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Optimization of methods for detecting norovirus on various fruit
Journal of Virological Methods 153 (2008) 104–110
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Optimization of methods for detecting norovirus on various fruit Hee-Yeon Kim a , In-Shin Kwak c , In-Gyun Hwang c , GwangPyo Ko a,b,∗ a
Department of Environmental Health and Institute of Health and Environment, School of Public Health, Seoul National University, Seoul, South Korea Institute of Microbiology, School of Biological Sciences, Seoul National University, Seoul, South Korea c Division of Food Microbiology, Department of Food Safety Evaluation, Korea Food and Drug Administration, Seoul, South Korea b
a b s t r a c t Article history: Received 6 May 2008 Received in revised form 23 July 2008 Accepted 29 July 2008 Available online 10 September 2008 Keywords: Norovirus Gastroenteritis Fruit Elution PEG concentration Nucleic acid extraction
Methods for detecting norovirus (NoV) in food are crucial for investigation and prevention of outbreaks caused by NoV-contaminated food. However, current NoV detection methods have not been well examined or optimized. In this study, the effectiveness of various methods for eluting NoV from various fruit, concentrating the virus using polyethylene glycol (PEG), and extracting the viral RNA for subsequent assay by RT-PCR was optimized. First, six different buffers previously described for eluting NoV from fruit surfaces were evaluated. A known amount of NoV was spiked onto the surface of grapes, strawberries, and raspberries, and the virus was recovered with distilled water, 0.05 M glycine–0.14 M NaCl (pH 7.5), 2.9% tryptose phosphate broth–6% glycine, 100 mM Tris–HCl (pH 9.5), 50 mM glycine–50 mM MgCl2 (pH 9.5), or 3% beef extract. Quantitation of the recovered virus using RT-PCR revealed that the most effective elution buffer was 3% beef extract. Secondly, to optimize a method for concentrating the recovered NoV, the key parameters of PEG precipitation, a typical method for concentrating enteric virus, were investigated. The influence of PEG molecular weight and the duration and temperature of the precipitation procedure were examined. NoV was concentrated most efficiently by precipitation when PEG10,000 was used for 4 h at room temperature. Finally, five different methods for nucleic acid extraction were evaluated. Among RNA extraction methods examined, QIAamp Viral RNA Mini kit showed the best recovery efficiency. Using the optimized method, approximately 6–80% of the seeded NoV was recovered from the various fruit. © 2008 Published by Elsevier B.V.
1. Introduction Food-borne viral infections are increasingly recognized as a major cause of illness in humans (Mullendore et al., 2001; Myrmel et al., 2000). The noroviruses (NoVs), a genetically and antigenically diverse group of positive-RNA viruses of the Caliciviridae family (Rutjes et al., 2006), have emerged as the single most important etiological agent in outbreaks of food-borne illness and in sporadic cases of acute gastroenteritis in children and adults worldwide (Mead et al., 1999; Vinje et al., 1997, 2003). NoV outbreaks have occurred in various settings, including nursing homes (Calderon-Margalit et al., 2005), hospitals, cruise ships, schools, restaurants, and catered events (Parashar et al., 1998; Rutjes et al., 2006). Contamination of all types of food products with NoV can occur either by contamination with sewage during growth or by food handlers during harvest or packaging. Typical food items implicated in
∗ Corresponding author at: Department of Environmental Health, School of Public Health, Seoul National University, 28 Yunkeun-Dong, Chongro-Ku, Seoul 110-799, South Korea. Tel.: +82 2 3668 7881; fax: +82 2 745 9104. E-mail address: [email protected] (G. Ko). 0166-0934/$ – see front matter © 2008 Published by Elsevier B.V. doi:10.1016/j.jviromet.2008.07.022
NoV outbreaks are raw or poorly cooked meat or seafood, such as shellfish, ready-to-eat food, and commodities such as fruit and vegetables, which are associated with a high risk of infection (Bosch et al., 2001; Daniels et al., 2000; Guevremont et al., 2006; Hutin et al., 1999; Le Guyader et al., 1994; Potasman et al., 2002; Rosenblum et al., 1990; Rutjes et al., 2006) because they are consumed typically without additional heat treatment. However, NoV contamination of food is demonstrated rarely during food-borne outbreaks, either because appropriate detection methods are lacking or because the culpable food samples are unavailable (Cliver, 1995). Many types of fruit, such as grapes, raspberries, and strawberries, are recognized increasingly being as the vehicles of many NoV outbreaks (Butot et al., 2007; Hill, 2003; Le Guyader et al., 2004). Therefore, the development and optimization of methods for detecting NoV on these fruit is important for preventing and investigating outbreaks of NoV. However, only a few methods have been well developed, and these have been applied only to limited types of food items, such as shellfish (Baert et al., 2007; Dubois et al., 2002; Heller et al., 1986; Jaykus et al., 1996; Kingsley and Richards, 2001; Schultz et al., 2007; Shieh et al., 1999; Sunen and Sobsey, 1999). In this study, each step in a procedure for detecting NoV on fruit was evaluated and optimized. The steps included elution of the virus from the fruit, concentration of the virus using
H.-Y. Kim et al. / Journal of Virological Methods 153 (2008) 104–110
polyethylene glycol (PEG) precipitation, and extraction of the viral RNA for subsequent assay by RT-PCR. The specific aims of this study were (1) to find the elution buffer providing the best recovery of NoV, (2) to determine the optimal conditions for virus concentration via PEG precipitation in the various elution buffers, and (3) to evaluate RNA extraction methods compatible with these elution and concentration procedures. 2. Methods and materials 2.1. Stool specimens and RT-PCR assay NoV/GII-4, the NoV genotype associated most often with outbreaks of food-borne illness, was obtained as a stool sample from the Korean Center for Disease Control and Prevention (KCDC). The stool sample was diluted serially, and the concentration of NoV in the stool sample was quantified using an RT-PCR assay. The viral RNA was extracted from 140 l of 10% fecal suspension in phosphate-buffered saline (PBS) using a QIAamp Viral RNA Mini Kit (Qiagen, Valencia, CA, USA) according to the manufacturer’s protocol and stored at −80 ◦ C. Two sets of primers for the capsid-encoding region of the target viral genome were used for RT-PCR, as described previously (Isakbaeva et al., 2005). Briefly, the G2-SKF/G2-SKR primer sets were used to amplify the NoV/GII-4 using a One-Step RT-PCR kit (Qiagen, Valencia, CA, USA), generating PCR products of 344 bp (Kojima et al., 2002). The viral RNA was reverse-transcribed for 60 min at 42 ◦ C and then incubated for 15 min at 95 ◦ C to activate the Taq polymerase. Thermocycling conditions for the reaction consisted of 40 cycles of 1 min at 94 ◦ C, 1 min at 40 ◦ C, and 1 min at 72 ◦ C, and a final extension step at 72 ◦ C for 10 min. Negative controls were included in each RT-PCR run. Amplified products were analyzed on ethidium bromide-stained 2% agarose gels. The resulting concentration of NoV/GII-4 in the stool sample was approximately 4 × 106 RT-PCR unit/ml. All stool specimens were stored at −70 ◦ C until use. 2.2. Optimization of NoV elution from various fruit Six different elution buffers used in previous studies were evaluated (Baert et al., 2008; Dubois et al., 2002; Guevremont et al., 2006; Lewis and Metcalf, 1988; Monpoeho et al., 2001; Mullendore et al., 2001). The tested elution buffers were: (1) distilled water, (2) 3% beef extract (pH 7.1), (3) 0.05 M glycine–0.14 M NaCl (pH 7.5), (4) 2.9% tryptose phosphate broth–6% glycine, (5) 100 mM Tris–HCl (pH 9.5), and (6) 50 mM glycine–50 mM MgCl2 (pH 9.5). A 10-l aliquot of NoV-containing stool suspension (4 × 106 RT-PCR units/ml) was inoculated onto 20 g of fresh grape, fresh strawberry, or frozen raspberry. After the inoculated fruit was airdried on a clean bench for 30 min, the NoV was eluted by shaking the fruit in elution buffer at 200 rpm for 5 h at room temperature. An equal volume of chloroform was added, and the mixture was vigorously vortexed (Monpoeho et al., 2001). The solution was centrifuged at 2000 × g at 4 ◦ C for 10 min, and the supernatant was recovered. For PEG precipitation, PEG with a molecular weight (MW) of 8000 (PEG8000 ) was added to the solution at a final concentration of 8%. This solution was mixed vigorously and incubated overnight at 4 ◦ C with occasional shaking. Then, the resulting flocculence was precipitated at 12,000 × g for 30 min at 4 ◦ C, and the pellet was resuspended in 2 ml of PBS. RNA was extracted from the suspension using a QIAamp kit according to the manufacturer’s protocol. The viral RNA was amplified using a OneStep RT-PCR kit. Recovery from the various elution buffers was estimated as the percentage of NoV recovered from
