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Real-time molecular beacon NASBA for rapid and sensitive detection of norovirus GII in clinical samples Safaa Lamhoujeb, Hugues Charest, Ismail Fliss, Solange Ngazoa, and Julie Jean

Abstract: To improve the sensitivity and efficiency of the real-time nucleic acid sequence based amplification (NASBA) assay targeting the open reading frame 1–2 (ORF1–ORF2) junction of the norovirus (NoV) genome, a selection of clinical samples were analyzed. The assay results were compared with those of TaqMan and conventional reverse transcription PCR (RT-PCR) and a commercial enzyme-linked immunoassay (ELISA) for the specific detection of GII NoV in 96 fecal samples. Based on end-point dilution, the two real-time assays had similar sensitivities (0.01 particle detectable units), two log10 cycles greater than that of conventional RT-PCR. GII NoV was detected in 88.54% of the samples by real-time NASBA, in 86.46% by TaqMan RT-PCR, in 81.25% by conventional RT-PCR, and in 65.7% by ELISA. The two realtime assays were in agreement for 88.5% of the samples. These results demonstrate that real-time NASBA with a molecular beacon probe is highly sensitive, accurate, and specific for NoV detection in clinical samples. Applying this technique to samples with complex matrix and low viral loads, such as food and environmental samples, could be useful for the detection of NoVs and will improve the prevention of NoV outbreaks. Key words: detection, norovirus, real-time NASBA, TaqMan RT-PCR, ELISA. Re´sume´ : Afin d’ame´liorer la sensibilite´ et l’efficacite´ de l’amplification en temps re´el par NASBA (« nucleic acid sequence based amplification ») de la jonction des cadres de lecture ouvert 1 et 2 (ORF1–ORF2) du ge´nome du norovirus (NoV), plusieurs e´chantillons cliniques ont e´te´ analyse´s. Ce test a e´te´ compare´ a` une amplification par TaqMan ou par RT-PCR (« reverse transcription PCR ») conventionnelle et a` un ELISA (« enzyme-linked immunoassay ») commercial utilise´ pour de´tecter spe´cifiquement le GII NoV dans 96 e´chantillons fe´caux. Par des dilutions se´quentielles, les deux essais en temps re´el montrent la meˆme sensibilite´ (0.01 UDP (« particle detectable units »)), deux log10 de plus qu’une RT-PCR conventionnelle. Le GII NoV a e´te´ de´tecte´ dans 88,45 % des e´chantillons par NASBA en temps re´el, dans 86,46 % par RT-PCR TaqMan, 81,25 % par RT-PCR conventionnelle et 65,7 % par ELISA. Les deux essais en temps re´el concordaient dans 88,5 % des e´chantillons. Ces re´sultats de´montrent que la NASBA en temps re´el a` l’aide d’une balise mole´culaire est hautement sensible, fiable et spe´cifique a` la de´tection du NoV dans les e´chantillons cliniques. L’application de cette technique a` des e´chantillons complexes dans lesquels le titre viral est faible, tels les e´chantillons alimentaires ou environnementaux, pourrait eˆtre utile pour de´tecter le NoV et ame´liorera la pre´vention d’e´pide´mies de NoV. Mots-cle´s : de´tection, norovirus, NASBA en temps re´el, RT-PCR TaqMan, ELISA. [Traduit par la Re´daction]

Introduction Noroviruses (NoVs) are the major cause of viral gastroenteritis in humans worldwide and are an important public health concern (Marshall et al. 2003). They are transmitted by the fecal–oral route or by fomites (Lopman et al. 2003). Human NoVs are genetically diverse and classified into five distinct genogroups: GI, GII, and GIV, which can be further divided into genotypes on the basis of sequence information from the genes encoding the viral RNA-dependent RNA polymerase (RdRp) and the capsid protein (Zheng et al. 2006).

