Enhanced Reverse Transcription-PCR Assay for Detection of Norovirus Ge
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《微生物临床杂志》
Institut für Laboratoriums und Transfusionsmedizin, Herz und Diabeteszentrum Nordrhein-Westfalen, Universittsklinik der Ruhr-Universitt Bochum, Bad Oeynhausen, Germany
Landesamt für Verbraucherschutz Sachsen-Anhalt, Halle, Germany
Berliner Betrieb für Zentrale Gesundheitliche Aufgaben (BBGes), Institut für Lebensmittel, Arzneimittel und Tierseuchen (ILAT), Berlin, Germany
ABSTRACT
We have developed a one-tube reverse transcription (RT)-PCR method using the real-time TaqMan PCR system for the detection of norovirus genogroup I (NV GGI). By introduction of a novel probe based on locked nucleic acid technology, we enhanced the sensitivity of the assay compared to those of conventional TaqMan probes. The sensitivity of the NV GGI RT-PCR was determined by probit analysis with defined RNA standards and quantified norovirus isolates to 711 copies/ml (95% detection limit). In order to detect PCR inhibition, we included a heterologous internal control (IC) system based on phage MS2. This internally controlled RT-PCR was tested on different real-time PCR platforms, LightCycler, Rotorgene, Mastercycler EP realplex, and ABI Prism. Compared to the assay without an IC, the duplex RT-PCR exhibited no reduction in sensitivity in clinical samples. In combination with an established NV GGII real-time RT-PCR, we used the novel assay in a routine assay for diagnosis of clinical and food-borne norovirus infection. We applied this novel assay to analyze outbreaks of nonbacterial acute gastroenteritis. Norovirus of GGI was detected in these outbreaks. Sequence and similarity plot analysis of open reading frame 1 (ORF1) and ORF2 showed two genotypes, GGI/2 and GGI/4, in semiclosed communities.
INTRODUCTION
Noroviruses (NVs) are one of the major causes of gastroenteritis in humans (2, 15, 25). They are transmitted both directly, via person-to-person contact, and indirectly by environmental contamination (31), via foods and drinking water (5, 13, 21, 22, 28, 32, 36, 37). The number of cases which can be attributed to food- or waterborne transmission is not known exactly (10). The percentage ranges between 16% (13) and 40% to 57% (14, 34).
The norovirus comprises five distinct genogroups (1, 20). The most prevalent circulating strains belong to genogroup II (GGII), responsible for up to 90% of NV outbreaks in Germany (16). The genogroup I (GGI) strains cause about one quarter (16) to 1/10 (18) of all NV-positive outbreaks.
In general, there are immunological, electron microscopic, tissue culture, and molecular methods to detect viruses in food. But, up to now, the major obstacles to laboratory diagnosis are the lack of a tissue culture system for propagating the viruses (12, 19) and the limited sensitivity of recently developed immunological assays. Therefore, molecular methods have to be the assay of choice for their detection. The aim should be a detection system which is applicable to stool samples as well as to food and environmental samples. The special sensitivity requirements are highly demanding because, on the one hand, the number of virus particles shed can vary between 102 and 1010 genome equivalents (16) and, on the other hand, the investigation of environmental samples requires a sensitivity corresponding to that of the low infectious dose. The minimal infectious dose is about 10 to 100 infectious virus particles (7, 13). Based on the minimal infectious dose, the detection limit should be fewer than 1,000 virus particles in food and environmental samples. At present, immunological and electron microscopic methods cannot be used because of their lack of sensitivity (34).
Real-time PCR systems using specific probes (16, 19, 20, 24, 33) or SYBR green (35) as a detection system have recently been developed. According to the European standard (3), a diagnostic PCR with food and environmental samples requires sequence-specific verification of the PCR product by restriction enzymes, probe hybridization, or sequencing. Melting curve analysis is not considered to be equivalent. For routine diagnostic applications, probe hybridization in real-time PCR seems to be the most promising system. Recent multilaboratory studies show that the sensitivity of real-time reverse transcription (RT)-PCR systems to detect GGI is lower than that of systems for the detection of GGII in environmental samples (27). This raises the need for a very sensitive real-time RT-PCR detection system using specific probes for the detection of NV GGI.
Parallel to the norovirus-specific detection reaction, the amplificability of the nucleic acids extracted shall be verified by an amplification control (4). This facilitates, on the one hand, a check for coextracted RT-PCR inhibitors and, on the other hand, a verification of the nucleic acid extraction efficiency when applied as a process control from the beginning of the nucleic acid extraction. Phage RNA can be used for this purpose (11). In this study, we present a real-time RT-PCR assay for the detection of NV GGI.
MATERIALS AND METHODS
Processing of stool specimens. We used real-time RT-PCR to screen stool samples from patients with nonbacterial acute gastroenteritis. All of the samples had already been proved to be negative for Salmonella, Shigella, Campylobacter, and Yersinia species by conventional bacterial culture procedure. Screening for group A rotaviruses was performed with the Ridascreen rotavirus enzyme-linked immunosorbent assay (R-Biopharm, Darmstadt, Germany). Fecal specimens from eight gastroenteritis outbreaks occurring in Berlin, Germany, in 2004 were used for this study. Samples were assayed for NV GGI and GGII by real-time RT-PCR. NV GGI RT-PCR was carried out using primers NV192 and NV193 (33) and probes Ring1(a) and Ring1(b) (19), as described previously (26). NV GGII RT-PCR was performed using primers NV107a and NV117 and probe TM6 (16) in a separate reaction. Positive screening results were confirmed by an alternative amplification system as described previously (6, 35) and by DNA sequencing of PCR products.
Nucleic acid isolation. A 10% (wt/vol) stool suspension was prepared with phosphate-buffered saline (PBS) and clarified by centrifugation at 3,000 x g for 20 min. For probing of food or environmental surfaces, we used sterile, rayon-tipped swabs with a plastic stem (Sarstedt, Nümbrecht, Germany). Spiked swabs, used for the validation of the application of the phage MS2 as a process control, were squeezed out three times in 500 μl of PBS before use as suspension in RNA extraction. RNA was extracted from 140 μl to 280 μl of PBS suspension with the QIAamp viral RNA mini kit (QIAGEN, Hilden, Germany) according to the manufacturer's protocol. The RNA was eluted with 60 μl AVE buffer (QIAGEN).
Food surfaces (from solid foods and fruits in which surface contamination is possible) were swabbed with a moistened sterile swab. Swabbing of an area with a size of about 100 cm2 was done very intensively to collect the majority of particles adhering to the surface. Virus particles adhering to the swabs were eluted in 500 μl PBS. The swab was used again to remove virus particles from the next 100 cm2 of the surface. In this way, particles from the whole surface were collected in 500 μl PBS and nucleic acid extraction was performed as described above.
To obtain virus particles from hackled foods, ground foods, or composed foods, 5 g of samples was placed into a stomacher bag including a net. After 5 to 10 ml of PBS was added, depending on the water content of the food, samples were processed for 1 min in a stomacher. The samples were incubated in PBS for 24 to 48 h at 4°C. The samples were processed several times in the stomacher bag. The liquid phase was transferred into a centrifugation tube. Coarse particles were sedimented by centrifugation for 10 min at 1,000 x g. The supernatant was filtered through a 0.2-μm syringe filter. Four milliliters of the clear filtrate was transferred into a 4-ml Vivaspin concentrator, 50000 MWCO (Vivasciences, Hannover, Germany). The samples were centrifuged at 15°C for 20 min at 3,000 x g. The centrifugation time was extended until a final volume of 150 μl was reached. A total of 140 μl of the concentrated solution was used for RNA extraction. In cases in which the samples could not be concentrated to the volume of 150 μl, 280 μl of the concentrated medium was used with double the amount of the extraction reagents of the QIAamp viral RNA mini kit.
Conventional RT-PCR. RT-PCR was performed to amplify sequences of the capsid gene of NV GGI and GGII, using Superscript III one-step RT-PCR with a Platinum Taq kit (Invitrogen, Karlsruhe, Germany). RT-PCR using the primer set Mon431, Mon432, Mon433, and Mon434 described by Beuret et al. (6) and Richards et al. (35) was carried out with the reaction mixture preparations as recommended by the manufacturer. The cycling conditions were as follows: reverse transcription at 50°C for 30 min and, for PCR, initial denaturation for 2 min at 95°C, 45 amplification cycles with denaturation for 30 s at 95°C, annealing for 1 min at 50°C, extension for 1 min at 72°C, and a final cycle of incubation at 72°C for 10 min.
For amplification of the RNA polymerase gene, RT-PCR using primers JV12 and JV13 (38) was performed under the same PCR conditions as those used for amplification of the capsid gene.
MS2 phage RT-PCR. Phage MS2 DSM13767 was purchased from DSMZ (Braunschweig, Germany). MS2 phage lysates were prepared as described previously (11), and the number of PFU was determined by plating assays. The MS2 RNA was quantified (copies per ml) with a genomic MS2 RNA purchased from Roche Diagnostics (Mannheim, Germany). Stool PBS suspensions were spiked with MS2 phage lysate containing 6 x 104 PFU per ml (final concentration, 3,000 PFU MS2 per ml of PBS), and RNA extraction was performed as described above. MS2 real-time RT-PCR was used for internal control of norovirus RT-PCR. The primer system MS2-TM2 with primers MS2-TM3-F and MS2-TM3-R and probe MS2-TM2JOE was used as described previously (11). For detection on the LightCycler platform, the MS2 probe was labeled with reporter dye DY521XL (Dyomics GmbH, Jena, Germany), and detection was performed in channel F3 (705 nm). This reporter fluorophor has an absorption maximum at 523 nm and an emission maximum at 668 nm.