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the fruit relative to the amount of NoV seeded onto the fruit. Two independent experiments were carried out. 2.3. Optimization of PEG precipitation To optimize PEG precipitation of NoV, the influence of PEG MW and the duration and temperature of incubation with PEG were examined. Each of three elution buffers (distilled water, 3% beef extract, and 0.05 M glycine–0.14 M NaCl) was tested with each fruit (grapes, strawberries, and raspberries). Each fruit was incubated with three different elution buffers at 200 rpm for 5 h. Ten l of NoV-containing stool suspension (4 × 104 RT-PCR units) was inoculated into each of the resulting elution buffers. Each of these inoculated suspensions was mixed with PEG of MW 6000, 8000, 10,000, or 20,000. The final concentration of PEG was adjusted to 8%, and the pH in the suspension was adjusted to 7.2. After vigorous mixing, the suspension was incubated with occasional shaking for either 4 h at room temperature or overnight at 4 ◦ C. The resulting flocculence was collected by centrifugation at 12,000 × g for 30 min at 4 ◦ C, and the pellets were resuspended in 2 ml of PBS. These solutions were serially diluted, and the viral RNA was extracted for use in an RT-PCR assay. Each precipitation experiment was performed in triplicate. The amount of NoV was estimated by the most probable number (MPN) method using serial 1:10 dilutions of the recovered viral RNA. The numbers of RT-PCR-positive or -negative dilution samples from three experiments were entered into an MPN computation program to calculate the concentration and confidence limits of recovered NoV RNA (De Man, 1983). The percentage recovery was calculated by dividing the number of RT-PCR units of NoV recovered by the number of units used for inoculation. 2.4. Optimization of RNA extraction Five different methods of viral RNA extraction were evaluated: (1) heat-release, (2) QIAamp Viral RNA Mini Kit (Qiagen, Valencia, CA, USA), (3) magnetic beads (MagExtractor Viral RNA Purification Kit, Toyobo, Osaka, Japan), (4) TRIzol reagent (Invitrogen, Carlsbad, CA, USA), and (5) immunomagnetic separation with magnetic Dynabeads (M-280 Tosylactivated, Dynal Biotech, Norway). A 10l aliquot of NoV-containing stool suspension was inoculated onto each of the tested fruit (grape, strawberry, and raspberry), and the virus was eluted using 3% beef extract as described above. After the eluted virus was precipitated with PEG10,000 for 4 h at 4 ◦ C, the viral RNA was extracted using one of the above methods, and 2.5 l of the extracted RNA was used in a 25 l RT-PCR reaction. The virus was stored at −80 ◦ C until use in the RT-PCR reaction. Each method was tested independently three times. For heat-release, the PEG-precipitated virus was heated to 95 ◦ C for 10 min and immediately chilled on ice. For the magnetic bead method, the viral RNA was extracted using a MagExtractor Viral RNA Purification Kit according to the manufacturer’s instructions. For RNA extraction using the QIAamp kit, the experiment was performed following the manufacturer’s protocol. Briefly, 140 l of eluted virus suspension was mixed with 560 l of guanidinium thiocyanate solution, followed by neutralization with an equal volume of 96–100% ethanol. The viral RNA was purified further using a QIAamp Mini Column (Qiagen, Valencia, CA, USA). For RNA extraction using TRIzol, the PEG-precipitated virus was resuspended in 2 ml of PBS, and 300 l of the suspension was added to 700 l of TRIzol reagent. After vortexing, the mixture was allowed to stand for 5 min at room temperature. Then, 150 l of chloroform was added to the solution, and the mixture was allowed to stand for 15 min at room temperature. After centrifugation at 108 × g for 15 min at 4 ◦ C, the supernatant fraction was transferred
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into a microtube with an equal volume of isopropanol, incubated for 2 h at −20 ◦ C, and centrifuged at 10,844 × g for 30 min at 4 ◦ C. The resulting pellet was washed with 1 ml of 75% ethanol, air-dried, and resuspended in 15 l of diethyl pyrocarbonate (DEPC)-treated water. For immunomagnetic separation, a 1 mg/ml stock suspension solution of Dynabeads was thoroughly resuspended by pipetting or vortexing for approximately 1 min, and then 33 l of beads were fixed in a magnetic particle concentrator (Invitrogen, Carlsbad, CA, USA) for ∼2 min until the beads migrated to the side of the tube and the liquid became clear. The supernatant fraction was removed carefully from the opposite side of the tube, leaving the beads undisturbed. This washing step was performed three times with 0.1 M Na-phosphate buffer (pH 7.4) at room temperature. After washing, the beads were resuspended gently in 1 ml of 0.1 M Na-phosphate buffer (pH 7.4) containing 5–10 l of rabbit norovirus capsid IgG antiserum. The beads were incubated with the antiserum for 16–24 h at room temperature with slow-tilt rotation on an MX4 sample mixer (Invitrogen, Carlsbad, CA, USA). The uncoupled IgG was removed by washing the beads four times with PBS (pH 7.4) containing 0.1% (w/v) bovine serum albumin (BSA) (Sigma, St. Louis, MO, USA) at 4 ◦ C. To prepare the eluted virus for immunomagnetic separation, the eluate was extracted with a 1/10th-volume of chloroform, PEG-precipitated as described above, and resuspended in 1.5 ml of PBS. The antibody-bound Dynabeads were gently mixed with 1.5 ml of resulting resuspension and incubated with rotation on the MX4 mixer for 1 h at room temperature. After incubation, the Dynabeads were separated using the MPC as described above, washed three times with PBS containing 0.1% (w/v) BSA, resuspended in 50 l of DEPC-treated water, and transferred to an Eppendorf tube for RT-PCR assays. 2.5. Data analysis PEG precipitation methods were compared using analysis of variance (ANOVA) or Kruskal–Wallis nonparametric tests. The different nucleic acid extraction methods were compared using the Mann–Whitney test. All statistical analysis was performed using SPSS software 11.0 (SPSS Inc., Chicago, IL, USA). Differences were considered statistically significant at P < 0.05. 3. Results 3.1. Elution buffers Six different elution buffers were tested for their ability to elute NoV from the surface of grapes, strawberries, and raspberries (Table 1). Overall, the virus was eluted more efficiently from grape than from strawberry. The strawberry recovery rate tended
to be at or below the limit of detection (8.5% recovery). For grapes, the 50 mM glycine–50 mM MgCl2 (pH 9.5) buffer was least efficient at recovering the virus. For strawberries, distilled water, 0.05 M glycine–0.14 M NaCl (pH 7.5), and 2.9% tryptose phosphate–6% glycine had recoveries below the detection limit (8.5%). For both grapes and strawberries, the highest recovery was obtained with 3% beef extract. 3.2. PEG precipitation The influence of PEG MW on the efficacy of NoV precipitation in various elution buffers is shown in Table 2. Of the four different PEGs tested, PEG10,000 recovered the most virus (17.8%). The other three PEGs (PEG6000 , PEG8000 , and PEG20,000 ) had average recoveries of 9.2%, 15.5%, and 7.2%, respectively. The average recovery ranges were 4.3–19.9% for grape, from below the limit of detection (2.6%) to 50.4% for strawberry, and 6.6–9.4% for raspberry. In all PEG MW experiments, the elution buffer with the highest recovery was 3% beef extract. The highest recovery from strawberry was achieved using 3% beef extract and PEG10,000 , but the recovery differences among the different PEGs were not statistically significant (ANOVA test, P = 0.67). The duration of the PEG precipitation incubation also affected the recovery of NoV (Table 3). Overall, better recovery was achieved from the 4 h process than from the overnight process. Again, 3% beef extract was the best elution buffer, regardless of conditions. For 4 h precipitation, PEG of lower MW was associated with less efficient recovery; in 3% beef extract, PEG6000 and PEG8000 recovered about 12.9% and 8.6% of the virus, respectively, whereas PEG10,000 and PEG20,000 recovered about 51.9% and 58.4% of the virus, respectively. However, for the lengthier precipitation (overnight), recovery was highest with PEG10,000 . The Kruskal–Wallis nonparametric comparisons test showed no significant difference between the overnight and 4 h averages (P = 0.543). 3.3. RNA extraction methods The NoV recovery rates achieved using five different viral RNA extraction methods are shown in Table 4. Of these methods, the QIAamp Viral RNA Mini Kit resulted in the best recovery; for grapes, this kit recovered 80% of the RNA (as determined using the MPN index to determine the most probable number of organisms per unit volume of the original sample), followed by the methods using heat-release, immunomagnetic separation, Toyobo magnetic beads, and TRIzol. Recovery rates from strawberries and raspberries were generally lower than those from grapes but similar to each other. For strawberry and raspberry, the QIAamp kit had recovery rates of 8% and 6%, respectively, and for the other RNA extraction methods, rates ranged from undetectable (TRI-
Table 1 Efficiency of norovirus (NoV) elution from grape and strawberry for six different elution buffers Elution buffer
Elution efficiency (%)a Grape
Strawberry
Distilled water 3% Beef extract 0.05 M Glycine–0.14 M NaCl (pH 7.5) 2.9% Tryptose phosphate–6% glycine 100 mM Tris–HCl (pH 9.5) 50 mM Glycine–50 mM MgCl2 (pH 9.5)
21 21 21 21 21 2.1
<8.5b 85 <8.5 <8.5 8.5 8.5
Overall average recovery
17.9
21.3
a
The elution efficiency was calculated as the percentage of NoV recovered compared with the initial amount of NoV seeded. Elution efficiencies shown are averages of duplicate experiments. b Beneath the limit of detection (8.5%).
