Multiplex PCR method for the detection of human norovirus, Salmonella spp., Shigella spp., and shiga toxin producing Escherichia coli in blackberry, coriander, lettuce and strawberry.

A multiplex PCR method was developed for the simultaneous detection of murine norovirus (MNV-1) as a surrogate for human norovirus (HuNoV) GI and GII, Salmonella spp., Shigella spp., and Shiga toxin producing Escherichia coli (STEC) in fresh produce. The toxicity of the glycine buffer on bacterial pathogens viability was evaluated. The growth of each of the three pathogens (previously stressed) was evaluated at 35 and 41.5 °C in modified buffered peptone water (mBPW) and trypticase soy broth (TSB), supplemented with vancomycin, novobiocin and brilliant green at two concentration levels. The selected conditions for simultaneous enrichment were: 41.5 °C/mBPW/supplemented with 8 ppm vancomycin, 0.6 ppm novobiocin and 0.2 ppm brilliant green. The pathogens and aerobic plate count (APC) growth was evaluated in the enrichment of lettuce, coriander, strawberry and blackberry under the best enrichment conditions. Starting from 1 to 10 CFU/mL, Salmonella reached from 7.63 to 8.91, Shigella 6.81 to 7.76 and STEC 7.43 to 9.27 log CFU/mL. The population reached for the APC was 5.11-6.56 log CFU/mL. Simultaneous detection by PCR was done using designed primers targeting invA, ipaH, stx1 and stx2 genes, and MNV-1. The detection sensitivity was 10-100 PFU for the MNV-1 and 1-10 CFU for each pathogenic bacteria. This protocol takes 6 h for MNV-1 and 24 h for Salmonella spp., Shigella spp., and STEC detection from the same food portion. In total, 200 samples were analyzed from retail markets from Queretaro, Mexico. Two strawberry samples were positive for HuNoV GI and one lettuce sample was positive for STEC. In conclusion, the method developed in this study is capable of detecting HuNoV GI and GII, Salmonella spp., Shigella spp and STEC from the same fresh produce sample.

Lactococcus lactis subsp. cremoris strain JFR1 attenuates Salmonella adhesion to human intestinal cells in vitro.

Lactococcus lactis subsp. cremoris JFR1 has been studied in reduced fat cheese due to its ability to produce exopolysaccharides (EPS) in situ, contributing to improved textural and organoleptic properties. In this study, the effect of strain JFR1 on virulence gene expression and attachment of Salmonella to HT-29 human colon carcinoma cells was investigated. Overnight cultures of L. lactis subsp. cremoris JFR1 containing EPS, grown in M17 media with 0.5% glucose supplementation, decreased attachment as well as down regulated virulence gene expression in Salmonella enterica subsp. enterica when tested on HT-29 cells. However, EPS isolated from milk fermented with L. lactis subsp. cremoris JFR1 did not affect Salmonella virulence gene expression or attachment to HT-29 cells. These results suggest that EPS does not contribute to the attachment of Salmonella to human intestinal cells. However, the possibility that the isolation process may have affected the structural features of EPS cannot be ruled out.

Stability of Secondary and Tertiary Structures of Virus-Like Particles Representing Noroviruses: Effects of pH, Ionic Strength, and Temperature and Implications for Adhesion to Surfaces.

Loss of ordered molecular structure in proteins is known to increase their adhesion to surfaces. The aim of this work was to study the stability of norovirus secondary and tertiary structures and its implications for viral adhesion to fresh foods and agrifood surfaces. The pH, ionic strength, and temperature conditions studied correspond to those prevalent in the principal vehicles of viral transmission (vomit and feces) and in the food processing and handling environment (pasteurization and refrigeration). The structures of virus-like particles representing GI.1, GII.4, and feline calicivirus (FCV) were studied using circular dichroism and intrinsic UV fluorescence. The particles were remarkably stable under most of the conditions. However, heating to 65°C caused losses of ß-strand structure, notably in GI.1 and FCV, while at 75°C the α-helix content of GII.4 and FCV decreased and tertiary structures unfolded in all three cases. Combining temperature with pH or ionic strength caused variable losses of structure depending on the particle type. Regardless of pH, heating to pasteurization temperatures or higher would be required to increase GII.4 and FCV adhesion, while either low or high temperatures would favor GI.1 adhesion. Regardless of temperature, increased ionic strength would increase GII.4 adhesion but would decrease GI.1 adhesion. FCV adsorption would be greater at refrigeration, pasteurization, or high temperature combined with a low salt concentration or at a higher NaCl concentration regardless of temperature. Norovirus adhesion mediated by hydrophobic interaction may depend on hydrophobic residues normally exposed on the capsid surface at pH 3, pH 8, physiological ionic strength, and low temperature, while at pasteurization temperatures it may rely more on buried hydrophobic residues exposed upon structural rearrangement.

