Viruses and viral stock preparation.
Norovirus material was obtained from a stool sample kindly provided by the Federal Institute for Risk Assessment (Berlin, Germany). The norovirus in this stool sample was classified as GII cluster 3 based on nucleotide sequence analyses. For storage, the stool sample was diluted 1:5 with phosphate-buffered saline (PBS) (137 mM NaCl, 2.7 mM KCl, 10.1 mM Na2HPO4, 1.8 mM KH2PO4; pH 7.4) and clarified by centrifugation at 3,000 × g for 15 min. The supernatant was mixed with 15% glycerol in PBS (final glycerol concentration, 10%) and filtered through a 0.45-μm syringe filter with a polyethersulfone (PES) membrane (VWR International, Darmstadt, Germany). The NV inoculation standard was determined to contain 4 × 109 copies/ml using real-time quantitative RT-PCR (qRT-PCR) as described below and was stored in aliquots at −150°C until it was used.
Bacteriophage MS2 strain DSM13767 was kindly provided by the Institut fuer Laboratoriums und Transfusionsmedizin, Herz und Diabeteszentrum Nordrhein-Westfalen, Universitätsklinik der Ruhr-Universität Bochum (Bad Oeynhausen, Germany). MS2 was propagated by the double-agar-layer plaque technique and was quantified by plating assays as previously described (29
). Aliquoted phage lysates were stored at −150°C until they were used.
Artificial inoculation of selected foods and sample processing.
Selected foods utilized in inoculation studies (Table ) were purchased from local commercial sources and temporarily stored according to the producers’ recommendations.
Foodstuffs, physicochemical treatments, and modes of inoculation used in this study
Composite or hackled foods, such as convenience foods, delicatessen salads, and mincemeat, were artificially inoculated internally with a defined number of NV GGII. For this purpose, the diverse food samples (7 g of sauces, 15 g of all other samples) were transferred to stomacher bags with a mesh filter (spiced tomato sauce, mincemeat) or to 50-ml centrifuge tubes (Greiner Bio One, Frickenhausen, Germany). The samples were inoculated by adding 1 ml of the standard NV inoculum containing 4 × 109 copies/ml. Delicatessen salad samples were carefully inverted, and the convenience food and mincemeat samples were repeatedly mixed thoroughly over a 25-min time period. For heat experiments mincemeat was manually formed into meatballs, and the virus inoculum was mixed with the mincemeat evenly. Solid foods (fruits, salad, frozen pizza, and frozen pizza baguette) were inoculated by dispensing 1 ml of the standard NV inoculum evenly across the surface. The inoculum was air dried for 20 min at room temperature. Fruits and salad (15 g) were not chopped or disrupted as much as possible to prevent the release of potential inhibitory substances into the samples. Frozen food (15 g) was briefly thawed for 15 min at room temperature before inoculation.
Product treatments performed in this study (Table ) were intended to simulate processes used in industrial food processing for preservation and during food storage and preparation by the consumer. Subsequent to processing, the samples were immediately brought to room temperature by chilling on ice or thawing.
The reference samples consisted of 1 ml of the standard NV inoculum and, instead of a food matrix, corresponding volumes of PBS. Reference samples were treated just like the food samples, being subjected to the same process.
Two replicates of each experimental sample were used, and each experiment was repeated under the same conditions.
Extraction of virus from experimentally inoculated food samples.
The virus recovery procedure was adapted from procedures described previously (30
), with minor modifications (Fig. ). The processed samples were transferred into stomacher bags with a mesh filter. Depending on the dryness and viscosity of the food matrix, 7 to 30 ml PBS was added to the samples, together with 5 μl MS2 phage lysate containing 1.5 × 1011
PFU/ml (final concentration, 7.5 × 108
PFU/ml of ultrafiltration concentrate) as an internal process control (see below). After this, the samples were incubated for 30 min at room temperature or for 24 h at 4°C (sauces); during this period the samples were mixed several times in the stomacher bags. Delicatessen salads were gently shaken in PBS to avoid disruption of the components. Solid surface foods (fruits and salad) were rinsed in 17 ml PBS including an MS2 process control, with agitation for 30 min at room temperature. Subsequently, each liquid phase was transferred into a 50-ml centrifuge tube. Sedimentation of particulate debris and phase separation were achieved by centrifugation at room temperature for 10 min at 1,000 × g
(fruits and salad) or for 30 min at 4,000 × g
(all other samples). The aqueous phase was purified by filtration through 1.2- to 0.45-μm syringe filters with PES membranes. Each filtrate was then transferred to a Vivaspin 20 concentrator (molecular weight cutoff, 50,000) with a PES membrane (Vivascience AG, Hannover, Germany). Ultrafiltration devices were centrifuged at room temperature and 4,000 × g
for 2 to 30 min. The centrifugation time was extended until the final retention volume was ca. 1 ml.
Flow chart of the experimental procedure.