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Table 2 Influence of PEG molecular weight on NoV precipitation from various elution buffers Type of Fruit
Elution buffera
Grape
A B C
Strawberry
A B C
Raspberry
A B C
PEG molecular weight 6000
8000
10,000
20,000
<2.6 (0.4–7.7) 19.7 (3.4–100) <2.6 (0.4–7.7)
7.7 (0.9–30.9) 19.7 (3.4–103) 3.4 (0.4–17.2)
3.4 (0.4–17.2) 19.7 (3.4–100) 3.4 (0.4–17.2)
12.9 (2.6–39.8) 18 (3.4–40.3) <2.6 (0.4–7.7)
6 (0.9–19.7) 79.8 (12.9–100) <2.6 (0.4–7.7)
3.4 (0.4–17.2) 85.8 (30.9–100) <2.6 (0.4–7.7)
7.7 (0.9–30.9) 18 (3.4–40.3) <2.6 (0.4–7.7)
18 (3.4–100) 9.4 (2.6–30.9) 7.7 (0.9–30.9)
12.9 (2.6–39.8) 6 (0.9–19.7) 7.7 (0.9–30.9)
3.4 (0.4–17.2) 9.4 (2.6–30.9) 7.7 (0.9–30.9)
3.4 (0.4–17.2) 3.4 (0.4–17.2) 3.4 (0.4–17.2)
3.4b (0.4–17.2)c 3.4 (0.4–17.2) 7.7 (0.9–30.9)
Overall a b c
9.2
15.5
17.8
7.2
A: distilled water; B: 3% beef extract; C: 0.05 M glycine–0.14 M NaCl. The percent recovery (%) of NoV recovered was calculated using the initial seeding of NoV as 100%. 95% confidence interval. Percent recovery of inoculated NoV was estimated from three independent experiments using the MPN table.
Table 3 Influence of PEG incubation period on NoV precipitation from various elution buffers PEG molecular weight
Elution buffera
6,000
A B C
8,000
Overnight precipitation of PEG
4 h precipitation of PEG
Strawberry
Raspberry
Strawberry
Raspberry
12.9b (2.6–37.8)c 18 (3.4–40.3) <2.6 (0.4–7.7)
18 (3.4–40.3) 9.4 (2.6–30.9) 7.7 (0.9–30.9)
3.4 (0.4–17.2) 12.9 (2.6–37.8) 18 (3.4–40.3)
12.9 (2.6–37.8) 12.9 (2.6–37.8) 12.9 (2.6–37.8)
A B C
6 (0.9–19.7) 79.8 (12.9–100) <2.6 (0.4–7.7)
12.9 (2.6–37.8) 6 (0.9–19.7) 7.7 (0.9–30.9)
3.4 (0.4–17.2) 7.7 (0.9–30.9) 12.9 (2.6–37.8)
7.7 (0.9–30.9) 9.4 (2.6–30.9) 6 (0.9–19.7)
10,000
A B C
3.4 (0.4–17.2) 85.8 (30.9–100) <2.6 (0.4–7.7)
3.4 (0.4–17.2) 9.4 (2.6–30.9) 7.7 (0.9–30.9)
3.4 (0.4–17.2) 18 (3.4–40.3) 12.9 (2.6–37.8)
3.4 (0.4–17.2) 85.8 (30.9–100) 3.4 (0.4–17.2)
20,000
A B C
7.7 (0.9–30.9) 18 (3.4–40.3) <2.6 (0.4–7.7)
3.4 (0.4–17.2) 3.4 (0.4–17.2) 3.4 (0.4–17.2)
<2.6 (0.4–7.7) 36.9 (6–100) 3.4 (0.4–17.2)
12.9 (2.6–37.8) 79.8 (12.9–100) 3.4 (0.4–17.2)
20.2
7.7
11.9
30
Overall a b c
A: distilled water; B: 3% beef extract; C: 0.05 M glycine–0.14 M NaCl. The percent recovery (%) of NoV recovered was calculated using the initial seeding of NoV as 100%. 95% confidence interval. Percent recovery of inoculated NoV was estimated from three independent experiments using the MPN table.
zol) to 8% (heat-release). Some of the RNA extraction methods differed significantly from the average in their recovery efficiencies (Kruskal–Wallis test, P = 0.025). Significant differences were observed between the QIAamp kit and both the Toyobo MagEx-
tractor kit (P = 0.046) and the TRIzol method (P = 0.05). However, no significant difference was observed between the QIAamp kit and the heat-release or immunomagnetic separation methods (P > 0.05).
Table 4 Recovery of NoV using five different RNA extraction methods RNA extraction methods
Fruit
Percentage recovery (%)a (95% confidence limits)
P-valueb
Grape Strawberry Raspberry
80 (12.9–100) 8 (0.9–19.7) 6 (2.6–9.3)
n/a
QIAampViral RNA Mini Kit
Grape Strawberry Raspberry
14 (2–42) 8 (1–40) 6 (1–26)
0.658
Heat-release
Grape Strawberry Raspberry
1.8 (0.2–7.2) 1.8 (0.2–7.2) 4.2 (0.8–9.4)
0.046
Toyobo Mag Extractor
Grape Strawberry Raspberry
0.4 (0.1–2) 1.5 (0.3–4.4) <0.3 (<0.3)
0.050
Trizol reagent
Grape Strawberry Raspberry
8.6 (1.4–42) 3 (0.6–8.8) 3 (0.6–8.8)
0.121
Immuno-magnetic separation a b
The percentage of NoV recovered was calculated using the initial seeding of NoV as 100%. Recovered NoV was estimated by MPN table from three independent experiments. P-value was estimated by Mann–Whitney test. Comparison was made between QIAampViral RNA Mini Kit and the other nucleic acid extraction methods.
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Fig. 1. Summary of optimized methods and their percent recoveries for detecting NoV on fruit.