Comparison of RNA extraction methods for the detection of a norovirus surrogate in ready-to-eat foods.

Four nucleic acid extraction methods were evaluated for the purpose of quantifying a norovirus surrogate (murine norovirus [MNV-1]) concentrated from different food samples. Simple (strawberries and lettuce) and complex (sliced turkey breast, soft-shell clams, and potato salad) food matrices were inoculated with a viral suspension containing high (4×10(5) PFU) or low (4×10(3) PFU) numbers of viral particles. MNV-1 was eluted using either the Pulsifier™ or repetitive pipetting. The four methods were based on using magnetic silica (MiniMAG), non-magnetic silica (bioMérieux Basic kit), silica membrane (Qiagen kit), and phenol (TriReagent) for RNA extraction. The greatest recovery of viral RNA from simple matrices was obtained using magnetic silica for both inoculation levels. For strawberries, the addition of pectinase during the elution step improved RNA recovery when the Pulsifier was used with silica membrane extraction and when repetitive pipetting was used with magnetic silica extraction. In the case of complex matrices, the extraction of high or low numbers of MNV-1 was highest overall using magnetic silica. The exception was soft-shell clams with a high viral load, in which the greatest recovery was obtained with the phenol-based method. In general, magnetic silica was the most effective for extracting both high and low numbers of MNV-1 particles from a wide range of foods.

Inactivation of hepatitis A virus and norovirus surrogate in suspension and on food-contact surfaces using pulsed UV light (pulsed light inactivation of food-borne viruses).

This study was conducted to evaluate the inactivation of murine norovirus (MNV-1) and hepatitis A virus (HAV) by pulsed ultraviolet (UV) light. MNV-1 was used as a model for human norovirus. Viral suspensions of about 10(6) PFU/ml were exposed to pulses of UV light for different times and at different distances in a Xenon Steripulse device (model RS-3000C). Inactivation studies were also carried out on 1-cm(2) stainless steel and polyvinyl chloride disks with 10(5) PFU/ml. Inactivation of MNV-1 and HAV at 10.5 cm from the UV source was greater on inert surfaces than in suspension. The presence of organic matter (fetal bovine serum) reduced the effectiveness of pulsed light both in suspension and on surfaces. However, 2-s treatment in the absence of FBS completely inactivated (5 log reduction) the viral load at different distances tested, whether in suspension (MNV-1) or on disks (MNV-1 and HAV). The same treatment in the presence of fetal bovine serum (5%) allowed a reduction of about 3 log. This study showed that short duration pulses represent an excellent alternative for inactivation of food-borne viruses. This technology could be used to inactivate viruses in drinking water or on food-handling surfaces.

Macromolecular trafficking between a vesicular arbuscular endomycorrhizal fungus and roots of transgenic tobacco.

The root system of transgenic tobacco plants expressing the enhanced green fluorescent protein (EGFP) under the control of the 35S cauliflower mosaic virus (CaMV) promoter, were colonized with the endomycorrhizal fungus Glomus intraradices. Translocation of EGFP protein from the root to the fungus was registered by light and confocal microscopy. Immunolocalization also showed the presence of EGFP in the mycelium of Glomus intraradices. Carboxyfluorescein feeding experiments on wild type mycorrhized plants evidenced the transport of fluorescein, a symplasmic tracer, from the plant to the fungus. Our results suggest that endomycorrhiza possess the capacity to exchange functional biological macromolecules as evidenced by the transport EGFP from the plant to the fungal symbiont.

Heat inactivation of hepatitis A virus and a norovirus surrogate in soft-shell clams (Mya arenaria).