Real-time PCR system. A Superscript III one-step RT-PCR with a Platinum Taq kit (Invitrogen, Karlsruhe, Germany) was used as the basis for the reaction mixture in the LightCycler RT-PCR assay. A volume of 20 μl was used in each reaction capillary, containing 5 μl of the extracted RNA and 15 μl of the reaction mixture: 1x reaction mix (including 3 mM MgSO4; Invitrogen), 500 ng per μl nonacetylated bovine serum albumin (Sigma-Aldrich, Taufkirchen, Germany), 400 nM of forward primer NV192, 400 nM of reverse primer NV193, 150 nM of probe NVGG1-LNA (6-carboxyfluorescein [FAM]-ATTCGGGCAGGAGAT-EclipseDQ [Eurogentec, Seraing, Belgium]; locked nucleic acid [LNA] nucleotides in underlined positions), and 0.6 μl RT/Platinum Taq mix (Invitrogen). In addition to the positive run control, each test run included one no-target control containing 15 μl of the reaction mixture and 5 μl PCR-grade water. The reaction capillaries were capped, centrifuged, and placed into the carousel of the LightCycler instrument (Roche Diagnostics). The LightCycler RT-PCR protocol included the following parameters: reverse transcription at 50°C for 10 min and, for PCR, initial denaturation and Taq DNA polymerase activation for 2 min at 95°C, subsequent 45 cycles at 95°C for 2 s, annealing and fluorescence detection at 56°C for 10 s, and extension at 72°C for 15 s. Data were obtained during the annealing period in the "single" mode with the channel setting F1.
The RT-PCR assays with the Rotorgene 2000 or 3000 cycler system (Corbett Research, Sydney, Australia), Mastercycler EP realplex (Eppendorf, Wesseling-Berzdorf, Germany), and ABI Prism 7700 (Applied Biosystems, Darmstadt, Germany) were performed in a volume of 50 μl, including 20 μl nucleic acid extract. The reaction mixture was as described above but without bovine serum albumin. Cycling conditions were 50°C for 10 min and 95°C for 2 min, followed by 45 cycles at 95°C for 5 s, annealing and fluorescence detection at 56°C for 20 s, and extension at 72°C for 20 s.
Construction of a norovirus RNA standard. NV GGI PCR products of 287 bp flanked by primers Mon431/Mon432 (6, 35) and NV193 (33) were cloned into Escherichia coli by use of the plasmid vector pCRII-TOPO, which was supplied by a TA-TOPO cloning kit (Invitrogen), according to the protocol provided by the supplier. NV GGII PCR products, generated with primers Mon431/Mon432 and NV117 (16), were cloned in E. coli using the TA-TOPO cloning kit.
Analysis of the recombinant plasmids was performed by extracting plasmid DNA from small-volume cultures grown overnight at 37°C using the QIAprep spin plasmid kit (QIAGEN). PCR with universal M13 primers was applied to amplify the inserted DNA fragment, including phage promoters T7 and SP6. Purified PCR products were used for runoff transcription using the SP6/T7 transcription kit (Roche Diagnostics). RNA concentrations were determined by a fluorometric analysis with the RiboGreen RNA quantitation reagent (Molecular Probes, Leiden, The Netherlands). Defined RNA copies were used to quantify NV isolates. For this purpose, RNA standards were extracted in parallel with NV dilutions, and nucleic acids were subjected to quantitative real-time RT-PCR. NV standards in the range from 10 to 1 x 107 copies per PCR were used as standards in real-time PCR assays.
Probit analysis of experimental data. The 95% detection limit was calculated by probit analysis using SPSS 11.0 software (SPSS GmbH Software, Munich, Germany).
DNA sequencing and phylogenetic analysis. PCR products of open reading frame 1 (ORF1) and ORF2 were sequenced directly with a BigDye Terminator v 1.1 cycle sequencing kit (Applied Biosystems) in both directions using an ABI Prism 310 sequencer (Applied Biosystems). Multiple sequence alignments were performed with the CLUSTAL W program, version 1.8. Phylogenetic dendrograms were constructed by the neighbor-joining method (PHYLIP), and phylogenetic trees were drawn using the TreeView program (30).
Nucleotide sequence accession numbers. Sequence data of ORF1 (RNA polymerase gene) and ORF2 (capsid gene) have been submitted to GenBank and assigned accession numbers DQ340076 to DQ340091.
RESULTS
Screening for NV and genogrouping by real-time RT-PCR. Fecal specimens from gastroenteritis outbreaks were screened for norovirus GGI and GGII by the use of real-time RT-PCR (16, 33). Because of reports of false-negative results with GGII/2 strains (Melksham) responsible for gastroenteritis outbreaks in two Bavarian youth hostels using this primer system (A. Carl, personal communication), we replaced the forward primer NV107a (16) with primer COG2F (19). DNA sequence analysis of GGII/2 isolates and sequence alignments revealed four mismatches in the primer NV107a (data not shown).
In order to identify inhibitions of the enzymatic reactions, we used the MS2 RT-PCR system as an external control reaction with the same nucleic acid extracts as those used for NV RT-PCR. For screening of food-borne norovirus contamination, the screening strategy was used as described above. Systematically, we evaluated the assay for food surfaces from solid foods and fruits in which surface contamination is possible (data not shown).
Development of novel real-time RT-PCR for NV genogroup I. Several NV GGI real-time RT-PCR systems were tested for use in routine NV screening (16, 19, 33). In our hands, the systems described exhibited low sensitivities. This was demonstrated in a multicenter collaborative trial (27). Furthermore, NV GGI-positive specimens were detected only with low fluorescence intensity (see Fig. 3).
We therefore designed a novel NV GGI-specific fluorescent probe in the NV ORF2 region for use in real-time RT-PCR with primers NV192 and NV193 (33). To increase the specificity and sensitivity having a higher specificity and higher melting temperature, we examined the use of LNA TaqMan probes that have a higher melting temperature than conventional TaqMan probes (23). To investigate the performance of the LNA probe, we compared the assay with recently described TaqMan assays (16, 19, 33). We compared the cycle threshold (CT) values and the levels of fluorescence intensity of the amplification curves (Fig. 1). The CT values vary up to 9.8 between the LNA and the conventional TaqMan probes. The TaqMan probes Ring1(a)/Ring1(b) and NV-TM6 revealed the same range of fluorescence intensities and CT values when tested in parallel (data not shown). Furthermore, the LNA probe produced a higher level of fluorescence intensity (Fig. 1). In parallel, a panel of NV GGI clinical samples representing 15 isolates of three different genotypes (GG1/1, GG1/2, and GG1/4) was analyzed with these TaqMan RT-PCR assays and confirmed the lower sensitivity of the conventional TaqMan assays (data not shown). Overall, the novel real-time RT-PCR assay with the LNA probe was 10- to 100-fold more sensitive. Therefore, the use of the LNA probe results in a slight increase in the sensitivity of the assay. To determine the specificity of the NV RT-PCR, we tested several NV GGI and GGII isolates. All NV GGI isolates tested were detected, while NV GGII gave no positive amplification signal. No cross-reaction with other viral pathogens, such as rotavirus, astrovirus, adenovirus, and enterovirus, was observed.
Internal amplification control for the norovirus real-time assay. We developed an internal amplification control (IC) by using E. coli phage MS2 as a target in duplex RT-PCR assays (11). This heterologous IC can be added before nucleic acid isolation as MS2 phage particles or directly prior to amplification as MS2 RNA. The MS2 RNA and norovirus RNA present in the sample were coamplified within the same reaction. In order to exclude competition between NV and MS2 amplification, we compared NV GGI (see Table 2) and NV GGII (data not shown) RT-PCRs with and without coamplification of MS2 RNA. For a process control, we performed MS2 spiking experiments using phage lysates that were quantified as PFU and copies per ml. Food and environmental specimens can be analyzed by using swab samples (26). Therefore, we used swabs to validate the application of the phage MS2 as a process control. Various MS2 phage concentrations were spiked to NV GGII-contaminated swabs. Swab resuspension, RNA extraction, and duplex NV-MS2 RT-PCR on the Rotorgene 3000 were performed (Table 1) to determine the optimal MS2 concentration. In all samples, NV GGII RNA was detected with approximate CT values (range, 29.59 to 30.98), independently of the MS2 phage concentration. The IC was detected only in samples spiked with more than 8,400 PFU per RNA preparation. Therefore, we used 8,400 PFU per extraction for routine testing.
The duplex NV-MS2 RT-PCR was used on different real-time PCR platforms. RT-PCR on the LightCycler was carried out with a DY521XL-labeled probe. This novel fluorophor permits the simultaneous detection of the IC in the F3 channel and reporter fluorophor FAM (for NV target) in the F1 channel without applying a color compensation (Fig. 2). Other fluorescence dyes usually used for TaqMan probes, e.g., JOE (6-carboxy-4',5'-dichloro-2',7'-dimethoxyfluorescein), VIC, and TET (tetrachloro-6-carboxyfluorescein), cannot be used because of the limited fluorescence excitation of the LightCycler's light-emitting diode with a peak emission of 470 nm.