4. Discussion Despite the importance of NoV in causing food-borne illness, methods for detecting NoV in foods have not been well studied. In most NoV outbreaks, investigators were unable to identify NoV in the suspected foods (Guevremont et al., 2006) because (1) the suspected food was unavailable; (2) NoV cannot be cultivated using typical methods; and (3) methods for concentrating NoV from food items are inefficient (Rutjes et al., 2006). Unlike the many bacterial food-borne pathogens that can be cultivated and enriched in culture media, NoV is neither cultivated nor enriched in vitro. For these reasons, detection and identification of NoV requires elution from contaminated foods and then concentrated. Typically, the concentration of NoV on foods is low, and various chemical components present in foods can inhibit molecular assays. All of these factors stress the importance of optimizing elution and concentration methods for detecting NoV in food. The typical procedure for detecting NoV on food involves elution, concentration by PEG precipitation, RNA extraction, and RT-PCR assay. In this study, the methods were characterized and optimized for elution of NoV contaminants from various fruit, as well as for concentration of, and RNA extraction from, eluted NoV. Most previous studies have focused on detecting NoV in shellfish. However, fruit and vegetables are among the most culpable food items in a number of food-borne outbreaks (Butot et al., 2007). Therefore, it is important to evaluate the efficiency of NoV recovery from various fruit. To identify the most effective elution buffer, all elution buffers that had been studied previously were tested (Baert et al., 2008; Dubois et al., 2002; Guevremont et al., 2006; Lewis and Metcalf, 1988; Monpoeho et al., 2001; Mullendore et al., 2001). Of these six elution buffers, 3% beef extract eluted NoV most effectively, probably because of its high protein concentration. In a recent study of NoV elution from fresh strawberries and frozen raspberries, the elution efficiency of 100 mM Tris buffer ranged from 0.42% to 11.2% (Butot et al., 2007), which is consistent with this study (8.5%). The addition of 1% beef extract to the Tris buffer increased the elution efficiency by 2.2- and 3.6-fold in strawberry and raspberry, respec-
tively. These results are consistent with current study suggesting that certain protein components of beef extract promote elution of NoV from berries. This study is the first to investigate the effect of both PEG MW and incubation period on viral precipitation using PEG. PEG precipitation is often used to concentrate viruses in solution because it is an easy and gentle method that can be performed at neutral pH and at high ionic concentrations without other organic materials (Lewis and Metcalf, 1988). Although it might be suspected that increasing the PEG MW might increase the recovery of NoV from eluates, the results do not support this idea. It was also found that increasing the duration of the precipitation incubation was unnecessary. The viral recovery rate provided by PEG precipitation in this study (12.4%) was lower than in a previous study of hepatitis A virus (13–27%) (Mullendore et al., 2001) but higher than in previous NoV studies (1–7%) (Butot et al., 2007; Rutjes et al., 2006). These differences are probably a result of differences in the protein and other components of the elution buffers. In this study, PEG precipitation was a highly effective method for concentrating NoV eluted in 3% beef extract, again probably because the high protein concentration in beef extract facilitates flocculation of NoV by PEG. Several food components are known to inhibit molecular assays such as RT-PCR (Lewis and Metcalf, 1988; Schwab and McDevitt, 2003). Viral detection on berries (such as raspberries and strawberries) can be particularly problematic due to the presence of both chemical inhibitors and a low pH (Butot et al., 2007). To date, NoV has been identified in only a few vegetables or berry samples contaminated naturally (Calder et al., 2003; Hernández et al., 1997; Kobayashi et al., 2004; Le Guyader et al., 2004). In this study, a procedure consisting of virus elution with 3% beef extract, virus precipitation with PEG10,000 for 4 h at room temperature, and extraction of the viral RNA using a QIAamp Viral RNA Mini Kit efficiently recovered and detected NoV on fruit (Fig. 1). In previous studies, beef extract was found to contain many inhibitors of RTPCR (Katayama et al., 2002; Schwab and McDevitt, 2003), but it did not significantly inhibit RT-PCR in this study, probably because the inhibitory factors were removed effectively in the viral RNA extraction procedure.
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Five different methods for extracting RNA from NoV were evaluated. The factors that determine the usefulness of viral RNA extraction methods are their compatibility with prior elution and concentration steps, their effectiveness for extracting RNA from the viral particles, and their ability to remove inhibitors of RT-PCR. Of the extraction methods tested in this study, the best recovery rate was obtained with the QIAamp Viral RNA Mini Kit, followed by immunomagnetic separation. However, the success of the latter method is dependent on the type and quality of the anti-NoV antibody used. Additional validation of antibodies against different types of NoV should be performed in the future. Although an RT-PCR assay to detect NoV was used in this study, other analytical methods, such as electron microscopy or enzyme-linked immunoassay, could also be used. Of the various possible analytical methods for NoV, RT-PCR is most commonly used because it is rapid and has high sensitivity and specificity. In this study, the percentage recovery was quantified by serial dilution and conventional RT-PCR assays, but a more quantitative method, such as real-time RT-PCR assay, could be used instead. One of the limitations of current method is that it provides no information about the infectivity of NoV, because the molecular assay does not differentiate infectious viruses from noninfectious ones. Straub et al. (2007) recently described a three-dimensional in vitro cultivation system for NoV, but this system is difficult and expensive to use. In addition, they reported that only a few types of NoV could be cultivated using this method. Alternatively, murine norovirus (MNV) might be used as a surrogate for human NoV. MNV is easily cultivated and plaque-assayed using the RAW264.7 cell line, which permits quantitation by infectivity (Karst et al., 2003; Thackray et al., 2007). However, MNV might have different surface characteristics, and surrogate viruses always have limitations. Therefore, finding elution and concentration methods for human NoV that are compatible with RT-PCR assays is of practical importance. Currently, there is no standardized method for detecting NoV contamination of various fruit. In light of the importance of NoV in food-borne illnesses, evaluation and optimization of methods for detecting NoV contamination of food items are matters of great urgency. In this study, optimal procedures were determined for detecting NoV on fruit often responsible for NoV-associated outbreaks (Fig. 1). This method will be useful for investigation of food-borne illness and for monitoring food. Further studies are warranted for optimizing the elution and concentration procedures for other important food items. Acknowledgment This study was partially funded by the Korean Food and Drug Administration (grant #06042). References Baert, L., Uyttendaele, M., Debevere, J., 2007. Evaluation of two viral extraction methods for the detection of human noroviruses in shellfish with conventional and real-time reverse transcriptase PCR. Lett. Appl. Microbiol. 44, 106–111. Baert, L., Uyttendaele, M., Debevere, J., 2008. Evaluation of viral extraction methods on a broad range of ready-to-eat foods with conventional and real-time RT-PCR for norovirus GII detection. Int. J. Food Microbiol. 123, 101–108. Bosch, A., Sanchez, G., Le Guyader, F., Vanaclocha, H., Haugarreau, L., Pinto, R.M., 2001. Human enteric viruses in Coquina clams associated with a large hepatitis A outbreak. Water Sci. Technol. 43, 61–65. Butot, S., Putallaz, T., Sanchez, G., 2007. Procedure for rapid concentration and detection of enteric viruses from berries and vegetables. Appl. Environ. Microbiol. 73, 186–192. Calder, L., Simmons, G., Thornley, C., Taylor, P., Pritchard, K., Greening, G., Bishop, J., 2003. An outbreak of hepatitis A associated with consumption of raw blueberries. Epidemiol. Infect. 131, 745–751.