The effectiveness of different thermal treatments for inactivating two viruses in clams was evaluated. Soft-shell clam digestive glands experimentally contaminated with hepatitis A virus (HAV) or murine norovirus (MNV) were heated for 90, 180, or 300 seconds at 85°C or 90°C in glass vials or plastic bags with 200 g of soft-shell clam meat. Inactivation was measured by plaque assay and real-time reverse-transcription (RT)-polymerase chain reaction assay. Measured inactivation was similar using both assays. The 90°C for 90 seconds treatment reduced MNV-1 titer by 3.33 log cycles and HAV by 2.66 log cycles. At 90°C for 180 seconds, both MNV-1 and HAV were completely inactivated (titer reduced by 5.47 log cycles) in glass vials. In the presence of clam meat as well, HAV inactivation was complete at 90°C for 180 seconds. In general, HAV was more resistant to heat treatment than MNV-1, suggesting that it would require a more severe treatment than human norovirus for inactivation in soft-shell clams. The results of the present study should contribute to the development of strategies for controlling the spread of enteric viral illness via shellfish.

Simultaneous separation and detection of hepatitis A virus and norovirus in produce.

Two sample preparation methods based on electrostatic binding were tested to simultaneously separate different viral particles from different food surfaces (lettuce, strawberry, raspberries and green onions). Both methods were evaluated using a multiplex real-time PCR assay designed for detection of hepatitis A virus and norovirus GI and GII. Single and multiplex detection limits were determined as 10(1) viral particles for HAV and norovirus GII, and 10(2) viral particles for norovirus GI using artificial templates, one HAV strain and different norovirus isolates. Manual extraction based on silica columns was found more suitable for viral RNA preparation than an automatic extraction technique. Consistent detection of infectious amounts (2-20viral particles/g) of HAV and norovirus in different food samples was achievable when the viruses were concentrated using cationically charged filters rather than with cationically charged beads in a flow-through system. Consequently, the developed multiplex detection protocol provides a promising alternative for rapid and simultaneous detection of viral pathogens in foods.

Anion-exchange filtration and real-time PCR for the detection of a norovirus surrogate in food.

In the present study, nanoalumina filters were used as a sample preparation step for the concentration of a norovirus surrogate (murine norovirus 1) from food, and this was coupled with a two-step, real-time reverse transcriptase PCR for quantification. The nanoalumina medium was provided in a syringe-filter format, and its binding and elution capacities were tested with different buffers. Among the binding buffers tested (0.1 M Tris-HCl [pH 7.0] with 0.1% Tween 80, 0.1% 3-[(3-cholamidopropyl)-dimethyl-ammonio]-1-propanesulfonate, or 1 M NaCl), no significant differences were found in the capture capacity of the nanoalumina filters, which was found to be as high as 99.8% of murine norovirus 1 present in the buffer. Elution of 50% of captured viral particles from the filters was possible by using glycine buffer. The desorption capacity of the binding buffers was tested on different inoculated food surfaces. Recoveries of up to 100% from lettuce, raspberries, strawberries, or mussels were obtained with 0.1 M Tris-HCl (pH 7.0) containing 1 M NaCl by using orbital shaking or pipetting. The latter method was more efficient and gave higher recoveries than did orbital shaking. The combination of an efficient desorption-binding-elution buffer with the high concentration capacity of the nanoalumina medium allowed the detection of 10(1) PFU from inoculated produce and 10(5) PFU from inoculated mussels.

Immunocapture and real-time PCR to detect Campylobacter spp.

In this work, the feasibility of using a large-volume immunocapture system as a sample pretreatment before detection of Campylobacter was studied. Real-time PCR was used for detection of captured cells after immunocapture. This immunocapture system is able to process high-volume samples by recirculation, increasing the possibility of capturing cells in low numbers. After 30 min of recirculation, the sample is concentrated from 250 ml to 200 microl. In this study, different parameters were compared in order to improve cell capture. The analysis of inoculated chicken skin showed that detection of Campylobacter at levels of 10(3) CFU/25 g was possible after 8 h of enrichment. The low recovery of Campylobacter cells (< 1%) makes this separation method qualitative rather than quantitative. The detection limit of the entire protocol was increased due to the low cell recovery of the sample pretreatment. Therefore, this immunoseparation is able to recover cells present in high concentration after enrichment but not cells present in low concentration. Isolation of Campylobacter cells is achievable using this separation method rather than rapid detection.