Quantitative detection of NV RNA. In order to estimate the detection limits of the NV RT-PCR assays, we constructed NV RNA standards. Plasmid constructs, which contained a region of the capsid gene, were used for in vitro transcription. The capsid gene region contains the primer sites used for conventional NV RT-PCR (6) and most of the previously described real-time RT-PCR systems (16, 18, 19, 24). After transcription, the integrity of the RNA was confirmed by gel electrophoresis and standardized by fluorometric quantitation. The number of molecules present was calculated by the use of Avogadro's number. No residual DNA was detected in the RNA preparations when PCR was performed without reverse transcription.
For calibration purposes, norovirus stool suspensions and PBS spiked with RNA standards were extracted and amplified in parallel. The NV suspension was quantified on the basis of an external standard curve created from the data produced by the individual amplification of the dilution series of the NV RNA standard.
To determine the analytical sensitivity of the RT-PCR assay, we used human stool samples spiked with different titers of NV GGI from 965 to 2.47 x 105 copies per ml, corresponding to 10 to 4,960 copies of NV GGI per RT-PCR. We compared MS2 internally controlled with uncontrolled RT-PCR. For this purpose, 1 x 104 MS2 phage were added to lysis buffer before RNA extraction. Three experiments with four replicates (n = 12) of each concentration were processed through all steps of nucleic acid isolation and RT-PCR (Table 2). The nucleic acid extracts were processed with the NV GGI RT-PCR assay on the Rotorgene 3000 with and without the addition of the MS2 primer-probe system. The 95% detection limit was calculated by probit analysis to 711 copies per PCR (range, 536 to 1117 copies/PCR) when NV GGI was used without an IC. No significant reduction in sensitivity was observed when NV GGI-MS2 duplex RT-PCR was performed with a 95% detection limit of 770 copies per PCR (range, 569 to 1,277 copies per PCR).
Outbreak investigation. During the year 2004, the Institut für Lebensmittel, Arzneimittel und Tierseuchen (ILAT), Berlin, received 86 fecal specimens from seven suspected outbreaks of NV-associated gastroenteritis. The majority of these outbreaks occurred in either residential homes or nursing homes (Table 3). NV GGI was detected as the etiological agent in all outbreaks by real-time RT-PCR. No other gastroenteritis-associated pathogen was identified in specimens collected from these outbreaks. Eight samples from these outbreaks were used to compare the GGI LNA real-time RT-PCR system, as described previously (27), using the oligonucleotides NV192/NV193 (33) and Ring1(a)/Ring1(b) as described by Kageyama et al. (19). With the LNA probe system, all suspect samples yielded positive results, whereas the RT-PCR using the Ring1(a)/Ring1(b) probe missed three of the eight strains (data not shown).
DNA sequence analyses of the RNA-dependent RNA polymerase gene (ORF1) and capsid gene (ORF2) regions were performed (Fig. 3). NV GGI strains were separated into two genotypes (GGI/2 [Southampton] and GGI/4 [Chiba]). Viral sequences determined from samples of patients in the same outbreak were highly homogenous. Single genotypes were observed in all outbreaks that occurred in semiclosed communities. Furthermore, three clusters of GGI/2 strain and one cluster of GGI/4 strain were detected in association with regional outbreaks. The local public health departments (BA 1 to 4) were responsible for sample collection. As an exception, isolates 91568.04-6 (BA 3) and 90068.04-8 (BA 4) were collected from different sources, but an association between these two cases cannot be excluded. In all cases, person-to-person spreading of the infection was obvious, while food-borne NV transmission was not observed.
DISCUSSION
Increasing attention has been paid to viral gastroenteritis outbreaks during the last few years. The development of molecular methods has shown that norovirus is one of the most common causes of gastroenteritis outbreaks in adults and children (16). The increasing number of outbreaks results in increasing numbers of samples that have to be handled in diagnostic laboratories. Therefore, RT-PCR seems to be the method of choice because it combines a potentially high throughput with reproducibility, sensitivity, and specificity.
Therefore, we established a sensitive NV RNA detection system using real-time RT-PCR to quantitatively detect NV RNA. The system does not require any other detection procedure, such as electrophoresis or nested PCR. We describe a real-time RT-PCR assay for NV GGI which is based on LNA probe technology. This assay should be applicable for both stool and food analyses. The introduction of LNA improves the hybridization affinity for complementary sequences, increases the melting temperature by several degrees, and therefore improves the specificity of the assay. LNA probes are ribonucleotides containing a methylene bridge that connects the 2' oxygen of ribose with the 4' carbon. This bridge results in a locked 3'-endo conformation that reduces the conformational flexibility of the ribose. The introduction of LNA into a DNA oligomer improves the hybridization affinity and increases the melting temperature. The increased stability allows the use of shorter probes which are more sensitive to single-base mismatches. The shorter length gave LNA probes better sequence specificity and lower fluorescent background than conventional probes (17). This observation may be attributed to a better efficiency of PCR when shorter detection probes are used, due to lower interference during the amplification process (9). Even for highly variable sequence regions, the stabilizing effect of LNA nucleotides can improve the detection of different isolates of NV GGI. The LNA nucleotides in primers or probes for PCR should be directed to highly conserved positions of the NV target.
An inherent problem in diagnostic PCR is the presence of amplification inhibitors which may cause false-negative results. Therefore, the amplificability of nucleic acids should be verified by an amplification control. In this study, the MS2 phage was successfully used as an internal amplification control in NV RT-PCRs. The use of intact viruses for external standards in absolute quantification assays or a positive control is preferred (8, 29), but the risk of infection for laboratory workers has to be considered when human- or animal-pathogenic viruses are used. Our approach avoids these disadvantages by using Escherichia coli F-specific phage MS2 as a target for the IC. The use of MS2 phage to control clinical diagnostic nucleic acid amplification tests was demonstrated (11). Feline calicivirus was discussed as well as process controls. Generally, we think that using an infectious human or animal pathogen is not applicable as a process control or amplification control in routine analysis. In analyzing food samples, MS2 phage is well suited as a process control. The vast majority of foods analyzed in routine diagnostics do not contain significant amounts of E. coli. Furthermore, legal requirements define an upper limit of 100 CFU per g; the number of E. coli strains carrying F pili is lower again. There is only a negligible risk for naturally occurring MS2 in food. Even the unlikely case of a higher amount of MS2 is acceptable, because this could lead to a gradual underestimation of RT-PCR inhibitors or to an overestimation of the extraction efficiency if the phage is used as a process control. Compared to the advantages of using phage MS2, this relatively low analytical uncertainty should be tolerable. Generally, high titers of noroviruses are observed with acute gastroenteritis.
The sensitivity of NV RT-PCR was determined using different approaches. Up to now, the detection limit of a quantitative NV RT-PCR assay has been calculated only by using plasmid standards (16, 19). Those methods, defining their analytical detection limit by using DNA, therefore seem to be highly sensitive. But, a quantitatively exact conclusion about the viral load of the sample cannot be reached because of the exclusion of important sensitivity-limiting steps of the assay, such as nucleic acid extraction and reverse transcription. For the first time, the sensitivity of an RT-PCR-based detection system was systematically calculated using a viral RNA standard.
By the introduction of a novel fluorophor, DY521XL, the application of duplex PCR with two TaqMan probes on the LightCycler platform is possible. Hitherto, because of limited excitation wavelength on this real-time thermocycler, common fluorophors, such as JOE, VIC, and TET, cannot be used. TaqMan probes have the advantage that they need shorter target sequences than LightCycler hybridization probes. This is of importance for variable targets, such as viral genomes, in which different subtypes or strains have to be detected.
The costs of the NV RT-PCR reagents are comparable with those of other real-time RT-PCR assays and are in the range of 2 to 5 U.S. dollars depending on the real-time platform used. At present, the costs for synthesis of LNA probes are in fact twice as much as those for synthesis of conventional TaqMan probes. Only small amounts of LNA probe are used per assay; consequently, this item has a minor influence on the total costs.
In conclusion, as long as NVs cannot be grown in cell culture, quantification by real-time PCR by using an appropriate primer-specific internal standard is the only option. Our real-time RT-PCR assay described above for the detection and quantification of norovirus in fecal and environmental specimens is fast, highly sensitive, reproducible, and highly specific. The evaluated primer and probe system makes it possible to use the assay on different real-time platforms. After preparation of the viral nucleic acids, amplification and detection can be achieved in less than 2 h.
ACKNOWLEDGMENTS
This study was supported by the German working group "Food-borne Viruses" (Arbeitsgruppe Lebensmittelassozierte Viren).
Norovirus isolates were kindly supplied by Kathrin Hartelt and Rainer Oehme (Landesgesundheitsamt Baden-Württemberg, Stuttgart, Germany). We thank Renate Josting and Anke Straeten-Barnbeck for their excellent technical assistance and Sarah L. Kirkby for her linguistic advice.
FOOTNOTES
Corresponding author. Mailing address: Institut für Laboratoriums und Transfusionsmedizin, Herz und Diabeteszentrum Nordrhein-Westfalen, Universittsklinik der Ruhr-Universitt Bochum, Georgstrasse 11, D-32545 Bad Oeynhausen, Germany. Phone: 49-5731-97 1390. Fax: 49-5731-97 2307. E-mail: jdreier@hdz-nrw.de.