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Development of a virus concentration method and its application to detection of enterovirus and Norwalk virus from coastal seawater. Appl. Environ. Microbiol. 68, 1033–1039. Kingsley, D.H., Richards, G.P., 2001. Rapid and efficient extraction method for reverse transcription-PCR detection of Hepatitis A and Norwalk-like viruses on shellfish. Appl. Environ. Microbiol. 67, 4152–4157. Kobayashi, S., Natori, K., Takeda, N., Sakae, K., 2004. Immunomagnetic capture RTPCR for detection of norovirus from foods implicated in a foodborne outbreak. Microbiol. Immunol. 48, 201–204. Kojima, S., Kageyama, T., Fukushi, S., Hoshino, F.B., Shinohara, M., Uchida, K., Natori, K., Takeda, N., Katayama, K., 2002. Genogroup-specific PCR primers for detection of Norwalk-like viruses. J. Virol. Methods 100, 107–114. Le Guyader, F., Dubois, E., Menard, D., Pommepuy, M., 1994. Detection of hepatitis A virus, rotavirus, and enterovirus on naturally contaminated shellfish and sediment by reverse transcription-seminested PCR. Appl. Environ. Microbiol. 60, 3665–3671. Le Guyader, F., Mittelholzer, C., Haugarreau, L., Hedlunf, K.O., Alsterlund, R., Pommepuy, M., Svensson, L., 2004. Detection of Noroviruses in raspberries associated with a gastroenteritis outbreak. Int. J. Food Microbiol. 97, 179–186. Lewis, G.D., Metcalf, T.G., 1988. Polyethylene glycol precipitation for recovery of pathogenic viruses including hepatitis A virus and human rotavirus, from oyster, water and sediment samples. Appl. Environ. Microbiol. 54, 1983–1988. Mead, P.S., Slutsker, L., Diets, V., McCaig, L.F., Bresee, J.S., Shapiro, C., Griffin, P.M., Tauxe, R.V., 1999. Food-related illness and death in the United States. Emerg. Infect. Dis. 5, 607–625. Monpoeho, S., Maul, A., Mignotte-Cadiergues, B., Schwartzbrod, L., Billaudel, S., Ferré, V., 2001. Best viral elution method available for quantification of enteroviruses in sludge by both cell culture and reverse transcription-PCR. Appl. Environ. Microbiol. 67, 2484–2488. Mullendore, J.L., Sobsey, M.D., Shieh, Y.C., 2001. Improved method for the recovery of hepatitis A virus from oysters. J. Virol. Methods 94, 25–35. Myrmel, M., Rimstad, E., Wasteson, Y., 2000. Immunomagnetic separation of a Norwalk-like virus (genogroup I) in artificially contaminated environmental water samples. Int. J. Food Microbiol. 62, 17–26. Parashar, U.D., Dow, L., Frankhauser, R., Humphrey, C.D., Miller, J., Ando, T., Williams, K.S., Eddy, C.R., Noel, J.S., Ingram, T., Bresee, J.S., Monroe, S.S., Glass, R.I., 1998. An outbreak of viral gastroenteritis associated with consumption of sandwiches: implications for the control of transmission by food handlers. Epidemiol. Infect. 121, 615–621. Potasman, I., Paz, A., Odeh, M., 2002. Infectious outbreaks associated with bivalve shellfish consumption: a worldwide perspective. Clin. Infect. Dis. 35, 921–928. Rosenblum, L.S., Mirkin, I.R., Allen, D.T., Safford, D., Hadler, S.C., 1990. A multifocal outbreak of hepatitis A traced to commercially distributed lettuce. Am. J. Public Health 80, 1075–1080.
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Journal of Virological Methods journal homepage: www.elsevier.com/locate/jviromet
Optimization of methods for detecting norovirus on various fruit Hee-Yeon Kim a , In-Shin Kwak c , In-Gyun Hwang c , GwangPyo Ko a,b,∗ a
Department of Environmental Health and Institute of Health and Environment, School of Public Health, Seoul National University, Seoul, South Korea Institute of Microbiology, School of Biological Sciences, Seoul National University, Seoul, South Korea c Division of Food Microbiology, Department of Food Safety Evaluation, Korea Food and Drug Administration, Seoul, South Korea b
a b s t r a c t Article history: Received 6 May 2008 Received in revised form 23 July 2008 Accepted 29 July 2008 Available online 10 September 2008 Keywords: Norovirus Gastroenteritis Fruit Elution PEG concentration Nucleic acid extraction
Methods for detecting norovirus (NoV) in food are crucial for investigation and prevention of outbreaks caused by NoV-contaminated food. However, current NoV detection methods have not been well examined or optimized. In this study, the effectiveness of various methods for eluting NoV from various fruit, concentrating the virus using polyethylene glycol (PEG), and extracting the viral RNA for subsequent assay by RT-PCR was optimized. First, six different buffers previously described for eluting NoV from fruit surfaces were evaluated. A known amount of NoV was spiked onto the surface of grapes, strawberries, and raspberries, and the virus was recovered with distilled water, 0.05 M glycine–0.14 M NaCl (pH 7.5), 2.9% tryptose phosphate broth–6% glycine, 100 mM Tris–HCl (pH 9.5), 50 mM glycine–50 mM MgCl2 (pH 9.5), or 3% beef extract. Quantitation of the recovered virus using RT-PCR revealed that the most effective elution buffer was 3% beef extract. Secondly, to optimize a method for concentrating the recovered NoV, the key parameters of PEG precipitation, a typical method for concentrating enteric virus, were investigated. The influence of PEG molecular weight and the duration and temperature of the precipitation procedure were examined. NoV was concentrated most efficiently by precipitation when PEG10,000 was used for 4 h at room temperature. Finally, five different methods for nucleic acid extraction were evaluated. Among RNA extraction methods examined, QIAamp Viral RNA Mini kit showed the best recovery efficiency. Using the optimized method, approximately 6–80% of the seeded NoV was recovered from the various fruit. © 2008 Published by Elsevier B.V.
1. Introduction Food-borne viral infections are increasingly recognized as a major cause of illness in humans (Mullendore et al., 2001; Myrmel et al., 2000). The noroviruses (NoVs), a genetically and antigenically diverse group of positive-RNA viruses of the Caliciviridae family (Rutjes et al., 2006), have emerged as the single most important etiological agent in outbreaks of food-borne illness and in sporadic cases of acute gastroenteritis in children and adults worldwide (Mead et al., 1999; Vinje et al., 1997, 2003). NoV outbreaks have occurred in various settings, including nursing homes (Calderon-Margalit et al., 2005), hospitals, cruise ships, schools, restaurants, and catered events (Parashar et al., 1998; Rutjes et al., 2006). Contamination of all types of food products with NoV can occur either by contamination with sewage during growth or by food handlers during harvest or packaging. Typical food items implicated in
∗ Corresponding author at: Department of Environmental Health, School of Public Health, Seoul National University, 28 Yunkeun-Dong, Chongro-Ku, Seoul 110-799, South Korea. Tel.: +82 2 3668 7881; fax: +82 2 745 9104. E-mail address: [email protected] (G. Ko). 0166-0934/$ – see front matter © 2008 Published by Elsevier B.V. doi:10.1016/j.jviromet.2008.07.022
NoV outbreaks are raw or poorly cooked meat or seafood, such as shellfish, ready-to-eat food, and commodities such as fruit and vegetables, which are associated with a high risk of infection (Bosch et al., 2001; Daniels et al., 2000; Guevremont et al., 2006; Hutin et al., 1999; Le Guyader et al., 1994; Potasman et al., 2002; Rosenblum et al., 1990; Rutjes et al., 2006) because they are consumed typically without additional heat treatment. However, NoV contamination of food is demonstrated rarely during food-borne outbreaks, either because appropriate detection methods are lacking or because the culpable food samples are unavailable (Cliver, 1995). Many types of fruit, such as grapes, raspberries, and strawberries, are recognized increasingly being as the vehicles of many NoV outbreaks (Butot et al., 2007; Hill, 2003; Le Guyader et al., 2004). Therefore, the development and optimization of methods for detecting NoV on these fruit is important for preventing and investigating outbreaks of NoV. However, only a few methods have been well developed, and these have been applied only to limited types of food items, such as shellfish (Baert et al., 2007; Dubois et al., 2002; Heller et al., 1986; Jaykus et al., 1996; Kingsley and Richards, 2001; Schultz et al., 2007; Shieh et al., 1999; Sunen and Sobsey, 1999). In this study, each step in a procedure for detecting NoV on fruit was evaluated and optimized. The steps included elution of the virus from the fruit, concentration of the virus using