Recently, real-time molecular technologies such as TaqMan reverse transcription PCR (RT-PCR) and molecular beacon nucleic acid sequence based amplification (NASBA) have been introduced into clinical diagnostic laboratories. These real-time approaches are time saving and provide more accurate and sensitive detection (Niesters 2002). TaqMan RT-PCR targeting the conserved open reading frame 1–2 (ORF1–ORF2) junction of the NoV genome provides high sensitivity and has allowed the detection of a larger variety of circulating NoV strains (Jothikumar et al. 2005; Loisy et al. 2005; Pang et al. 2005). However, although sen-

Received 22 May 2009. Revision received 24 September 2009. Accepted 5 October 2009. Published on the NRC Research Press Web site at cjm.nrc.ca on 18 December 2009. S. Lamhoujeb. Bureau of Microbial Hazards, Health Canada, Ottawa, ON K1A 0K9, Canada. H. Charest. Laboratoire de sante´ publique du Que´bec, 20045, chemin Sainte-Marie, Sainte-Anne-de-Bellevue, QC H9X 3R5, Canada. I. Fliss, S. Ngazoa, and J. Jean.1 Institut des Nutraceutiques et des aliments fonctionnels, Universite´ Laval, Que´bec, QC G1K 7P4, Canada. 1Corresponding

author (e-mail: [email protected]).

Can. J. Microbiol. 55: 1375–1380 (2009)

doi:10.1139/W09-105

Published by NRC Research Press

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Table 1. Primer and probe oligonucleotides used in this study. Oligonuleotide

Sequence (5’–3’)

Polarity

Location

Reference(s)

Conventional RT-PCR SR46 TGGAATTCCATCGCCCACTGG SR33 TGTCACGATCTCATCATCACC

+ –

4493–4513 4615–4595

Ando et al. 1995 Ando et al. 1995

Real-time RT-PCR COG2F CARGARBCNATGTTYAGRTGGATGAG COG2R TCGACGCCATCTTCATTCACA RING2-TP FAM-TGGGAGGGCGATCGCAATCT-TAMRA

+ – +

5003–5028 5100–5080 5048–5067

Kageyama et al. 2003 Kageyama et al. 2003 Kageyama et al. 2003

Real-time NASBA JJV2F CAAGAGTCAATGTTTAGGTGGATGAG COG2-NasR aattctaatacgactcactatagggagaTCGACGCCATCTTCATTCACA

+ –

5003–5028 5100–5080

Saf1-MBP

+

Jothikumar et al. 2005 Modified from Kageyama et al. 2003; Lamhoujeb et al. 2008 Lamhoujeb et al. 2008

FAM-CCAAGCGGAGGGCGATCGCAATCTGGGCTTGG-Dabcyl

Note: FAM, 6-carboxyfluorescein as the reporter dye; TAMRA, 6-carboxytetramethylrhodamine as the quencher dye; Dabcyl, 4-(4’-dimethylaminophenylazo)benzoic acid as the quencher dye. IUPAC code: R, A or G; B, C, T, U, or G (not A); Y, C, T, or U; N, any base. Lowercase letters indicate the T7 RNA promoter sequence. Underlined nucleotide designations represent the stem structure of the molecular beacon. Nucleotide position (location) based on the Lordsdale (GII) sequence (accession No. X86557).

Fig. 1. TaqMan RT-PCR standard curve for NoV GII detection. Data shown are representative of triplicate experiments. The threshold cycle is the time required for fluorescence to exceed the negative control level. PDU, particle detectable units.