REFERENCES
Ando, T., J. S. Noel, and R. L. Fankhauser. 2000. Genetic classification of "Norwalk-like viruses." J. Infect. Dis. 181(Suppl. 2):336-348.
Ando, T., S. S. Monroe, J. R. Gentsch, Q. Jin, D. C. Lewis, and R. I. Glass. 1995. Detection and differentiation of antigenically distinct small round-structured viruses (Norwalk-like viruses) by reverse transcription-PCR and Southern hybridization. J. Clin. Microbiol. 33:64-71.
Anonymous. 2005. Microbiology of food and animal feeding stuffs. Polymerase chain reaction (PCR) for the detection of food-borne pathogens. Requirements for amplification and detection for qualitative methods. Draft International Standard ISO/FDIS 20838:2005. DIN, Berlin, Germany.
Anonymous. 2005. Microbiology of food and animal feeding stuffs. Polymerase chain reaction (PCR) for the detection of food-borne pathogens. General requirements and definitions. Draft International Standard ISO 22174:2005. DIN, Berlin, Germany.
Atmar, R. L., F. H. Neill, J. L. Romalde, F. Le Guyader, C. M. Woodley, T. G. Metcalf, and M. K. Estes. 1995. Detection of Norwalk virus and hepatitis A virus in shellfish with the PCR. Appl. Environ. Microbiol. 61:3014-3018.
Beuret, C., D. Kohler, A. Baumgartner, and T. M. Luthi. 2002. Norwalk-like virus sequences in mineral waters: one-year monitoring of three brands. Appl. Environ. Microbiol. 68:1925-1931.
Bresee, J. S., M. A. Widdowson, S. S. Monroe, and R. I. Glass. 2002. Foodborne viral gastroenteritis: challenges and opportunities. Clin. Infect. Dis. 35:748-753.
Cleland, A., P. Nettleton, L. Jarvis, and P. Simmonds. 1999. Use of bovine viral diarrhoea virus as an internal control for amplification of hepatitis C virus. Vox Sang. 76:170-174.
Costa, J. M., P. Ernault, M. Olivi, T. Gaillon, and K. Arar. 2004. Chimeric LNA/DNA probes as a detection system for real-time PCR. Clin. Biochem. 37:930-932.
Cromeans, T. L. 1997. Understanding and preventing virus transmissions via foods. Food Technol. 51:20.
Dreier, J., M. Strmer, and K. Kleesiek. 2005. Use of bacteriophage MS2 as an internal control in viral reverse transcription-PCR assays. J. Clin. Microbiol. 43:4551-4557.
Duizer, E., K. J. Schwab, F. H. Neill, R. L. Atmar, M. P. G. Koopmans, and M. K. Estes. 2004. Laboratory efforts to cultivate noroviruses. J. Gen. Virol. 85:79-87.
European Commission. 2002. Opinion of the Scientific Committee on Veterinary Measures Relating to Public Health on Norwalk-like Viruses. [Online.] http://www.crlcefas.org/InformationCentre/docs/scvnorwalklikevirus.pdf.
Fankhauser, R. L., S. S. Monroe, J. S. Noel, C. D. Humphrey, J. S. Breeze, U. D. Parashar, T. Ando, and R. I. Glass. 2002. Epidemiologic and molecular trends of "Norwalk-like viruses" associated with outbreaks of gastroenteritis in the United States. J. Infect. Dis. 186:1-7.
Green, K. Y., G. Belliot, J. L. Taylor, J. Valdesuso, J. F. Lew, A. Z. Kapikian, and F. Y. C. Lin. 2002. A predominant role for Norwalk-like viruses as agents of epidemic gastroenteritis in Maryland nursing homes for the elderly. J. Infect. Dis. 185:133-146.
Hhne, M., and E. Schreier. 2004. Detection and characterization of norovirus outbreaks in Germany: application of a one-tube RT-PCR using a fluorogenic real-time detection system. J. Med. Virol. 72:312-319.
Hummelshoj, L., L. P. Ryder, H. O. Madsen, and L. K. Poulsen. 2005. Locked nucleic acid inhibits amplification of contaminating DNA in real-time PCR. BioTechniques 38:605-610.
Jothikumar, N., J. A. Lowther, K. Henshilwood, D. N. Lees, V. R. Hill, and J. Vinje. 2005. Rapid and sensitive detection of noroviruses by using TaqMan-based one-step reverse transcription-PCR assays and application to naturally contaminated shellfish samples. Appl. Environ. Microbiol. 71:1870-1875.
Kageyama, T., S. Kojima, M. Shinohara, K. Uchida, S. Fukushi, F. B. Hoshino, N. Takeda, and K. Katayama. 2003. Broadly reactive and highly sensitive assay for Norwalk-like viruses based on real-time quantitative reverse transcription-PCR. J. Clin. Microbiol. 41:1548-1557.
Karst, S. M., C. E. Wobus, M. Lay, J. Davidson, and H. W. Virgin IV. 2003. STAT1-dependent innate immunity to a Norwalk-like virus. Science 299:1575-1578.
Kingsley, D. H., G. K. Meade, and G. P. Richards. 2002. Detection of both hepatitis A virus and Norwalk-like virus in imported clams associated with food-borne illness. Appl. Environ. Microbiol. 68:3914-3918.
Le Guyader, F., M. K. Estes, S. S. Monroe, T. Ando, and R. L. Atmar. 1996. Detection and analysis of a small round-structured virus strain in oysters implicated in an outbreak of acute gastroenteritis. Appl. Environ. Microbiol. 62:4268-4272.
Letertre, C., S. Perelle, F. Dilasser, K. Arar, and P. Fach. 2003. Evaluation of the performance of LNA and MGB probes in 5'-nuclease PCR assays. Mol. Cell. Probes 17:307-311.
Loisy, F., R. L. Atmar, P. Guillon, P. Le Cann, M. Pommepuy, and F. S. Le Guyader. 2005. Real-time RT-PCR for norovirus screening in shellfish. J. Virol. Methods 123:1-7.
Lopman, B. A., M. H. Reacher, Y. van Duijnhoven, F. X. Hanon, D. Brown, and M. Koopmans. 2003. Viral gastroenteritis outbreaks in Europe, 1995-2000. Emerg. Infect. Dis. 9:90-96.
Mde, D., S. Kahle, and K. Trübner. 2005. Detection of norovirus in food and environmental samples by RT-PCR. Application in routine diagnostics. Arch. Lebensmittelhyg. 56:1-24.
Mde, D., R. Reiting, J. Dreier, B. Heun-Münch, U. Loss, M. Hagen, S. Burkhardt, C. Schrader, and B. Becker. 2006. Detection of norovirus in environmental samples. Results of a ring trial for method development. Arch. Lebensmittelhyg. 57:29-35.
Mead, P. S., L. Slutsker, V. Dietz, L. F. McCaig, J. S. Breese, C. Shapiro, P. M. Griffin, and R. V. Tauxe. 1999. Food-related illness and death in the United States. Emerg. Infect. Dis. 5:607-625.
Neisters, H. G. M. 2002. Clinical virology in real time. J. Clin. Virol. 25:S3-S12.
Page, R. D. 1996. TreeView: an application to display phylogenetic trees on personal computers. Comput. Appl. Biosci. 12:357-358.
Patterson, W., P. Haswell, P. T. Fryers, and J. Green. 1997. Outbreak of small round structured virus gastroenteritis arose after kitchen assistant vomited. Commun. Dis. Rep. CDR Rev. 7:101-103.
Pnk, A., L. Maunula, C. H. von Bonsdorff, and O. Lyytikinen. 1999. Outbreak of calicivirus gastroenteritis associated with eating frozen raspberries. Eurosurveillance 4:66-69.
Pusch, D., D.-Y. Oh, S. Wolf, R. Dumke, U. Schrter-Bobsin, M. Hhne, I. Rske, and E. Schreier. 2005. Detection of enteric viruses and bacterial indicators in German environmental waters. Arch. Virol. 150:929-947.
Richards, A. F., B. Lopman, A. Gunn, A. Curry, D. Ellis, H. Cotterill, S. Ratcliffe, M. Jenkins, H. Appleton, C. I. Gallimore, J. J. Gray, and D. W. Brown. 2003. Evaluation of a commercial ELISA for detecting Norwalk-like virus antigen in faeces. J. Clin. Virol. 26:109-115.
Richards, G. P., M. A. Watson, R. L. Fankhauser, and S. S. Monroe. 2004. Genogroup I and II noroviruses detected in stool samples by real-time reverse transcription-PCR using highly degenerate universal primers. Appl. Environ. Microbiol. 70:7179-7184.
Schwab, K. J., F. H. Neill, R. L. Fankhauser, N. A. Daniels, S. S. Monroe, D. A. Bergmire-Sweat, M. K. Estes, and R. L. Atmar. 2000. Development of methods to detect "Norwalk-like viruses" (NLVs) and hepatitis A virus in delicatessen foods: application to a food-borne NLV outbreak. Appl. Environ. Microbiol. 66:213-218.
Schwab, K. J., F. H. Neill, F. Le Guyader, M. K. Estes, and R. L. Atmar. 2001. Development of a reverse transcription-PCR-DNA enzyme immunoassay for detection of "Norwalk-like" viruses and hepatitis A virus in stool and shellfish. Appl. Environ. Microbiol. 67:742-749.
