Rapid and Quantitative Detection of Hepatitis A Virus from Green Onion and Strawberry Rinses by Use of Real-Time Reverse Transcription-PCR

In this study, an immunomagnetic capture method and a real-timereverse transcription-PCR assay were used to quantify hepatitisA virus (HAV) in green onion and strawberry rinses. This combinedprotocol detected as low as 0.5 PFU HAV in produce rinses andconcentrated HAV levels up to 20-fold.

INTRODUCTION

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Introduction

Hepatitis A virus (HAV) is responsible for acute infectioushepatitis, which is endemic worldwide. Recent studies suggestedthat HAV is one of the most common causes of illness throughoral-fecal infection (16). The major routes of food contaminationinclude shellfish, fruits, and vegetables, which often becomecontaminated through contaminated water in their growing areaor during preparation through contact with fecally contaminatedsurfaces or infected food handlers (12). Recent food-borne outbreaksof hepatitis A have been associated with strawberries (2, 11,19) and green onions (9). Poor growth of most wild-type HAVstrains in vitro makes cell culture propagation of HAV difficultto perform (10). Real-time PCR is one of the most promisingdetection methods due to its sensitivity, specificity, speed,and possibility to deliver quantitative data. However, its usehas thus far been limited to clinical and environmental samples(1, 5, 22). The main obstacles concerning routine detectionof HAV in food include the presence of inhibitory substancesin the samples and the low concentration of virus recovered.Immunomagnetic separation (IMS) is one of the sample treatmentmethods that can address these limitations. It is the purposeof this study to apply a real-time reverse transcription-PCR(RT-PCR) assay combined with IMS pretreatment for quantificationof HAV in fresh produce.

Fresh produce was purchased locally. Twenty-five grams of samplewas vigorously shaken with 250 ml phosphate-buffered saline(PBS; pH 7.4) for 2 min. After centrifugation at 1,000 xg for15 min to remove heavy materials, the rinse was stored at –20°C.In all experiments, nonseeded samples were used as negativecontrols. FRhK-4 (fetal rhesus monkey kidney-derived) cellsand cytopathic HAV strain HM-175 24A were used (provided byT. Cromeans, Centers for Disease Control and Prevention, Atlanta,Ga.). HAV was propagated as described by Cromeans et al. (6),and the virus was enumerated by plaque assay following the methodof Mullendore et al. (18). Viral RNA was extracted using TRIzolreagent (Invitrogen, Rockville, MD) according to the manufacturer'sinstructions. A real-time RT-PCR assay was performed using theLightCycler HAV quantification kit (catalog no. 3246795; RocheDiagnostics, Germany) and LightCycler equipment (Roche Diagnostics)according to the manufacturer's instructions. Monoclonal antibodiesagainst HAV (anti-HAV 1009; Argene, New York) were coupled withstreptavidin-coated Dynabeads M-280 (2.8 µm in diameter;10 mg/ml) and Dynabeads MyOne (1.05 µm in diameter; 10mg/ml) (Dynal, Great Neck, NY). Antibodies were first biotinylatedusing the EZ-Link Sulfo-NHS-LC biotinylation kit (Pierce Biotechnology,Rockford, IL) and then attached to Dynabeads according to themanufacturer's instructions. To optimize the IMS conditions,different quantities of Dynabeads (12.5, 25, 37.5, 50, 67.5,75, and 100 µl) were added to 1 ml PBS seeded with 10⁵PFU HAV. After incubation at room temperature for 1.5 h thebeads were collected and washed three times with 1 ml PBS. Finally,the collected beads were resuspended in 50 µl PBS, boiledfor 5 min, and chilled on ice. The beads were harvested by centrifugationat 12,000 x g for 1 min. The supernatant was analyzed usingreal-time PCR. The same procedure was used to compare the antigencapture abilities of both bead types.

In this study, both PFU and RNA copy number were used as themeasures to indicate the amount of HAV in the real-time RT-PCRassay. In the present study PFU as a detection unit was usedto compare our data with data from previous studies (14), butsince its use is not applicable for noncytopathic wild-typeHAV strains and also because there is a variation in viral RNAcopies per PFU in different HAV strains, the use of RNA copynumber as the detection unit was chosen as an alternative wayto quantify the amount of HAV. Statistical significance wastested by one-way analysis of variance and Fisher's protectedleast significant differences (LSD) test.