Fig. 2. Real-time NASBA standard curve for Nov GII detection. Data shown are representative of triplicate experiments. The threshold cycle is the time required for fluorescence to exceed the negative control level. PDU, particle detectable units.

sitive and specific for NoV, quantitation of viral RNA by real-time RT-PCR depends mainly on the efficiency of cDNA synthesis by reverse transcriptase (Rutjes et al. 2006). Moreover, this assay requires the Taq polymerase enzyme, which is sensitive to environmental inhibitory factors (Tebbe and Vahjen 1993). Real-time NASBA (bioMe´rieux, Boxtel, The Netherlands) has been used for NoV detection (Patterson et al. 2006; Rutjes et al. 2006). As described in other studies (Cook 2003; Monis and Giglio 2006), NASBA is a technique specially designed for amplifying RNA genomes. The reaction is performed at 41 8C (at which temperature DNA contamination of samples does not affect the efficiency of the process) and does not require Taq polymerase. The commercial Amplification Nuclisens basic kit is easy to use and time saving (bioMe´rieux, Boxtel). Real-time NASBA coupled with a molecular beacon probe has been described as a sensitive and specific assay for NoV detection in clinical and environmental samples (Patterson et al. 2006; Rutjes et al. 2006). Rutjes et al. (2006) reported that factors inhibitory to RT-PCR had little or no effect on the perform-

ance of molecular beacon NASBA for NoV detection in environmental samples. However, the RdRp gene was the amplification target, even though the ORF1–ORF2 junction has been described as the most conserved region for NoV detection (Katayama et al. 2002). More recently, molecular beacon NASBA targeting the most conserved junction (ORF1–ORF2) has been combined with an enzymatic pretreatment for distinguishing infectious from noninfectious NoVs in ready-to-eat food (Lamhoujeb et al. 2008). The authors demonstrated that this technique is very sensitive for NoV detection in food with a complex matrix such as turkey. Since NoVs are so diverse, this real-time NASBA system should be evaluated for NoV detection in fecal samples to detect all genetic variations. The aim of this study was to improve the sensitivity and efficiency of the molecular beacon NASBA assay by targeting the ORF1–ORF2 junction and to compare the method with those commonly used for the detection of NoV in fecal samples, namely TaqMan RT-PCR, conventional RT-PCR, and enzyme-linked immunoassay (ELISA). Published by NRC Research Press

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Table 2. Comparison of the results for NoV GII detection using molecular approaches and ELISA. ELISA Province Que´bec Newfoundland and Labrador Nova Scotia New Brunswick British Columbia Manitoba Total %

No. of specimens 29 13 18 8 19 9 96

Conventional RT-PCR

Real-time RT-PCR

Real-time NASBA

Positive nd 12

Negative nd 1

Positive 22 12

Negative 7 1

Positive 19 13

Negative 10 0

Positive 23 12

Negative 6 1

5 5 16 6 44 65.7

13 3 3 3 23 34.3

15 7 18 4 78 81.25

3 1 1 5 18 18.75

18 8 19 6 83 86.46

0 0 0 3 13 13.54

16 8 19 7 85 88.54

2 0 0 2 11 11.46

Note: nd, not determined. Institutions that provided the analyzed samples: Laboratoire de Sante´ Public du Que´bec (Sainte-Anne de Bellevue, Que´bec), Newfoundland and Labrador Public Health Laboratory (St. John’s, Newfoundland), British Columbia Centre for Disease Control (Vancouver, British Columbia), and Cadham Provincial Laboratory (Winnipeg, Manitoba).