Vinje, J., and M. P. Koopmans. 1996. Molecular detection and epidemiology of small round-structured viruses in outbreaks of gastroenteritis in the Netherlands. J. Infect. Dis. 174:610-615.(Jens Dreier, Melanie Strmer, Dietrich Md)
Landesamt für Verbraucherschutz Sachsen-Anhalt, Halle, Germany
Berliner Betrieb für Zentrale Gesundheitliche Aufgaben (BBGes), Institut für Lebensmittel, Arzneimittel und Tierseuchen (ILAT), Berlin, Germany
ABSTRACT
We have developed a one-tube reverse transcription (RT)-PCR method using the real-time TaqMan PCR system for the detection of norovirus genogroup I (NV GGI). By introduction of a novel probe based on locked nucleic acid technology, we enhanced the sensitivity of the assay compared to those of conventional TaqMan probes. The sensitivity of the NV GGI RT-PCR was determined by probit analysis with defined RNA standards and quantified norovirus isolates to 711 copies/ml (95% detection limit). In order to detect PCR inhibition, we included a heterologous internal control (IC) system based on phage MS2. This internally controlled RT-PCR was tested on different real-time PCR platforms, LightCycler, Rotorgene, Mastercycler EP realplex, and ABI Prism. Compared to the assay without an IC, the duplex RT-PCR exhibited no reduction in sensitivity in clinical samples. In combination with an established NV GGII real-time RT-PCR, we used the novel assay in a routine assay for diagnosis of clinical and food-borne norovirus infection. We applied this novel assay to analyze outbreaks of nonbacterial acute gastroenteritis. Norovirus of GGI was detected in these outbreaks. Sequence and similarity plot analysis of open reading frame 1 (ORF1) and ORF2 showed two genotypes, GGI/2 and GGI/4, in semiclosed communities.
INTRODUCTION
Noroviruses (NVs) are one of the major causes of gastroenteritis in humans (2, 15, 25). They are transmitted both directly, via person-to-person contact, and indirectly by environmental contamination (31), via foods and drinking water (5, 13, 21, 22, 28, 32, 36, 37). The number of cases which can be attributed to food- or waterborne transmission is not known exactly (10). The percentage ranges between 16% (13) and 40% to 57% (14, 34).
The norovirus comprises five distinct genogroups (1, 20). The most prevalent circulating strains belong to genogroup II (GGII), responsible for up to 90% of NV outbreaks in Germany (16). The genogroup I (GGI) strains cause about one quarter (16) to 1/10 (18) of all NV-positive outbreaks.
In general, there are immunological, electron microscopic, tissue culture, and molecular methods to detect viruses in food. But, up to now, the major obstacles to laboratory diagnosis are the lack of a tissue culture system for propagating the viruses (12, 19) and the limited sensitivity of recently developed immunological assays. Therefore, molecular methods have to be the assay of choice for their detection. The aim should be a detection system which is applicable to stool samples as well as to food and environmental samples. The special sensitivity requirements are highly demanding because, on the one hand, the number of virus particles shed can vary between 102 and 1010 genome equivalents (16) and, on the other hand, the investigation of environmental samples requires a sensitivity corresponding to that of the low infectious dose. The minimal infectious dose is about 10 to 100 infectious virus particles (7, 13). Based on the minimal infectious dose, the detection limit should be fewer than 1,000 virus particles in food and environmental samples. At present, immunological and electron microscopic methods cannot be used because of their lack of sensitivity (34).
Real-time PCR systems using specific probes (16, 19, 20, 24, 33) or SYBR green (35) as a detection system have recently been developed. According to the European standard (3), a diagnostic PCR with food and environmental samples requires sequence-specific verification of the PCR product by restriction enzymes, probe hybridization, or sequencing. Melting curve analysis is not considered to be equivalent. For routine diagnostic applications, probe hybridization in real-time PCR seems to be the most promising system. Recent multilaboratory studies show that the sensitivity of real-time reverse transcription (RT)-PCR systems to detect GGI is lower than that of systems for the detection of GGII in environmental samples (27). This raises the need for a very sensitive real-time RT-PCR detection system using specific probes for the detection of NV GGI.
Parallel to the norovirus-specific detection reaction, the amplificability of the nucleic acids extracted shall be verified by an amplification control (4). This facilitates, on the one hand, a check for coextracted RT-PCR inhibitors and, on the other hand, a verification of the nucleic acid extraction efficiency when applied as a process control from the beginning of the nucleic acid extraction. Phage RNA can be used for this purpose (11). In this study, we present a real-time RT-PCR assay for the detection of NV GGI.
MATERIALS AND METHODS
Processing of stool specimens. We used real-time RT-PCR to screen stool samples from patients with nonbacterial acute gastroenteritis. All of the samples had already been proved to be negative for Salmonella, Shigella, Campylobacter, and Yersinia species by conventional bacterial culture procedure. Screening for group A rotaviruses was performed with the Ridascreen rotavirus enzyme-linked immunosorbent assay (R-Biopharm, Darmstadt, Germany). Fecal specimens from eight gastroenteritis outbreaks occurring in Berlin, Germany, in 2004 were used for this study. Samples were assayed for NV GGI and GGII by real-time RT-PCR. NV GGI RT-PCR was carried out using primers NV192 and NV193 (33) and probes Ring1(a) and Ring1(b) (19), as described previously (26). NV GGII RT-PCR was performed using primers NV107a and NV117 and probe TM6 (16) in a separate reaction. Positive screening results were confirmed by an alternative amplification system as described previously (6, 35) and by DNA sequencing of PCR products.
Nucleic acid isolation. A 10% (wt/vol) stool suspension was prepared with phosphate-buffered saline (PBS) and clarified by centrifugation at 3,000 x g for 20 min. For probing of food or environmental surfaces, we used sterile, rayon-tipped swabs with a plastic stem (Sarstedt, Nümbrecht, Germany). Spiked swabs, used for the validation of the application of the phage MS2 as a process control, were squeezed out three times in 500 μl of PBS before use as suspension in RNA extraction. RNA was extracted from 140 μl to 280 μl of PBS suspension with the QIAamp viral RNA mini kit (QIAGEN, Hilden, Germany) according to the manufacturer's protocol. The RNA was eluted with 60 μl AVE buffer (QIAGEN).
Food surfaces (from solid foods and fruits in which surface contamination is possible) were swabbed with a moistened sterile swab. Swabbing of an area with a size of about 100 cm2 was done very intensively to collect the majority of particles adhering to the surface. Virus particles adhering to the swabs were eluted in 500 μl PBS. The swab was used again to remove virus particles from the next 100 cm2 of the surface. In this way, particles from the whole surface were collected in 500 μl PBS and nucleic acid extraction was performed as described above.
To obtain virus particles from hackled foods, ground foods, or composed foods, 5 g of samples was placed into a stomacher bag including a net. After 5 to 10 ml of PBS was added, depending on the water content of the food, samples were processed for 1 min in a stomacher. The samples were incubated in PBS for 24 to 48 h at 4°C. The samples were processed several times in the stomacher bag. The liquid phase was transferred into a centrifugation tube. Coarse particles were sedimented by centrifugation for 10 min at 1,000 x g. The supernatant was filtered through a 0.2-μm syringe filter. Four milliliters of the clear filtrate was transferred into a 4-ml Vivaspin concentrator, 50000 MWCO (Vivasciences, Hannover, Germany). The samples were centrifuged at 15°C for 20 min at 3,000 x g. The centrifugation time was extended until a final volume of 150 μl was reached. A total of 140 μl of the concentrated solution was used for RNA extraction. In cases in which the samples could not be concentrated to the volume of 150 μl, 280 μl of the concentrated medium was used with double the amount of the extraction reagents of the QIAamp viral RNA mini kit.
Conventional RT-PCR. RT-PCR was performed to amplify sequences of the capsid gene of NV GGI and GGII, using Superscript III one-step RT-PCR with a Platinum Taq kit (Invitrogen, Karlsruhe, Germany). RT-PCR using the primer set Mon431, Mon432, Mon433, and Mon434 described by Beuret et al. (6) and Richards et al. (35) was carried out with the reaction mixture preparations as recommended by the manufacturer. The cycling conditions were as follows: reverse transcription at 50°C for 30 min and, for PCR, initial denaturation for 2 min at 95°C, 45 amplification cycles with denaturation for 30 s at 95°C, annealing for 1 min at 50°C, extension for 1 min at 72°C, and a final cycle of incubation at 72°C for 10 min.
For amplification of the RNA polymerase gene, RT-PCR using primers JV12 and JV13 (38) was performed under the same PCR conditions as those used for amplification of the capsid gene.
MS2 phage RT-PCR. Phage MS2 DSM13767 was purchased from DSMZ (Braunschweig, Germany). MS2 phage lysates were prepared as described previously (11), and the number of PFU was determined by plating assays. The MS2 RNA was quantified (copies per ml) with a genomic MS2 RNA purchased from Roche Diagnostics (Mannheim, Germany). Stool PBS suspensions were spiked with MS2 phage lysate containing 6 x 104 PFU per ml (final concentration, 3,000 PFU MS2 per ml of PBS), and RNA extraction was performed as described above. MS2 real-time RT-PCR was used for internal control of norovirus RT-PCR. The primer system MS2-TM2 with primers MS2-TM3-F and MS2-TM3-R and probe MS2-TM2JOE was used as described previously (11). For detection on the LightCycler platform, the MS2 probe was labeled with reporter dye DY521XL (Dyomics GmbH, Jena, Germany), and detection was performed in channel F3 (705 nm). This reporter fluorophor has an absorption maximum at 523 nm and an emission maximum at 668 nm.