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polyethylene glycol (PEG) precipitation, and extraction of the viral RNA for subsequent assay by RT-PCR. The specific aims of this study were (1) to find the elution buffer providing the best recovery of NoV, (2) to determine the optimal conditions for virus concentration via PEG precipitation in the various elution buffers, and (3) to evaluate RNA extraction methods compatible with these elution and concentration procedures. 2. Methods and materials 2.1. Stool specimens and RT-PCR assay NoV/GII-4, the NoV genotype associated most often with outbreaks of food-borne illness, was obtained as a stool sample from the Korean Center for Disease Control and Prevention (KCDC). The stool sample was diluted serially, and the concentration of NoV in the stool sample was quantified using an RT-PCR assay. The viral RNA was extracted from 140 l of 10% fecal suspension in phosphate-buffered saline (PBS) using a QIAamp Viral RNA Mini Kit (Qiagen, Valencia, CA, USA) according to the manufacturer’s protocol and stored at −80 ◦ C. Two sets of primers for the capsid-encoding region of the target viral genome were used for RT-PCR, as described previously (Isakbaeva et al., 2005). Briefly, the G2-SKF/G2-SKR primer sets were used to amplify the NoV/GII-4 using a One-Step RT-PCR kit (Qiagen, Valencia, CA, USA), generating PCR products of 344 bp (Kojima et al., 2002). The viral RNA was reverse-transcribed for 60 min at 42 ◦ C and then incubated for 15 min at 95 ◦ C to activate the Taq polymerase. Thermocycling conditions for the reaction consisted of 40 cycles of 1 min at 94 ◦ C, 1 min at 40 ◦ C, and 1 min at 72 ◦ C, and a final extension step at 72 ◦ C for 10 min. Negative controls were included in each RT-PCR run. Amplified products were analyzed on ethidium bromide-stained 2% agarose gels. The resulting concentration of NoV/GII-4 in the stool sample was approximately 4 × 106 RT-PCR unit/ml. All stool specimens were stored at −70 ◦ C until use. 2.2. Optimization of NoV elution from various fruit Six different elution buffers used in previous studies were evaluated (Baert et al., 2008; Dubois et al., 2002; Guevremont et al., 2006; Lewis and Metcalf, 1988; Monpoeho et al., 2001; Mullendore et al., 2001). The tested elution buffers were: (1) distilled water, (2) 3% beef extract (pH 7.1), (3) 0.05 M glycine–0.14 M NaCl (pH 7.5), (4) 2.9% tryptose phosphate broth–6% glycine, (5) 100 mM Tris–HCl (pH 9.5), and (6) 50 mM glycine–50 mM MgCl2 (pH 9.5). A 10-l aliquot of NoV-containing stool suspension (4 × 106 RT-PCR units/ml) was inoculated onto 20 g of fresh grape, fresh strawberry, or frozen raspberry. After the inoculated fruit was airdried on a clean bench for 30 min, the NoV was eluted by shaking the fruit in elution buffer at 200 rpm for 5 h at room temperature. An equal volume of chloroform was added, and the mixture was vigorously vortexed (Monpoeho et al., 2001). The solution was centrifuged at 2000 × g at 4 ◦ C for 10 min, and the supernatant was recovered. For PEG precipitation, PEG with a molecular weight (MW) of 8000 (PEG8000 ) was added to the solution at a final concentration of 8%. This solution was mixed vigorously and incubated overnight at 4 ◦ C with occasional shaking. Then, the resulting flocculence was precipitated at 12,000 × g for 30 min at 4 ◦ C, and the pellet was resuspended in 2 ml of PBS. RNA was extracted from the suspension using a QIAamp kit according to the manufacturer’s protocol. The viral RNA was amplified using a OneStep RT-PCR kit. Recovery from the various elution buffers was estimated as the percentage of NoV recovered from
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the fruit relative to the amount of NoV seeded onto the fruit. Two independent experiments were carried out. 2.3. Optimization of PEG precipitation To optimize PEG precipitation of NoV, the influence of PEG MW and the duration and temperature of incubation with PEG were examined. Each of three elution buffers (distilled water, 3% beef extract, and 0.05 M glycine–0.14 M NaCl) was tested with each fruit (grapes, strawberries, and raspberries). Each fruit was incubated with three different elution buffers at 200 rpm for 5 h. Ten l of NoV-containing stool suspension (4 × 104 RT-PCR units) was inoculated into each of the resulting elution buffers. Each of these inoculated suspensions was mixed with PEG of MW 6000, 8000, 10,000, or 20,000. The final concentration of PEG was adjusted to 8%, and the pH in the suspension was adjusted to 7.2. After vigorous mixing, the suspension was incubated with occasional shaking for either 4 h at room temperature or overnight at 4 ◦ C. The resulting flocculence was collected by centrifugation at 12,000 × g for 30 min at 4 ◦ C, and the pellets were resuspended in 2 ml of PBS. These solutions were serially diluted, and the viral RNA was extracted for use in an RT-PCR assay. Each precipitation experiment was performed in triplicate. The amount of NoV was estimated by the most probable number (MPN) method using serial 1:10 dilutions of the recovered viral RNA. The numbers of RT-PCR-positive or -negative dilution samples from three experiments were entered into an MPN computation program to calculate the concentration and confidence limits of recovered NoV RNA (De Man, 1983). The percentage recovery was calculated by dividing the number of RT-PCR units of NoV recovered by the number of units used for inoculation. 2.4. Optimization of RNA extraction Five different methods of viral RNA extraction were evaluated: (1) heat-release, (2) QIAamp Viral RNA Mini Kit (Qiagen, Valencia, CA, USA), (3) magnetic beads (MagExtractor Viral RNA Purification Kit, Toyobo, Osaka, Japan), (4) TRIzol reagent (Invitrogen, Carlsbad, CA, USA), and (5) immunomagnetic separation with magnetic Dynabeads (M-280 Tosylactivated, Dynal Biotech, Norway). A 10l aliquot of NoV-containing stool suspension was inoculated onto each of the tested fruit (grape, strawberry, and raspberry), and the virus was eluted using 3% beef extract as described above. After the eluted virus was precipitated with PEG10,000 for 4 h at 4 ◦ C, the viral RNA was extracted using one of the above methods, and 2.5 l of the extracted RNA was used in a 25 l RT-PCR reaction. The virus was stored at −80 ◦ C until use in the RT-PCR reaction. Each method was tested independently three times. For heat-release, the PEG-precipitated virus was heated to 95 ◦ C for 10 min and immediately chilled on ice. For the magnetic bead method, the viral RNA was extracted using a MagExtractor Viral RNA Purification Kit according to the manufacturer’s instructions. For RNA extraction using the QIAamp kit, the experiment was performed following the manufacturer’s protocol. Briefly, 140 l of eluted virus suspension was mixed with 560 l of guanidinium thiocyanate solution, followed by neutralization with an equal volume of 96–100% ethanol. The viral RNA was purified further using a QIAamp Mini Column (Qiagen, Valencia, CA, USA). For RNA extraction using TRIzol, the PEG-precipitated virus was resuspended in 2 ml of PBS, and 300 l of the suspension was added to 700 l of TRIzol reagent. After vortexing, the mixture was allowed to stand for 5 min at room temperature. Then, 150 l of chloroform was added to the solution, and the mixture was allowed to stand for 15 min at room temperature. After centrifugation at 108 × g for 15 min at 4 ◦ C, the supernatant fraction was transferred