Materials and methods Fecal specimens A panel of 96 fecal samples collected in Canada between 2004 and 2005 were kindly provided by four laboratories: Laboratoire de Sante´ Public du Que´bec (Sainte-Anne de Bellevue, Que´bec), British Columbia Centre for Disease Control (Vancouver, British Columbia), Newfoundland and Labrador Public Health Laboratory (St. John’s, Newfoundland), and Cadham Provincial Laboratory (Winnipeg, Manitoba). This panel of clinical samples was tested in the listed laboratories by conventional RT-PCR and (or) electron microscopy and consisted of positive and negative NoV samples. Each sample was stored as several aliquots frozen at – 80 8C until use. Viral RNA extraction Thawed samples were diluted in an equal volume of minimum essential medium (Sigma-Aldrich Canada Ltd., Oakville, Ontario), mixed using a vortex device, and centrifuged at 4500g for 5 min. The supernatant was mixed with an equal volume of 1,1,2-trichloro-1,2,2-trifluoroethane (Sigma-Aldrich Canada Ltd.) and centrifuged for 5 min at 4500g. Viral RNA was extracted from the aqueous supernatant using the QIAamp Viral RNA minikit (QIAGEN, Mississauga, Ontario) according to the manufacturer’s protocol. The RNA extract was immediately stored at –80 8C until use in RT-PCR assays (using the Eppendorf mastercycler; Eppendorf Canada Inc., Mississauga, Ontario). Conventional RT-PCR amplification RT-PCR was performed in a one-step reaction format using primer set SR33/SR46 (Table 1) described by Ando et al. (1995) as the gold standard. This primer set amplifies an 81 bp region of nucleotides 4513 and 4595 within the RNA polymerase gene of NoV GII (Lordsdale, GenBank accession No. X86557). The RT-PCR amplification was performed in 50 mL of reaction mixture containing 2 mL of viral RNA extract. The reaction was carried out using a QIAGEN One-Step RT-PCR kit (QIAGEN) under the following conditions: 50 8C for 45 min, 95 8C for 15 min, and then 40 cycles of 94 8C for 30 s, 51 8C for 1 min, and 72 8C

for 40 s. The RT-PCR products were separated by electrophoresis in 3% agarose gel and visualized after staining using ethidium bromide. Real-time TaqMan RT-PCR amplification Real-time RT-PCR was carried out in the 7500 Real-Time PCR system (Applied Biosystems, Foster City, California, USA) using a QuantiTect Probe RT-PCR kit (QIAGEN). The amplification was performed in 25 mL of reaction mixture containing 5 mL of viral RNA extract, 12.5 mL of 2 QuantiTect Probe RT-PCR master mix, 0.5 mL of uracyl Nglycosylase, 10 mmol/L each primer (COG2F and COG2R) (Table 1), 0.25 mL of QuantiTect Probe RT mix, 4 mmol/L RING2 TaqMan probe (Table 1), and 5.25 mL of nucleasefree water. The amplification conditions used were as follows: 50 8C for 30 min, 95 8C for 15 min, and then 45 cycles of 95 8C for 15 s, 56 8C for 1 min, and 4 8C for 30 s. Real-time NASBA A real-time NASBA reaction was carried out in a NucliSens EasyQ reader using an Amplification Nuclisens basic kit as described in the manufacturer’s instructions (bioMe´rieux, Boxtel, The Netherlands) with some modifications. Briefly, the reagent sphere was reconstituted in the diluent and KCl was added to a final concentration of 80 mmol/L. Primers JJV2F and COG2R and the molecular beacon probe (Table 1) were then added to obtain previously optimized concentrations of 0.16 and 0.08 mmol/L, respectively. NASBA reactions were initiated by mixing 5 mL of template RNA and 10 mL of primer–probe–beacon – reagent mixture. A 5 mL NoV-free fecal sample served as the negative control. The reaction mixtures were then incubated at 65 8C for 2 min followed by incubation at 41 8C for 2 min. Finally, 5 mL of the reconstituted enzyme mixture was added to the reaction. The final mixture was then incubated at 41 8C for 90 min to allow exponential amplification. ELISA The Dako kit (IDEIA Norwalk-Like virus; Dakocytomation Ltd., New York) based on monoclonal and polyclonal antibodies in 96-well microtiter plates and solid-phase sandwich enzyme immunoassay was used to detect NoV genogroups GI and GII. One hundred microlitres of a 10% Published by NRC Research Press