Real-time PCR system. A Superscript III one-step RT-PCR with a Platinum Taq kit (Invitrogen, Karlsruhe, Germany) was used as the basis for the reaction mixture in the LightCycler RT-PCR assay. A volume of 20 μl was used in each reaction capillary, containing 5 μl of the extracted RNA and 15 μl of the reaction mixture: 1x reaction mix (including 3 mM MgSO4; Invitrogen), 500 ng per μl nonacetylated bovine serum albumin (Sigma-Aldrich, Taufkirchen, Germany), 400 nM of forward primer NV192, 400 nM of reverse primer NV193, 150 nM of probe NVGG1-LNA (6-carboxyfluorescein [FAM]-ATTCGGGCAGGAGAT-EclipseDQ [Eurogentec, Seraing, Belgium]; locked nucleic acid [LNA] nucleotides in underlined positions), and 0.6 μl RT/Platinum Taq mix (Invitrogen). In addition to the positive run control, each test run included one no-target control containing 15 μl of the reaction mixture and 5 μl PCR-grade water. The reaction capillaries were capped, centrifuged, and placed into the carousel of the LightCycler instrument (Roche Diagnostics). The LightCycler RT-PCR protocol included the following parameters: reverse transcription at 50°C for 10 min and, for PCR, initial denaturation and Taq DNA polymerase activation for 2 min at 95°C, subsequent 45 cycles at 95°C for 2 s, annealing and fluorescence detection at 56°C for 10 s, and extension at 72°C for 15 s. Data were obtained during the annealing period in the "single" mode with the channel setting F1.
The RT-PCR assays with the Rotorgene 2000 or 3000 cycler system (Corbett Research, Sydney, Australia), Mastercycler EP realplex (Eppendorf, Wesseling-Berzdorf, Germany), and ABI Prism 7700 (Applied Biosystems, Darmstadt, Germany) were performed in a volume of 50 μl, including 20 μl nucleic acid extract. The reaction mixture was as described above but without bovine serum albumin. Cycling conditions were 50°C for 10 min and 95°C for 2 min, followed by 45 cycles at 95°C for 5 s, annealing and fluorescence detection at 56°C for 20 s, and extension at 72°C for 20 s.
Construction of a norovirus RNA standard. NV GGI PCR products of 287 bp flanked by primers Mon431/Mon432 (6, 35) and NV193 (33) were cloned into Escherichia coli by use of the plasmid vector pCRII-TOPO, which was supplied by a TA-TOPO cloning kit (Invitrogen), according to the protocol provided by the supplier. NV GGII PCR products, generated with primers Mon431/Mon432 and NV117 (16), were cloned in E. coli using the TA-TOPO cloning kit.
Analysis of the recombinant plasmids was performed by extracting plasmid DNA from small-volume cultures grown overnight at 37°C using the QIAprep spin plasmid kit (QIAGEN). PCR with universal M13 primers was applied to amplify the inserted DNA fragment, including phage promoters T7 and SP6. Purified PCR products were used for runoff transcription using the SP6/T7 transcription kit (Roche Diagnostics). RNA concentrations were determined by a fluorometric analysis with the RiboGreen RNA quantitation reagent (Molecular Probes, Leiden, The Netherlands). Defined RNA copies were used to quantify NV isolates. For this purpose, RNA standards were extracted in parallel with NV dilutions, and nucleic acids were subjected to quantitative real-time RT-PCR. NV standards in the range from 10 to 1 x 107 copies per PCR were used as standards in real-time PCR assays.
Probit analysis of experimental data. The 95% detection limit was calculated by probit analysis using SPSS 11.0 software (SPSS GmbH Software, Munich, Germany).
DNA sequencing and phylogenetic analysis. PCR products of open reading frame 1 (ORF1) and ORF2 were sequenced directly with a BigDye Terminator v 1.1 cycle sequencing kit (Applied Biosystems) in both directions using an ABI Prism 310 sequencer (Applied Biosystems). Multiple sequence alignments were performed with the CLUSTAL W program, version 1.8. Phylogenetic dendrograms were constructed by the neighbor-joining method (PHYLIP), and phylogenetic trees were drawn using the TreeView program (30).
Nucleotide sequence accession numbers. Sequence data of ORF1 (RNA polymerase gene) and ORF2 (capsid gene) have been submitted to GenBank and assigned accession numbers DQ340076 to DQ340091.
RESULTS
Screening for NV and genogrouping by real-time RT-PCR. Fecal specimens from gastroenteritis outbreaks were screened for norovirus GGI and GGII by the use of real-time RT-PCR (16, 33). Because of reports of false-negative results with GGII/2 strains (Melksham) responsible for gastroenteritis outbreaks in two Bavarian youth hostels using this primer system (A. Carl, personal communication), we replaced the forward primer NV107a (16) with primer COG2F (19). DNA sequence analysis of GGII/2 isolates and sequence alignments revealed four mismatches in the primer NV107a (data not shown).
In order to identify inhibitions of the enzymatic reactions, we used the MS2 RT-PCR system as an external control reaction with the same nucleic acid extracts as those used for NV RT-PCR. For screening of food-borne norovirus contamination, the screening strategy was used as described above. Systematically, we evaluated the assay for food surfaces from solid foods and fruits in which surface contamination is possible (data not shown).
Development of novel real-time RT-PCR for NV genogroup I. Several NV GGI real-time RT-PCR systems were tested for use in routine NV screening (16, 19, 33). In our hands, the systems described exhibited low sensitivities. This was demonstrated in a multicenter collaborative trial (27). Furthermore, NV GGI-positive specimens were detected only with low fluorescence intensity (see Fig. 3).
We therefore designed a novel NV GGI-specific fluorescent probe in the NV ORF2 region for use in real-time RT-PCR with primers NV192 and NV193 (33). To increase the specificity and sensitivity having a higher specificity and higher melting temperature, we examined the use of LNA TaqMan probes that have a higher melting temperature than conventional TaqMan probes (23). To investigate the performance of the LNA probe, we compared the assay with recently described TaqMan assays (16, 19, 33). We compared the cycle threshold (CT) values and the levels of fluorescence intensity of the amplification curves (Fig. 1). The CT values vary up to 9.8 between the LNA and the conventional TaqMan probes. The TaqMan probes Ring1(a)/Ring1(b) and NV-TM6 revealed the same range of fluorescence intensities and CT values when tested in parallel (data not shown). Furthermore, the LNA probe produced a higher level of fluorescence intensity (Fig. 1). In parallel, a panel of NV GGI clinical samples representing 15 isolates of three different genotypes (GG1/1, GG1/2, and GG1/4) was analyzed with these TaqMan RT-PCR assays and confirmed the lower sensitivity of the conventional TaqMan assays (data not shown). Overall, the novel real-time RT-PCR assay with the LNA probe was 10- to 100-fold more sensitive. Therefore, the use of the LNA probe results in a slight increase in the sensitivity of the assay. To determine the specificity of the NV RT-PCR, we tested several NV GGI and GGII isolates. All NV GGI isolates tested were detected, while NV GGII gave no positive amplification signal. No cross-reaction with other viral pathogens, such as rotavirus, astrovirus, adenovirus, and enterovirus, was observed.
Internal amplification control for the norovirus real-time assay. We developed an internal amplification control (IC) by using E. coli phage MS2 as a target in duplex RT-PCR assays (11). This heterologous IC can be added before nucleic acid isolation as MS2 phage particles or directly prior to amplification as MS2 RNA. The MS2 RNA and norovirus RNA present in the sample were coamplified within the same reaction. In order to exclude competition between NV and MS2 amplification, we compared NV GGI (see Table 2) and NV GGII (data not shown) RT-PCRs with and without coamplification of MS2 RNA. For a process control, we performed MS2 spiking experiments using phage lysates that were quantified as PFU and copies per ml. Food and environmental specimens can be analyzed by using swab samples (26). Therefore, we used swabs to validate the application of the phage MS2 as a process control. Various MS2 phage concentrations were spiked to NV GGII-contaminated swabs. Swab resuspension, RNA extraction, and duplex NV-MS2 RT-PCR on the Rotorgene 3000 were performed (Table 1) to determine the optimal MS2 concentration. In all samples, NV GGII RNA was detected with approximate CT values (range, 29.59 to 30.98), independently of the MS2 phage concentration. The IC was detected only in samples spiked with more than 8,400 PFU per RNA preparation. Therefore, we used 8,400 PFU per extraction for routine testing.
The duplex NV-MS2 RT-PCR was used on different real-time PCR platforms. RT-PCR on the LightCycler was carried out with a DY521XL-labeled probe. This novel fluorophor permits the simultaneous detection of the IC in the F3 channel and reporter fluorophor FAM (for NV target) in the F1 channel without applying a color compensation (Fig. 2). Other fluorescence dyes usually used for TaqMan probes, e.g., JOE (6-carboxy-4',5'-dichloro-2',7'-dimethoxyfluorescein), VIC, and TET (tetrachloro-6-carboxyfluorescein), cannot be used because of the limited fluorescence excitation of the LightCycler's light-emitting diode with a peak emission of 470 nm.