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into a microtube with an equal volume of isopropanol, incubated for 2 h at −20 ◦ C, and centrifuged at 10,844 × g for 30 min at 4 ◦ C. The resulting pellet was washed with 1 ml of 75% ethanol, air-dried, and resuspended in 15 l of diethyl pyrocarbonate (DEPC)-treated water. For immunomagnetic separation, a 1 mg/ml stock suspension solution of Dynabeads was thoroughly resuspended by pipetting or vortexing for approximately 1 min, and then 33 l of beads were fixed in a magnetic particle concentrator (Invitrogen, Carlsbad, CA, USA) for ∼2 min until the beads migrated to the side of the tube and the liquid became clear. The supernatant fraction was removed carefully from the opposite side of the tube, leaving the beads undisturbed. This washing step was performed three times with 0.1 M Na-phosphate buffer (pH 7.4) at room temperature. After washing, the beads were resuspended gently in 1 ml of 0.1 M Na-phosphate buffer (pH 7.4) containing 5–10 l of rabbit norovirus capsid IgG antiserum. The beads were incubated with the antiserum for 16–24 h at room temperature with slow-tilt rotation on an MX4 sample mixer (Invitrogen, Carlsbad, CA, USA). The uncoupled IgG was removed by washing the beads four times with PBS (pH 7.4) containing 0.1% (w/v) bovine serum albumin (BSA) (Sigma, St. Louis, MO, USA) at 4 ◦ C. To prepare the eluted virus for immunomagnetic separation, the eluate was extracted with a 1/10th-volume of chloroform, PEG-precipitated as described above, and resuspended in 1.5 ml of PBS. The antibody-bound Dynabeads were gently mixed with 1.5 ml of resulting resuspension and incubated with rotation on the MX4 mixer for 1 h at room temperature. After incubation, the Dynabeads were separated using the MPC as described above, washed three times with PBS containing 0.1% (w/v) BSA, resuspended in 50 l of DEPC-treated water, and transferred to an Eppendorf tube for RT-PCR assays. 2.5. Data analysis PEG precipitation methods were compared using analysis of variance (ANOVA) or Kruskal–Wallis nonparametric tests. The different nucleic acid extraction methods were compared using the Mann–Whitney test. All statistical analysis was performed using SPSS software 11.0 (SPSS Inc., Chicago, IL, USA). Differences were considered statistically significant at P < 0.05. 3. Results 3.1. Elution buffers Six different elution buffers were tested for their ability to elute NoV from the surface of grapes, strawberries, and raspberries (Table 1). Overall, the virus was eluted more efficiently from grape than from strawberry. The strawberry recovery rate tended
to be at or below the limit of detection (8.5% recovery). For grapes, the 50 mM glycine–50 mM MgCl2 (pH 9.5) buffer was least efficient at recovering the virus. For strawberries, distilled water, 0.05 M glycine–0.14 M NaCl (pH 7.5), and 2.9% tryptose phosphate–6% glycine had recoveries below the detection limit (8.5%). For both grapes and strawberries, the highest recovery was obtained with 3% beef extract. 3.2. PEG precipitation The influence of PEG MW on the efficacy of NoV precipitation in various elution buffers is shown in Table 2. Of the four different PEGs tested, PEG10,000 recovered the most virus (17.8%). The other three PEGs (PEG6000 , PEG8000 , and PEG20,000 ) had average recoveries of 9.2%, 15.5%, and 7.2%, respectively. The average recovery ranges were 4.3–19.9% for grape, from below the limit of detection (2.6%) to 50.4% for strawberry, and 6.6–9.4% for raspberry. In all PEG MW experiments, the elution buffer with the highest recovery was 3% beef extract. The highest recovery from strawberry was achieved using 3% beef extract and PEG10,000 , but the recovery differences among the different PEGs were not statistically significant (ANOVA test, P = 0.67). The duration of the PEG precipitation incubation also affected the recovery of NoV (Table 3). Overall, better recovery was achieved from the 4 h process than from the overnight process. Again, 3% beef extract was the best elution buffer, regardless of conditions. For 4 h precipitation, PEG of lower MW was associated with less efficient recovery; in 3% beef extract, PEG6000 and PEG8000 recovered about 12.9% and 8.6% of the virus, respectively, whereas PEG10,000 and PEG20,000 recovered about 51.9% and 58.4% of the virus, respectively. However, for the lengthier precipitation (overnight), recovery was highest with PEG10,000 . The Kruskal–Wallis nonparametric comparisons test showed no significant difference between the overnight and 4 h averages (P = 0.543). 3.3. RNA extraction methods The NoV recovery rates achieved using five different viral RNA extraction methods are shown in Table 4. Of these methods, the QIAamp Viral RNA Mini Kit resulted in the best recovery; for grapes, this kit recovered 80% of the RNA (as determined using the MPN index to determine the most probable number of organisms per unit volume of the original sample), followed by the methods using heat-release, immunomagnetic separation, Toyobo magnetic beads, and TRIzol. Recovery rates from strawberries and raspberries were generally lower than those from grapes but similar to each other. For strawberry and raspberry, the QIAamp kit had recovery rates of 8% and 6%, respectively, and for the other RNA extraction methods, rates ranged from undetectable (TRI-
Table 1 Efficiency of norovirus (NoV) elution from grape and strawberry for six different elution buffers Elution buffer
Elution efficiency (%)a Grape
Strawberry
Distilled water 3% Beef extract 0.05 M Glycine–0.14 M NaCl (pH 7.5) 2.9% Tryptose phosphate–6% glycine 100 mM Tris–HCl (pH 9.5) 50 mM Glycine–50 mM MgCl2 (pH 9.5)
21 21 21 21 21 2.1
<8.5b 85 <8.5 <8.5 8.5 8.5
Overall average recovery
17.9
21.3
a
The elution efficiency was calculated as the percentage of NoV recovered compared with the initial amount of NoV seeded. Elution efficiencies shown are averages of duplicate experiments. b Beneath the limit of detection (8.5%).
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Table 2 Influence of PEG molecular weight on NoV precipitation from various elution buffers Type of Fruit
Elution buffera
Grape
A B C
Strawberry
A B C
Raspberry
A B C
PEG molecular weight 6000
8000
10,000
20,000
<2.6 (0.4–7.7) 19.7 (3.4–100) <2.6 (0.4–7.7)
7.7 (0.9–30.9) 19.7 (3.4–103) 3.4 (0.4–17.2)
3.4 (0.4–17.2) 19.7 (3.4–100) 3.4 (0.4–17.2)
12.9 (2.6–39.8) 18 (3.4–40.3) <2.6 (0.4–7.7)
6 (0.9–19.7) 79.8 (12.9–100) <2.6 (0.4–7.7)
3.4 (0.4–17.2) 85.8 (30.9–100) <2.6 (0.4–7.7)
7.7 (0.9–30.9) 18 (3.4–40.3) <2.6 (0.4–7.7)
18 (3.4–100) 9.4 (2.6–30.9) 7.7 (0.9–30.9)
12.9 (2.6–39.8) 6 (0.9–19.7) 7.7 (0.9–30.9)
3.4 (0.4–17.2) 9.4 (2.6–30.9) 7.7 (0.9–30.9)
3.4 (0.4–17.2) 3.4 (0.4–17.2) 3.4 (0.4–17.2)
3.4b (0.4–17.2)c 3.4 (0.4–17.2) 7.7 (0.9–30.9)
Overall a b c
9.2
15.5
17.8
7.2
A: distilled water; B: 3% beef extract; C: 0.05 M glycine–0.14 M NaCl. The percent recovery (%) of NoV recovered was calculated using the initial seeding of NoV as 100%. 95% confidence interval. Percent recovery of inoculated NoV was estimated from three independent experiments using the MPN table.
Table 3 Influence of PEG incubation period on NoV precipitation from various elution buffers PEG molecular weight
Elution buffera
6,000
A B C
8,000
Overnight precipitation of PEG
4 h precipitation of PEG
Strawberry
Raspberry
Strawberry
Raspberry
12.9b (2.6–37.8)c 18 (3.4–40.3) <2.6 (0.4–7.7)
18 (3.4–40.3) 9.4 (2.6–30.9) 7.7 (0.9–30.9)
3.4 (0.4–17.2) 12.9 (2.6–37.8) 18 (3.4–40.3)
12.9 (2.6–37.8) 12.9 (2.6–37.8) 12.9 (2.6–37.8)
A B C
6 (0.9–19.7) 79.8 (12.9–100) <2.6 (0.4–7.7)
12.9 (2.6–37.8) 6 (0.9–19.7) 7.7 (0.9–30.9)
3.4 (0.4–17.2) 7.7 (0.9–30.9) 12.9 (2.6–37.8)
7.7 (0.9–30.9) 9.4 (2.6–30.9) 6 (0.9–19.7)
10,000
A B C
3.4 (0.4–17.2) 85.8 (30.9–100) <2.6 (0.4–7.7)
3.4 (0.4–17.2) 9.4 (2.6–30.9) 7.7 (0.9–30.9)
3.4 (0.4–17.2) 18 (3.4–40.3) 12.9 (2.6–37.8)
3.4 (0.4–17.2) 85.8 (30.9–100) 3.4 (0.4–17.2)
20,000
A B C
7.7 (0.9–30.9) 18 (3.4–40.3) <2.6 (0.4–7.7)
3.4 (0.4–17.2) 3.4 (0.4–17.2) 3.4 (0.4–17.2)
<2.6 (0.4–7.7) 36.9 (6–100) 3.4 (0.4–17.2)
12.9 (2.6–37.8) 79.8 (12.9–100) 3.4 (0.4–17.2)
20.2
7.7
11.9
30
Overall a b c
A: distilled water; B: 3% beef extract; C: 0.05 M glycine–0.14 M NaCl. The percent recovery (%) of NoV recovered was calculated using the initial seeding of NoV as 100%. 95% confidence interval. Percent recovery of inoculated NoV was estimated from three independent experiments using the MPN table.
zol) to 8% (heat-release). Some of the RNA extraction methods differed significantly from the average in their recovery efficiencies (Kruskal–Wallis test, P = 0.025). Significant differences were observed between the QIAamp kit and both the Toyobo MagEx-
tractor kit (P = 0.046) and the TRIzol method (P = 0.05). However, no significant difference was observed between the QIAamp kit and the heat-release or immunomagnetic separation methods (P > 0.05).