Quantification with the real-time RT-PCR assay.

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Quantification with the real...

To evaluate the quantitative real-time PCR assay for HAV, serial10-fold dilutions of the HAV external standards were amplifiedby real-time RT-PCR to generate a standard curve. The standardcurve was linear over the range from 10⁶ to 10¹ copies, witha slope of -3.41 (R² = 0.996). The real-time RT-PCR assay coulddistinguish as few as 100 copies of the HAV RNA with a highdegree of confidence, as indicated by the low coefficient ofvariation (CV; <10%) (Table 1). Since the CV increased to173% at low standard RNA copy number, the linear slope between10² and 10⁶ RNA copies was used for quantification of HAV. Thedetection limit of the assay was 10 copies of viral RNA perreaction mixture, as this was the lowest dilution that couldbe consistently detected. However, a single copy of viral RNAwas also occasionally detected. These results were similar toor better than the lowest HAV detection limits reported so far(4, 5, 8, 13, 20).

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TABLE 1. Detection of HAV RNA standards in serial 10-fold dilutions using real-time RT-PCR

Effects of food components on real-time RT-PCR assay.

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Effects of food components...

The numbers of viral RNA copies detected in green onion rinseand strawberry rinse seeded with 10⁴ and 10³ PFU/ml of HAV weresignificantly lower than those in PBS controls (Table 2). Endpoint detection results showed that real-time RT-PCR could detect100 PFU/ml HAV (0.5 PFU per PCR) in all seeded green onion andstrawberry rinses, but only one-third of samples seeded with10 PFU/ml HAV were detectable, suggesting a 10-fold-lower sensitivitythan that in PBS (Table 3). These results confirm the need foradditional sample treatment prior to quantification to removePCR inhibitors (21).

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TABLE 2. HAV RNA copies detected in seeded buffer and samples using real-time RT-PCR

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TABLE 3. Detection of HAV seeded in samples by real-time RT-PCR

Detection of HAV in seeded green onion and strawberry rinses using IMS/real-time RT-PCR.

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Detection of hav in...

During the development of the IMS methods, the optimal amountof beads for binding virus in 1-ml samples was found to be 250µg for both bead types: Dynabeads M280 and MyOne (datanot shown). Data from repeated experiments showed that the amountof HAV captured by both beads was not significantly different(up to 21% for M280 and up to 27% for MyOne). Bidawid et al.(4) reported that the IMS system using M280 beads coated withK3-2F2 antibodies specifically captured 40.6% of the virus suspendedin PBS (10³ PFU/ml). Differences between the present study andothers may be a result of a number of factors, such as the typeof antibodies, the way that antibodies were coated on the beads,and the components in the samples (15, 17).

The ability of IMS to remove PCR-inhibitory materials from thesamples and concentrate virus for PCR analysis and, thus, improvethe sensitivity of detection has been reported in previous studies(1, 3, 4, 7, 13). In the present study, the sensitivity of theIMS/real-time PCR assay on food samples was determined in freshproduce rinses seeded with HAV. In both green onion and strawberrysamples, the sensitivity attained with IMS treatment was 10PFU/ml, which was 10-fold higher than in samples without IMStreatment (Table 3). Using quantification, in 1 ml strawberryrinse seeded with 10⁵ PFU of HAV, IMS-treated samples capturedmore than 20 times more HAV particles than those without IMStreatments (Table 4).

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TABLE 4. Quantification of HAV RNA in green onion and strawberry rinses by direct real-time RT-PCR and IMS/real-time RT-PCR

In summary, this study demonstrated for the first time the applicationof IMS combined with real-time RT-PCR for quantification ofHAV in food rinses. This procedure can be completed within 6hours and has the potential to be applied for routine surveillanceof HAV in fresh produce and environmental samples.

ACKNOWLEDGMENTS

This project was supported by the Ontario Ministry of Agricultureand Food.

We thank T. Cromeans and N. Jothikumar at the Centers for DiseaseControl and Prevention and A. Blake and E. Nagy at the Universityof Guelph for their helpful discussion and technical support.

FOOTNOTES

* Corresponding author. Mailing address: Department of Food Science, University of Guelph, Guelph, Ontario N1G 2W1, Canada. Phone: (519) 824-4120. Fax: (519) 824-6631. E-mail: mgriffit{at}uoguelph.ca.

REFERENCES

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