Quantitative detection of NV RNA. In order to estimate the detection limits of the NV RT-PCR assays, we constructed NV RNA standards. Plasmid constructs, which contained a region of the capsid gene, were used for in vitro transcription. The capsid gene region contains the primer sites used for conventional NV RT-PCR (6) and most of the previously described real-time RT-PCR systems (16, 18, 19, 24). After transcription, the integrity of the RNA was confirmed by gel electrophoresis and standardized by fluorometric quantitation. The number of molecules present was calculated by the use of Avogadro's number. No residual DNA was detected in the RNA preparations when PCR was performed without reverse transcription.
For calibration purposes, norovirus stool suspensions and PBS spiked with RNA standards were extracted and amplified in parallel. The NV suspension was quantified on the basis of an external standard curve created from the data produced by the individual amplification of the dilution series of the NV RNA standard.
To determine the analytical sensitivity of the RT-PCR assay, we used human stool samples spiked with different titers of NV GGI from 965 to 2.47 x 105 copies per ml, corresponding to 10 to 4,960 copies of NV GGI per RT-PCR. We compared MS2 internally controlled with uncontrolled RT-PCR. For this purpose, 1 x 104 MS2 phage were added to lysis buffer before RNA extraction. Three experiments with four replicates (n = 12) of each concentration were processed through all steps of nucleic acid isolation and RT-PCR (Table 2). The nucleic acid extracts were processed with the NV GGI RT-PCR assay on the Rotorgene 3000 with and without the addition of the MS2 primer-probe system. The 95% detection limit was calculated by probit analysis to 711 copies per PCR (range, 536 to 1117 copies/PCR) when NV GGI was used without an IC. No significant reduction in sensitivity was observed when NV GGI-MS2 duplex RT-PCR was performed with a 95% detection limit of 770 copies per PCR (range, 569 to 1,277 copies per PCR).
Outbreak investigation. During the year 2004, the Institut für Lebensmittel, Arzneimittel und Tierseuchen (ILAT), Berlin, received 86 fecal specimens from seven suspected outbreaks of NV-associated gastroenteritis. The majority of these outbreaks occurred in either residential homes or nursing homes (Table 3). NV GGI was detected as the etiological agent in all outbreaks by real-time RT-PCR. No other gastroenteritis-associated pathogen was identified in specimens collected from these outbreaks. Eight samples from these outbreaks were used to compare the GGI LNA real-time RT-PCR system, as described previously (27), using the oligonucleotides NV192/NV193 (33) and Ring1(a)/Ring1(b) as described by Kageyama et al. (19). With the LNA probe system, all suspect samples yielded positive results, whereas the RT-PCR using the Ring1(a)/Ring1(b) probe missed three of the eight strains (data not shown).
DNA sequence analyses of the RNA-dependent RNA polymerase gene (ORF1) and capsid gene (ORF2) regions were performed (Fig. 3). NV GGI strains were separated into two genotypes (GGI/2 [Southampton] and GGI/4 [Chiba]). Viral sequences determined from samples of patients in the same outbreak were highly homogenous. Single genotypes were observed in all outbreaks that occurred in semiclosed communities. Furthermore, three clusters of GGI/2 strain and one cluster of GGI/4 strain were detected in association with regional outbreaks. The local public health departments (BA 1 to 4) were responsible for sample collection. As an exception, isolates 91568.04-6 (BA 3) and 90068.04-8 (BA 4) were collected from different sources, but an association between these two cases cannot be excluded. In all cases, person-to-person spreading of the infection was obvious, while food-borne NV transmission was not observed.
DISCUSSION
Increasing attention has been paid to viral gastroenteritis outbreaks during the last few years. The development of molecular methods has shown that norovirus is one of the most common causes of gastroenteritis outbreaks in adults and children (16). The increasing number of outbreaks results in increasing numbers of samples that have to be handled in diagnostic laboratories. Therefore, RT-PCR seems to be the method of choice because it combines a potentially high throughput with reproducibility, sensitivity, and specificity.
Therefore, we established a sensitive NV RNA detection system using real-time RT-PCR to quantitatively detect NV RNA. The system does not require any other detection procedure, such as electrophoresis or nested PCR. We describe a real-time RT-PCR assay for NV GGI which is based on LNA probe technology. This assay should be applicable for both stool and food analyses. The introduction of LNA improves the hybridization affinity for complementary sequences, increases the melting temperature by several degrees, and therefore improves the specificity of the assay. LNA probes are ribonucleotides containing a methylene bridge that connects the 2' oxygen of ribose with the 4' carbon. This bridge results in a locked 3'-endo conformation that reduces the conformational flexibility of the ribose. The introduction of LNA into a DNA oligomer improves the hybridization affinity and increases the melting temperature. The increased stability allows the use of shorter probes which are more sensitive to single-base mismatches. The shorter length gave LNA probes better sequence specificity and lower fluorescent background than conventional probes (17). This observation may be attributed to a better efficiency of PCR when shorter detection probes are used, due to lower interference during the amplification process (9). Even for highly variable sequence regions, the stabilizing effect of LNA nucleotides can improve the detection of different isolates of NV GGI. The LNA nucleotides in primers or probes for PCR should be directed to highly conserved positions of the NV target.
An inherent problem in diagnostic PCR is the presence of amplification inhibitors which may cause false-negative results. Therefore, the amplificability of nucleic acids should be verified by an amplification control. In this study, the MS2 phage was successfully used as an internal amplification control in NV RT-PCRs. The use of intact viruses for external standards in absolute quantification assays or a positive control is preferred (8, 29), but the risk of infection for laboratory workers has to be considered when human- or animal-pathogenic viruses are used. Our approach avoids these disadvantages by using Escherichia coli F-specific phage MS2 as a target for the IC. The use of MS2 phage to control clinical diagnostic nucleic acid amplification tests was demonstrated (11). Feline calicivirus was discussed as well as process controls. Generally, we think that using an infectious human or animal pathogen is not applicable as a process control or amplification control in routine analysis. In analyzing food samples, MS2 phage is well suited as a process control. The vast majority of foods analyzed in routine diagnostics do not contain significant amounts of E. coli. Furthermore, legal requirements define an upper limit of 100 CFU per g; the number of E. coli strains carrying F pili is lower again. There is only a negligible risk for naturally occurring MS2 in food. Even the unlikely case of a higher amount of MS2 is acceptable, because this could lead to a gradual underestimation of RT-PCR inhibitors or to an overestimation of the extraction efficiency if the phage is used as a process control. Compared to the advantages of using phage MS2, this relatively low analytical uncertainty should be tolerable. Generally, high titers of noroviruses are observed with acute gastroenteritis.
The sensitivity of NV RT-PCR was determined using different approaches. Up to now, the detection limit of a quantitative NV RT-PCR assay has been calculated only by using plasmid standards (16, 19). Those methods, defining their analytical detection limit by using DNA, therefore seem to be highly sensitive. But, a quantitatively exact conclusion about the viral load of the sample cannot be reached because of the exclusion of important sensitivity-limiting steps of the assay, such as nucleic acid extraction and reverse transcription. For the first time, the sensitivity of an RT-PCR-based detection system was systematically calculated using a viral RNA standard.
By the introduction of a novel fluorophor, DY521XL, the application of duplex PCR with two TaqMan probes on the LightCycler platform is possible. Hitherto, because of limited excitation wavelength on this real-time thermocycler, common fluorophors, such as JOE, VIC, and TET, cannot be used. TaqMan probes have the advantage that they need shorter target sequences than LightCycler hybridization probes. This is of importance for variable targets, such as viral genomes, in which different subtypes or strains have to be detected.
The costs of the NV RT-PCR reagents are comparable with those of other real-time RT-PCR assays and are in the range of 2 to 5 U.S. dollars depending on the real-time platform used. At present, the costs for synthesis of LNA probes are in fact twice as much as those for synthesis of conventional TaqMan probes. Only small amounts of LNA probe are used per assay; consequently, this item has a minor influence on the total costs.
In conclusion, as long as NVs cannot be grown in cell culture, quantification by real-time PCR by using an appropriate primer-specific internal standard is the only option. Our real-time RT-PCR assay described above for the detection and quantification of norovirus in fecal and environmental specimens is fast, highly sensitive, reproducible, and highly specific. The evaluated primer and probe system makes it possible to use the assay on different real-time platforms. After preparation of the viral nucleic acids, amplification and detection can be achieved in less than 2 h.
ACKNOWLEDGMENTS
This study was supported by the German working group "Food-borne Viruses" (Arbeitsgruppe Lebensmittelassozierte Viren).
Norovirus isolates were kindly supplied by Kathrin Hartelt and Rainer Oehme (Landesgesundheitsamt Baden-Württemberg, Stuttgart, Germany). We thank Renate Josting and Anke Straeten-Barnbeck for their excellent technical assistance and Sarah L. Kirkby for her linguistic advice.
FOOTNOTES
Corresponding author. Mailing address: Institut für Laboratoriums und Transfusionsmedizin, Herz und Diabeteszentrum Nordrhein-Westfalen, Universittsklinik der Ruhr-Universitt Bochum, Georgstrasse 11, D-32545 Bad Oeynhausen, Germany. Phone: 49-5731-97 1390. Fax: 49-5731-97 2307. E-mail: jdreier@hdz-nrw.de.
REFERENCES
Ando, T., J. S. Noel, and R. L. Fankhauser. 2000. Genetic classification of "Norwalk-like viruses." J. Infect. Dis. 181(Suppl. 2):336-348.