Reference samples (without a food matrix) were diluted with 15 ml PBS inoculated with the MS2 internal process control (final concentration, 7.5 × 108 PFU/ml of ultrafiltration concentrate), analogous to the food samples (see above). After 30 min of incubation, each suspension was directly transferred into an ultrafiltration device and concentrated to a final volume of 1 ml by centrifugation at room temperature and 4,000 × g for approximately 2 min.
A total of 140 μl of each sample concentrate was used for further experimental procedures.
Precautions were taken to prevent false-positive and false-negative results. In addition to spatial separation of workspaces at crucial experimental points (e.g., during RNase treatment and RNA extraction), each experiment included several overall control samples.
The internal process control with phage MS2 (29
) was used to monitor the efficiency and reproducibility of recovery of virus from foods and to rule out false-negative results. Each sample except the amplification control samples (see below) was spiked with 5 μl MS2 phage lysate containing 1.5 × 1011
PFU/ml (final concentration, 7.5 × 108
PFU/ml of ultrafiltration concentrate) in an appropriate amount of PBS at the beginning of the virus recovery procedure (as described above). Thus, MS2 and NV were corecovered from the same food sample and concentrated. RNA was also coextracted from both NV and MS2, but the viruses were detected by performing individual monoplex real-time RT-PCRs (see below) in separate tubes.
An extraction-negative control was performed for both NV detection and MS2 detection by extracting food samples like the other samples but without any experimental virus contamination. This control was carried out to uncover potential false-positive results caused by cross contamination during the virus and RNA extraction or by contaminated kits, reagents, and foods.
External amplification controls were carried out to monitor the intensity of inhibitory effects on the PCRs mediated by the food matrices. Furthermore, amplification controls could reveal any suboptimal composition of the PCR mixture or the presence of residual RNase activity. An RNA eluate from a sample was spiked with defined concentrations of purchased genomic MS2 RNA (Roche Diagnostics, Mannheim, Germany); 1 μl of a 10−7 dilution of the MS2 stock solution was mixed with 9 μl RNA eluate from a corresponding sample. The solution was subsequently used as a template in real-time RT-PCR MS2 detection assays, as described below. The amplification control was included for each of the sample replicates which were without an MS2 process control.
Amplification-positive controls were applied for both NV detection and MS2 detection. The NV amplification control consisted of 10 μl RNA eluate isolated from 140 μl of an NV inoculation standard (see above). The MS2 control template comprised 1 μl of a 10−7 dilution of the MS2 stock solution mixed with 9 μl buffer AVE (Qiagen, Hilden, Germany).
Amplification-negative controls were also carried out for the NV and MS2 detection assays. These controls revealed potential contamination of the PCR mixture that would lead to false-positive results. The negative control PCR samples contained 10 μl RNase-free water instead of the RNA template.
Degradation of free RNA and RNase inhibition.
Digestion of free RNA was carried out by adding 35 μg RNase A (Qiagen) per sample and incubating the mixture at 37°C for 1 h. The RNase activity in the samples containing a food matrix was verified using the RNaseAlert lab test kit (Applied Biosystems, Darmstadt, Germany) according to the manufacturer's instructions. Fluorescence was excited using a transilluminator. After this, the RNase activity was inhibited by adding 140 U/sample of Qiagen RNase inhibitor (Qiagen) and incubating the mixture for 30 min at room temperature.
Nucleic acid isolation.
Virus RNA was extracted by the column centrifugation method, using the commercially available QIAamp viral RNA mini kit (Qiagen) according to the manufacturer's protocol. In brief, 140 μl of sample was denatured, adsorbed to a silica gel column, and washed twice. The RNA was finally eluted with 60 μl buffer AVE after 2 min of incubation at room temperature. To avoid residual RNase activity, 50 U/sample of Protector RNase inhibitor (Roche Diagnostics) was added, and the mixture was incubated for 30 min at room temperature. Suspensions were tested for residual RNase activity by using the RNaseAlert lab test kit as described above. The nucleic acid was immediately used in downstream PCR application or stored at −19°C until it was used.
Primer and probe design.
Primer and probe sequences for norovirus detection were selected based on a consensus sequence resulting from a multiple sequence alignment. The sequences aligned were obtained by performing sequence homology searches with nucleotide databases (GenBank, RefSeq Nucleotides, EMBL, and DDBJ) using a 450-bp sequence of a highly conserved region (ORF1-ORF2 junction) of the norovirus genome (49
) as a reference sequence.
Sequence similarity database searches were performed with the BLAST algorithm (1
) and the multiple alignment using CLUSTAL W (85
). The oligonucleotides were designed using CLC Combined Workspace (version 3.01; CLC bio, Aarhus, Denmark) and Clone Manager Suite (version 6; Scientific & Educational Software, Cary, NC). The oligonucleotides were purchased from MWG Biotech AG (Ebersberg, Germany).
TaqMan real-time RT-PCR assays.