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fecal sample or positive control with 100 mL of conjugate was incubated in microwells for 120 min at 20–30 8C. Wells were then washed five times with 300 mL of workingstrength wash buffer and incubated with 100 mL of 3,3’,5,5’tetramethylbenzidine substrate. After 30 min of incubation at room temperature, the reaction was stopped by addition of 100 mL of stop solution and absorbance was measured at 450 nm. Analysis of results The sensitivities of the molecular beacon NASBA and TaqMan RT-PCR assay were determined by analyzing 10fold serial dilutions of known NoV GII RNA that was kindly provided by the Newfoundland and Labrador Public Health Laboratory (St. John’s, Newfoundland). The number of viral genomes was estimated as RT-PCR particle detectable units (PDU). One PDU is the highest dilution showing a positive result by conventional RT-PCR. Assay reproducibility was evaluated by testing serial dilutions of the standard sample in triplicate. The specificity of the molecular beacon and TaqMan probe was evaluated by testing NoV GII-negative and NoV GI-positive clinical samples. To establish the cutoff for the real-time NASBA assay, the signals for six NoV-negative samples were measured. A fluorescence value greater than the mean of the negative controls plus 20% was considered as NoV positive (Loens et al. 2006). For TaqMan RT-PCR, a sample was considered NoV positive when the RNA extract showed a positive response at less than 37 cycles. The cutoff value for ELISA was calculated by adding 0.10 absorbance unit to the negative control value.

Results Sensitivity and specificity of real-time RT-PCR and molecular beacon NASBA systems No difference in the limit of detection between real-time RT-PCR and molecular beacon NASBA was observed, with both assays allowing the detection of 0.01 PDU (Figs. 1 and 2). Both assays also showed high specificity for GII NoV (data not shown). Comparison of molecular beacon NASBA, TaqMan RTPCR, conventional RT-PCR, and ELISA for the detection of NoV in fecal samples Of the 96 clinical samples tested, 85 (88.5%), 83 (86.5%), and 78 (81.2%) were diagnosed as NoV positive by molecular beacon NASBA, TaqMan RT-PCR, and conventional RT-PCR, respectively (Table 2). Of the 67 clinical samples tested by ELISA, 44 (65.7%) were found to be NoV positive. Among the 96 clinical samples, 78 tested positive and seven negative using both TaqMan RT-PCR and molecular beacon NASBA. Eleven samples gave discordant results for a coefficient of agreement of 88.5% between these two methods (data not shown). Only one sample was positive using molecular beacon NASBA and negative using the two molecular assays (RT-PCR assays). Three samples that were negative according to real-time NASBA and conventional RT-PCR gave positive results when tested using TaqMan RT-PCR. Furthermore, these samples were also

Can. J. Microbiol. Vol. 55, 2009

negative when other sets of primer were used including G2SKF/G2SKR (G1SKF/G1SKR) (Kojima et al. 2002) and CVF/CVR (Gonin et al. 2000) targeting a capsid N/S and a large part of RdRp genes, respectively.