Ando, T., S. S. Monroe, J. R. Gentsch, Q. Jin, D. C. Lewis, and R. I. Glass. 1995. Detection and differentiation of antigenically distinct small round-structured viruses (Norwalk-like viruses) by reverse transcription-PCR and Southern hybridization. J. Clin. Microbiol. 33:64-71.
Anonymous. 2005. Microbiology of food and animal feeding stuffs. Polymerase chain reaction (PCR) for the detection of food-borne pathogens. Requirements for amplification and detection for qualitative methods. Draft International Standard ISO/FDIS 20838:2005. DIN, Berlin, Germany.
Anonymous. 2005. Microbiology of food and animal feeding stuffs. Polymerase chain reaction (PCR) for the detection of food-borne pathogens. General requirements and definitions. Draft International Standard ISO 22174:2005. DIN, Berlin, Germany.
Atmar, R. L., F. H. Neill, J. L. Romalde, F. Le Guyader, C. M. Woodley, T. G. Metcalf, and M. K. Estes. 1995. Detection of Norwalk virus and hepatitis A virus in shellfish with the PCR. Appl. Environ. Microbiol. 61:3014-3018.
Beuret, C., D. Kohler, A. Baumgartner, and T. M. Luthi. 2002. Norwalk-like virus sequences in mineral waters: one-year monitoring of three brands. Appl. Environ. Microbiol. 68:1925-1931.
Bresee, J. S., M. A. Widdowson, S. S. Monroe, and R. I. Glass. 2002. Foodborne viral gastroenteritis: challenges and opportunities. Clin. Infect. Dis. 35:748-753.
Cleland, A., P. Nettleton, L. Jarvis, and P. Simmonds. 1999. Use of bovine viral diarrhoea virus as an internal control for amplification of hepatitis C virus. Vox Sang. 76:170-174.
Costa, J. M., P. Ernault, M. Olivi, T. Gaillon, and K. Arar. 2004. Chimeric LNA/DNA probes as a detection system for real-time PCR. Clin. Biochem. 37:930-932.
Cromeans, T. L. 1997. Understanding and preventing virus transmissions via foods. Food Technol. 51:20.
Dreier, J., M. Strmer, and K. Kleesiek. 2005. Use of bacteriophage MS2 as an internal control in viral reverse transcription-PCR assays. J. Clin. Microbiol. 43:4551-4557.
Duizer, E., K. J. Schwab, F. H. Neill, R. L. Atmar, M. P. G. Koopmans, and M. K. Estes. 2004. Laboratory efforts to cultivate noroviruses. J. Gen. Virol. 85:79-87.
European Commission. 2002. Opinion of the Scientific Committee on Veterinary Measures Relating to Public Health on Norwalk-like Viruses. [Online.] http://www.crlcefas.org/InformationCentre/docs/scvnorwalklikevirus.pdf.
Fankhauser, R. L., S. S. Monroe, J. S. Noel, C. D. Humphrey, J. S. Breeze, U. D. Parashar, T. Ando, and R. I. Glass. 2002. Epidemiologic and molecular trends of "Norwalk-like viruses" associated with outbreaks of gastroenteritis in the United States. J. Infect. Dis. 186:1-7.
Green, K. Y., G. Belliot, J. L. Taylor, J. Valdesuso, J. F. Lew, A. Z. Kapikian, and F. Y. C. Lin. 2002. A predominant role for Norwalk-like viruses as agents of epidemic gastroenteritis in Maryland nursing homes for the elderly. J. Infect. Dis. 185:133-146.
Hhne, M., and E. Schreier. 2004. Detection and characterization of norovirus outbreaks in Germany: application of a one-tube RT-PCR using a fluorogenic real-time detection system. J. Med. Virol. 72:312-319.
Hummelshoj, L., L. P. Ryder, H. O. Madsen, and L. K. Poulsen. 2005. Locked nucleic acid inhibits amplification of contaminating DNA in real-time PCR. BioTechniques 38:605-610.
Jothikumar, N., J. A. Lowther, K. Henshilwood, D. N. Lees, V. R. Hill, and J. Vinje. 2005. Rapid and sensitive detection of noroviruses by using TaqMan-based one-step reverse transcription-PCR assays and application to naturally contaminated shellfish samples. Appl. Environ. Microbiol. 71:1870-1875.
Kageyama, T., S. Kojima, M. Shinohara, K. Uchida, S. Fukushi, F. B. Hoshino, N. Takeda, and K. Katayama. 2003. Broadly reactive and highly sensitive assay for Norwalk-like viruses based on real-time quantitative reverse transcription-PCR. J. Clin. Microbiol. 41:1548-1557.
Karst, S. M., C. E. Wobus, M. Lay, J. Davidson, and H. W. Virgin IV. 2003. STAT1-dependent innate immunity to a Norwalk-like virus. Science 299:1575-1578.
Kingsley, D. H., G. K. Meade, and G. P. Richards. 2002. Detection of both hepatitis A virus and Norwalk-like virus in imported clams associated with food-borne illness. Appl. Environ. Microbiol. 68:3914-3918.
Le Guyader, F., M. K. Estes, S. S. Monroe, T. Ando, and R. L. Atmar. 1996. Detection and analysis of a small round-structured virus strain in oysters implicated in an outbreak of acute gastroenteritis. Appl. Environ. Microbiol. 62:4268-4272.
Letertre, C., S. Perelle, F. Dilasser, K. Arar, and P. Fach. 2003. Evaluation of the performance of LNA and MGB probes in 5'-nuclease PCR assays. Mol. Cell. Probes 17:307-311.
Loisy, F., R. L. Atmar, P. Guillon, P. Le Cann, M. Pommepuy, and F. S. Le Guyader. 2005. Real-time RT-PCR for norovirus screening in shellfish. J. Virol. Methods 123:1-7.
Lopman, B. A., M. H. Reacher, Y. van Duijnhoven, F. X. Hanon, D. Brown, and M. Koopmans. 2003. Viral gastroenteritis outbreaks in Europe, 1995-2000. Emerg. Infect. Dis. 9:90-96.
Mde, D., S. Kahle, and K. Trübner. 2005. Detection of norovirus in food and environmental samples by RT-PCR. Application in routine diagnostics. Arch. Lebensmittelhyg. 56:1-24.
Mde, D., R. Reiting, J. Dreier, B. Heun-Münch, U. Loss, M. Hagen, S. Burkhardt, C. Schrader, and B. Becker. 2006. Detection of norovirus in environmental samples. Results of a ring trial for method development. Arch. Lebensmittelhyg. 57:29-35.
Mead, P. S., L. Slutsker, V. Dietz, L. F. McCaig, J. S. Breese, C. Shapiro, P. M. Griffin, and R. V. Tauxe. 1999. Food-related illness and death in the United States. Emerg. Infect. Dis. 5:607-625.
Neisters, H. G. M. 2002. Clinical virology in real time. J. Clin. Virol. 25:S3-S12.
Page, R. D. 1996. TreeView: an application to display phylogenetic trees on personal computers. Comput. Appl. Biosci. 12:357-358.
Patterson, W., P. Haswell, P. T. Fryers, and J. Green. 1997. Outbreak of small round structured virus gastroenteritis arose after kitchen assistant vomited. Commun. Dis. Rep. CDR Rev. 7:101-103.
Pnk, A., L. Maunula, C. H. von Bonsdorff, and O. Lyytikinen. 1999. Outbreak of calicivirus gastroenteritis associated with eating frozen raspberries. Eurosurveillance 4:66-69.
Pusch, D., D.-Y. Oh, S. Wolf, R. Dumke, U. Schrter-Bobsin, M. Hhne, I. Rske, and E. Schreier. 2005. Detection of enteric viruses and bacterial indicators in German environmental waters. Arch. Virol. 150:929-947.
Richards, A. F., B. Lopman, A. Gunn, A. Curry, D. Ellis, H. Cotterill, S. Ratcliffe, M. Jenkins, H. Appleton, C. I. Gallimore, J. J. Gray, and D. W. Brown. 2003. Evaluation of a commercial ELISA for detecting Norwalk-like virus antigen in faeces. J. Clin. Virol. 26:109-115.
Richards, G. P., M. A. Watson, R. L. Fankhauser, and S. S. Monroe. 2004. Genogroup I and II noroviruses detected in stool samples by real-time reverse transcription-PCR using highly degenerate universal primers. Appl. Environ. Microbiol. 70:7179-7184.
Schwab, K. J., F. H. Neill, R. L. Fankhauser, N. A. Daniels, S. S. Monroe, D. A. Bergmire-Sweat, M. K. Estes, and R. L. Atmar. 2000. Development of methods to detect "Norwalk-like viruses" (NLVs) and hepatitis A virus in delicatessen foods: application to a food-borne NLV outbreak. Appl. Environ. Microbiol. 66:213-218.
Schwab, K. J., F. H. Neill, F. Le Guyader, M. K. Estes, and R. L. Atmar. 2001. Development of a reverse transcription-PCR-DNA enzyme immunoassay for detection of "Norwalk-like" viruses and hepatitis A virus in stool and shellfish. Appl. Environ. Microbiol. 67:742-749.
Vinje, J., and M. P. Koopmans. 1996. Molecular detection and epidemiology of small round-structured viruses in outbreaks of gastroenteritis in the Netherlands. J. Infect. Dis. 174:610-615.(Jens Dreier, Melanie Strmer, Dietrich Md)