The real-time RT-PCRs were performed as one-step monoplex assays with TaqMan hydrolysis probes, using the QuantiTect Virus +ROX vial kit (Qiagen). Each PCR mixture (final volume, 15 μl) consisted of 5 μl QuantiTect Virus NR Master Mix, 0.25 μl QuantiTect Virus RT-Mix, 500 nM (final concentration) of each primer, 250 nM (final concentration) of a probe, and nuclease-free water. The primers and probes used for NV and MS2 detection are listed in Table . The final volume of each reaction mixture was adjusted to 25 μl by adding 10 μl of RNA solution (template) or RNase-free distilled water (control).
Primers and probes used for real-time RT-PCR in this study
Amplification and detection were performed with the Rotor-Gene 3000 system (Corbett Research, Sydney, Australia). After the thermocycler rotor was loaded with the sample tubes, the run was initiated immediately. The thermal cycling conditions were reverse transcription at 50°C for 30 min, denaturation at 95°C for 5 min, and amplification for 65 cycles with denaturation at 95°C for 15 s, annealing, and fluorescence detection at 60°C for 45 s. Raw data were obtained during the annealing step of each cycle in the 510-nm channel (NV) and the 555-nm channel (MS2).
The cycle threshold (CT
) is defined as the fractional cycle at which the fluorescence rises above a given threshold value (41
values were calculated from the raw fluorescence data by using CAmpER (“c
alculation of a
fficiencies for real-time RT-PCR experiments”), an open web-based application for automatic analysis of real-time PCR experiments (http://www.cebitec.uni-bielefeld.de/groups/brf/software/camper_info/index.html
), allowing the standardized determination for single samples. After the Rotor-Gene output file was uploaded to the CAmpER web server, the CT
value of each sample was calculated by using the implemented DART algorithm (71
). The threshold is calculated as a multiple of the mean standard deviation of the first 10 cycles of the automated background-corrected fluorescence curve of all samples of the experiment.
Samples with CT values below 43 and with a sigmoidal amplification plot were considered suitable for analysis.
For determination of recovery rate, protective effect, and inactivation, the quantity of NV was calculated as described above using the average CT value derived from four replicates from two independent experiments. NV recovery, expressed as percent recovery, was calculated by using the following formula: % recovery rate = (virus quantity in untreated food sample)/(virus quantity in untreated reference sample) × 100. The food-specific protective effect, expressed as percent survival rate, was calculated by using the following formula: % survival rate = [(virus quantity in processed food sample) − (virus quantity in processed reference sample)]/(virus quantity in untreated food sample) × 100. Virus inactivation, expressed as logarithmic titer reduction, was calculated as follows: log10 (Nt/N0), where N0 is the quantity of virus in the untreated food sample and Nt is the quantity of virus in the processed food sample.
Mean values, standard deviations, and coefficients of variation were calculated by using the office application suite OpenOffice (version 3.01; Sun Microsystems free software community). Statistical analyses regarding significant differences between the mean numbers of viruses found in the treated and untreated samples were performed with Student's t test, and a P value of ≤0.05 was considered significant.
Quantification of norovirus.
Absolute quantification of NV RNA was performed by means of an external standard curve which displays the relationship between a given CT value and the corresponding number of genome equivalents per PCR assay. The external standard curve was derived from real-time RT-PCRs performed with known quantities of the NV target, a recombinant RNA (recRNA) standard.
Construction of the norovirus RNA standard was performed by using a previously described method (30
), with some modifications. In brief, a recombinant plasmid (kindly provided by the Institut fuer Laboratoriums und Transfusionsmedizin, Herz und Diabeteszentrum Nordrhein-Westfalen, Universitätsklinik der Ruhr-Universität Bochum, Bad Oeynhausen, Germany), which harbors a cloned norovirus GGII sequence from the ORF1-ORF2 junction, was subjected to a PCR assay with the universal M13 primers to amplify the inserted DNA fragment, including phage promoter T7. The PCR product was purified with the QIAquick PCR purification kit (Qiagen) and subsequently used for runoff transcription using a T7 transcription kit (Fermentas, St. Leon-Rot, Germany) according to the manufacturer's protocols. The remaining DNA was digested with DNase I (RNase-Free DNase set; Qiagen) before the RNA transcripts were purified with a QIAamp viral mini kit (Qiagen) used according to the manufacturer's instructions, except for the addition of carrier RNA.
To generate the standard curve, a 10-fold dilution series of the recombinant RNA standard was prepared. RNA concentrations were obtained by spectroscopy at a wavelength of 260 nm. Quantification of RNA standard dilutions was carried out by using a formula that considers the RNA concentration, the fragment size, and the Avogadro constant (37
). RNA dilutions from 10−1
were used as templates in real-time RT-PCR assays under the conditions described above. The standard curve was finally obtained by plotting the calculated quantities of the RNA standard dilutions (in genome equivalents per PCR assay) against the corresponding CT