Table 4 Recovery of NoV using five different RNA extraction methods RNA extraction methods
Fruit
Percentage recovery (%)a (95% confidence limits)
P-valueb
Grape Strawberry Raspberry
80 (12.9–100) 8 (0.9–19.7) 6 (2.6–9.3)
n/a
QIAampViral RNA Mini Kit
Grape Strawberry Raspberry
14 (2–42) 8 (1–40) 6 (1–26)
0.658
Heat-release
Grape Strawberry Raspberry
1.8 (0.2–7.2) 1.8 (0.2–7.2) 4.2 (0.8–9.4)
0.046
Toyobo Mag Extractor
Grape Strawberry Raspberry
0.4 (0.1–2) 1.5 (0.3–4.4) <0.3 (<0.3)
0.050
Trizol reagent
Grape Strawberry Raspberry
8.6 (1.4–42) 3 (0.6–8.8) 3 (0.6–8.8)
0.121
Immuno-magnetic separation a b
The percentage of NoV recovered was calculated using the initial seeding of NoV as 100%. Recovered NoV was estimated by MPN table from three independent experiments. P-value was estimated by Mann–Whitney test. Comparison was made between QIAampViral RNA Mini Kit and the other nucleic acid extraction methods.
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Fig. 1. Summary of optimized methods and their percent recoveries for detecting NoV on fruit.
4. Discussion Despite the importance of NoV in causing food-borne illness, methods for detecting NoV in foods have not been well studied. In most NoV outbreaks, investigators were unable to identify NoV in the suspected foods (Guevremont et al., 2006) because (1) the suspected food was unavailable; (2) NoV cannot be cultivated using typical methods; and (3) methods for concentrating NoV from food items are inefficient (Rutjes et al., 2006). Unlike the many bacterial food-borne pathogens that can be cultivated and enriched in culture media, NoV is neither cultivated nor enriched in vitro. For these reasons, detection and identification of NoV requires elution from contaminated foods and then concentrated. Typically, the concentration of NoV on foods is low, and various chemical components present in foods can inhibit molecular assays. All of these factors stress the importance of optimizing elution and concentration methods for detecting NoV in food. The typical procedure for detecting NoV on food involves elution, concentration by PEG precipitation, RNA extraction, and RT-PCR assay. In this study, the methods were characterized and optimized for elution of NoV contaminants from various fruit, as well as for concentration of, and RNA extraction from, eluted NoV. Most previous studies have focused on detecting NoV in shellfish. However, fruit and vegetables are among the most culpable food items in a number of food-borne outbreaks (Butot et al., 2007). Therefore, it is important to evaluate the efficiency of NoV recovery from various fruit. To identify the most effective elution buffer, all elution buffers that had been studied previously were tested (Baert et al., 2008; Dubois et al., 2002; Guevremont et al., 2006; Lewis and Metcalf, 1988; Monpoeho et al., 2001; Mullendore et al., 2001). Of these six elution buffers, 3% beef extract eluted NoV most effectively, probably because of its high protein concentration. In a recent study of NoV elution from fresh strawberries and frozen raspberries, the elution efficiency of 100 mM Tris buffer ranged from 0.42% to 11.2% (Butot et al., 2007), which is consistent with this study (8.5%). The addition of 1% beef extract to the Tris buffer increased the elution efficiency by 2.2- and 3.6-fold in strawberry and raspberry, respec-
tively. These results are consistent with current study suggesting that certain protein components of beef extract promote elution of NoV from berries. This study is the first to investigate the effect of both PEG MW and incubation period on viral precipitation using PEG. PEG precipitation is often used to concentrate viruses in solution because it is an easy and gentle method that can be performed at neutral pH and at high ionic concentrations without other organic materials (Lewis and Metcalf, 1988). Although it might be suspected that increasing the PEG MW might increase the recovery of NoV from eluates, the results do not support this idea. It was also found that increasing the duration of the precipitation incubation was unnecessary. The viral recovery rate provided by PEG precipitation in this study (12.4%) was lower than in a previous study of hepatitis A virus (13–27%) (Mullendore et al., 2001) but higher than in previous NoV studies (1–7%) (Butot et al., 2007; Rutjes et al., 2006). These differences are probably a result of differences in the protein and other components of the elution buffers. In this study, PEG precipitation was a highly effective method for concentrating NoV eluted in 3% beef extract, again probably because the high protein concentration in beef extract facilitates flocculation of NoV by PEG. Several food components are known to inhibit molecular assays such as RT-PCR (Lewis and Metcalf, 1988; Schwab and McDevitt, 2003). Viral detection on berries (such as raspberries and strawberries) can be particularly problematic due to the presence of both chemical inhibitors and a low pH (Butot et al., 2007). To date, NoV has been identified in only a few vegetables or berry samples contaminated naturally (Calder et al., 2003; Hernández et al., 1997; Kobayashi et al., 2004; Le Guyader et al., 2004). In this study, a procedure consisting of virus elution with 3% beef extract, virus precipitation with PEG10,000 for 4 h at room temperature, and extraction of the viral RNA using a QIAamp Viral RNA Mini Kit efficiently recovered and detected NoV on fruit (Fig. 1). In previous studies, beef extract was found to contain many inhibitors of RTPCR (Katayama et al., 2002; Schwab and McDevitt, 2003), but it did not significantly inhibit RT-PCR in this study, probably because the inhibitory factors were removed effectively in the viral RNA extraction procedure.
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Five different methods for extracting RNA from NoV were evaluated. The factors that determine the usefulness of viral RNA extraction methods are their compatibility with prior elution and concentration steps, their effectiveness for extracting RNA from the viral particles, and their ability to remove inhibitors of RT-PCR. Of the extraction methods tested in this study, the best recovery rate was obtained with the QIAamp Viral RNA Mini Kit, followed by immunomagnetic separation. However, the success of the latter method is dependent on the type and quality of the anti-NoV antibody used. Additional validation of antibodies against different types of NoV should be performed in the future. Although an RT-PCR assay to detect NoV was used in this study, other analytical methods, such as electron microscopy or enzyme-linked immunoassay, could also be used. Of the various possible analytical methods for NoV, RT-PCR is most commonly used because it is rapid and has high sensitivity and specificity. In this study, the percentage recovery was quantified by serial dilution and conventional RT-PCR assays, but a more quantitative method, such as real-time RT-PCR assay, could be used instead. One of the limitations of current method is that it provides no information about the infectivity of NoV, because the molecular assay does not differentiate infectious viruses from noninfectious ones. Straub et al. (2007) recently described a three-dimensional in vitro cultivation system for NoV, but this system is difficult and expensive to use. In addition, they reported that only a few types of NoV could be cultivated using this method. Alternatively, murine norovirus (MNV) might be used as a surrogate for human NoV. MNV is easily cultivated and plaque-assayed using the RAW264.7 cell line, which permits quantitation by infectivity (Karst et al., 2003; Thackray et al., 2007). However, MNV might have different surface characteristics, and surrogate viruses always have limitations. Therefore, finding elution and concentration methods for human NoV that are compatible with RT-PCR assays is of practical importance. Currently, there is no standardized method for detecting NoV contamination of various fruit. In light of the importance of NoV in food-borne illnesses, evaluation and optimization of methods for detecting NoV contamination of food items are matters of great urgency. In this study, optimal procedures were determined for detecting NoV on fruit often responsible for NoV-associated outbreaks (Fig. 1). This method will be useful for investigation of food-borne illness and for monitoring food. Further studies are warranted for optimizing the elution and concentration procedures for other important food items. Acknowledgment This study was partially funded by the Korean Food and Drug Administration (grant #06042). References Baert, L., Uyttendaele, M., Debevere, J., 2007. Evaluation of two viral extraction methods for the detection of human noroviruses in shellfish with conventional and real-time reverse transcriptase PCR. Lett. Appl. Microbiol. 44, 106–111. Baert, L., Uyttendaele, M., Debevere, J., 2008. Evaluation of viral extraction methods on a broad range of ready-to-eat foods with conventional and real-time RT-PCR for norovirus GII detection. Int. J. Food Microbiol. 123, 101–108. Bosch, A., Sanchez, G., Le Guyader, F., Vanaclocha, H., Haugarreau, L., Pinto, R.M., 2001. Human enteric viruses in Coquina clams associated with a large hepatitis A outbreak. Water Sci. Technol. 43, 61–65. Butot, S., Putallaz, T., Sanchez, G., 2007. Procedure for rapid concentration and detection of enteric viruses from berries and vegetables. Appl. Environ. Microbiol. 73, 186–192. Calder, L., Simmons, G., Thornley, C., Taylor, P., Pritchard, K., Greening, G., Bishop, J., 2003. An outbreak of hepatitis A associated with consumption of raw blueberries. Epidemiol. Infect. 131, 745–751.
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