visible) cell lysis. However, semiquantitative PCR showed up to a 1,000-fold increase in intracellular DNA availability from ultrasonically disrupted spores, and liberated DNA was intact and available for subsequent detection.

Automated immunomagnetic separation and microarray detection of E. coli O157:H7 from poultry carcass rinse.

We describe the development and application of an electromagnetic flow cell and fluidics system for automated immunomagnetic separation (IMS) of Escherichia coli O157:H7 directly from poultry carcass rinse. We further describe the biochemical coupling of automated sample preparation with nucleic acid microarrays. Both the cell concentration system and microarray detection method did not require cell growth or enrichment from the poultry carcass rinse prior to IMS. Highly porous Ni foam was used to enhance the magnetic field gradient within the flow path, providing a mechanism for immobilizing immunomagnetic particles throughout the fluid rather than the tubing wall. A maximum of 32% recovery efficiency of non-pathogenic E. coli was achieved within the automated system with 6 s cell contact times using commercially available antibodies targeted against the O and K antigens. A 15-min protocol (from sample injection though elution) provided a cell recovery efficiency that was statistically similar to > I h batch captures. O157:H7 cells were reproducibly isolated directly from poultry carcass rinse with 39% recovery efficiency at 10(3) CFU ml(-1) inoculum. Direct plating of washed beads showed positive recovery of O157:H7 directly from poultry carcass rinse at an inoculum of 10 CFU ml(-1). Recovered beads were used for direct polymerase chain reaction (PCR) amplification and microarray detection, with a process-level detection limit (automated cell concentration though microarray detection) of < 10(3)CFU ml(-1) in poultry carcass rinse.

Rotating rod renewable microcolumns for automated, solid-phase DNA hybridization studies.

The development of a new temperature-controlled renewable microcolumn flow cell for solid-phase nucleic acid hybridization in an automated sequential injection system is described. The flow cell included a stepper motor-driven rotating rod with the working end cut to a 45 degrees angle. In one position, the end of the rod prevented passage of microbeads while allowing fluid flow; rotation of the rod by 180 degrees releases the beads. This system was used to rapidly test many hybridization and elution protocols to examine the temperature and solution conditions required for sequence-specific nucleic acid hybridization. Target nucleic acids labeled with a near-infrared fluorescent dye were detected immediately postcolumn during all column perfusion and elution steps using a flow-through fluorescence detector. Temperature control of the column and the presence of Triton X-100 surfactant were critical for specific hybridization. Perfusion of the column with complementary oligonucleotide (200 microL, 10 nM) resulted in hybridization with 8% of the DNA binding sites on the microbeads with a solution residence time of less than 1 s and a total sample perfusion time of 40 s. The use of the renewable column system for detection of an unlabeled PCR product in a sandwich assay was also demonstrated.

Automated nucleic acid isolation and purification from soil extracts using renewable affinity microcolumns in a sequential injection system.

We have combined affinity purification concepts with novel renewable-surface microcolumns in a sequential injection system for the automated and rapid isolation and purification of nucleic acids directly from crude soil extracts. Geobacter chapellii DNA was spiked at femtomolar concentrations into clean solutions or crude soil extracts containing picomolar concentrations of competitive DNA, humic acids and other soluble soil constituents. The 16S rDNA targets (indigenous and spiked) were purified and eluted in less than 20 min in a form suitable for direct polymerase chain reaction (PCR) amplification and detection. The extraction efficiency of the automated system was equivalent to a 4-h batch reaction using identical reagents. The estimated efficiency of isolation and purification was maximally 30% under the conditions employed here, with levels comparable to those obtained with soils/sediments processed by standard techniques, and a detection limit of 1.7 attamoles (10(6) copies) Geobacter target in a soil extract containing a competitive background of 10(9) genomes. This manuscript represents the first report of automated nucleic acid purification from an environmental sample using sequential injection fluidic systems and renewable microcolumn technology, and provides an excellent platform from which to optimize and accelerate the development of an integrated microbial/nucleic acid detector.

Investigation of AC-DC magnetic field effects in planar phospholipid bilayers.

Observations recently reported by others indicate that a combination of a weak dc magnetic field and extremely-low-frequency ac magnetic field can produce resonant effects in biological systems. We report measurements of the effects of combined dc and ac magnetic fields on the dc current through channel-free planar phospholipid membranes. The combined dc-ac magnetic fields did affect the dc current through planar phospholipid membranes, but not in every membrane, and not consistently at the same values of magnetic flux density and frequency. None of our measurements showed resonant response akin to the cyclotron-like resonance reported in diatoms [Smith et al., 1987] and lymphocytes [Liboff et al., 1987].

Calcium binding to metallochromic dyes and calmodulin in the presence of combined, AC-DC magnetic fields.

The possibility that weak, ac and dc magnetic fields in combination may affect binding equilibria of calcium-ions (Ca2+) was investigated with two metallochromic dyes as calcium-binding molecules: murexide and arsenazo III. Calcium-dye equilibria were followed by measuring solution absorbances with a fiber-optic spectrophotometer. A Ca(2+)-arsenazo solution was also used indirectly to monitor the binding of Ca2+ to calmodulin. Parallel, ac and dc magnetic fields were applied to each preparation. The ac magnetic field was held constant during each of a series of experiments at a frequency in the range between 50 and 120 Hz (sine wave) or at 50 pps (square wave) and at an rms flux density in the range between 65 and 156 microT. The dc magnetic field was then varied from 0 to 299 microT at 1.3 microT increments. The magnetic fields did not measurably affect equilibria in the binding of metallochromic dyes or calmodulin to Ca2+.