Discussion Human NoVs are the viral agents most frequently implicated in outbreaks of nonbacterial gastroenteritis worldwide (Marshall et al. 2003). Conventional techniques used for NoV detection, such as electron microscopy, immunoelectron microscopy, enzyme immunoassay, and conventional RT-PCR, each have their shortcomings. Consequently, there is still much effort devoted to developing a novel molecular approach, such as real-time amplification technology, to obtain more rapid, sensitive, and specific detection of these human viruses. Real-time NASBA has been shown to be accurate, sensitive, and specific for detecting human pathogens such as Mycoplasma pneumoniae (Templeton et al. 2003), Chlamydophila pneumoniae (Loens et al. 2006), human immunodeficiency virus type 1 (McClernon et al. 2006), hepatitis B virus (Yates et al. 2001), and enteroviruses (Landry et al. 2005). Recently, Patterson et al. (2006) and Rutjes et al. (2006) developed real-time NASBA detection assays for human NoV using primers and molecular beacon probes that target the RdRp gene. Molecular beacon based NASBA has several advantages over real-time RT-PCR, including integration of the reverse transcription portion of the entire amplification without any need for Taq polymerase (Rutjes et al. 2006). Moreover, real-time NASBA performance and accuracy are not affected by genomic DNA from microbial contaminants (Monis and Giglio 2006). However, while the RdRp gene is appropriate for genotyping, the ORF1–ORF2 junction is much more conserved, making it more suitable for detection of emergent variant NoV strains (Ando et al. 1995; Rutjes et al. 2006). Because of the high genetic diversity of NoVs, many primers and probes have been designed to allow sensitive and specific detection of cocirculating NoV genotypes. To improve sensitivity, several studies have explored the ORF1– ORF2 junction, the most conserved part of the NoV genome (Katayama et al. 2002, 2003; Jothikumar et al. 2005; Rutjes et al. 2006), which corresponds to the C-terminal polymerase to N-terminal capsid region (Katayama et al. 2002). Recently, Jothikumar et al. (2005) designed and evaluated the efficiency of the forward GII primer JJV2F, which targets the ORF1–ORF2 domain and contains eight bases at the 3’ end that are identical to the corresponding sequence in the majority of GII NoV strains. In the present study, a realtime NoV NASBA targeting the ORF1–ORF2 domain was evaluated for specific NoV GII detection in fecal samples. The results presented in this study clearly indicate that the ELISA was less sensitive (65.7%) than the three RNA sequence based molecular approaches. This is in agreement with results reported by de Bruin et al. (2006), who compared two ELISA kits with RT-PCR for detecting NoV in clinical samples. About half of the 15 samples found to be NoV positive by RT-PCR were detected by ELISA. This low sensitivity of immunological detection of NoV may be attributed to factors such as low viral concentration in the Published by NRC Research Press

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sample and the antigenic variability of NoV strains (Parashar et al. 2001; Gonza´lez et al. 2006). ELISA-negative samples would therefore require confirmation by molecular assays such as RT-PCR to compensate for the lack of sensitivity of the ELISA approach (Rabenau et al. 2003; Richards et al. 2003). The dilution end-point detection limit for NoV RNA in clinical samples was 10–4 for conventional RT-PCR (data not shown). Both real-time NASBA and TaqMan RT-PCR detect 0.01 PDU. An estimate of assay sensitivity is also provided by the detection of the various NoV genotypes represented by the 96 clinical samples, as shown in Table 2. On this basis, real-time NASBA (88.5%) was found to be more sensitive than conventional RT-PCR (81.2%). Rutjes et al. (2006) found a similar greater sensitivity of real-time NASBA for NoV detection in large-volume river water and suggested that this assay is less affected by inhibitory factors. More importantly, the designed molecular beacon NASBA system has the same limit of detection as the TaqMan RT-PCR targeting the same sequence. Patterson et al. (2006) reported a high correlation between real-time NASBA and TaqMan RT-PCR for the NoV genome region RdRp; however, their molecular beacon probe design was unable to detect NoV in all samples and gave false-positive results, which may be attributed to the targeted regions in the NoV genome. These finding suggest that the efficiency and accuracy of the molecular beacon NASBA designed in the present study are due to the combination of a robust and specific primer – molecular beacon probe system with realtime technology. This sensitivity is comparable with the recently developed TaqMan RT-PCR approach. In conclusion, the real-time molecular beacon NASBA approach allows rapid, sensitive, and selective detection of a wide variety of human NoV strains and may be useful for limiting or preventing outbreaks of waterborne and foodborne gastroenteritis caused by this virus. This method shows promise for improved quantitative detection in environmental samples where very low levels of virus contamination are anticipated. Moreover, the results reported in the present study suggest implementing an ORF1–ORF2 junction targeting molecular beacon NASBA for GII NoV detection on a routine basis in samples of environmental origin. This developed technique might be combined easily with other concentration and separation methods for the effective recovery of low numbers of virus in foods and water.

Acknowledgements This study was funded by a grant from the Natural Sciences and Engineering Research Council of Canada and Fonds Que´be´cois de la recherche sur la nature et les technologies. The authors are especially grateful to Rene´e Lamirande, Jasmine Chamberland, and Maryline Girard for technical support. Nuclisens basic kit reagents were kindly provided by bioMe´rieux Inc.

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