Skip to main content

Advertisement

SpringerLink
Log in

A Rapid Single-Tube Protocol for HAV Detection by Nested Real-Time PCR

  • Original Paper
  • Published:
Food and Environmental Virology Aims and scope Submit manuscript

Abstract

Infections by food-borne viruses such as hepatitis A virus (HAV) and norovirus are significant public health concerns worldwide. Since food-borne viruses are rarely confirmed through direct isolation from contaminated samples, highly sensitive molecular techniques remain the methods of choice for the detection of viral genetic material. Our group has previously developed a specific nested real-time PCR (NRT-PCR) assay for HAV detection that improved overall sensitivity. Furthermore in this study, we have developed a single-tube NRT-PCR approach for HAV detection in food samples that reduces the likelihood of cross contamination between tubes during sample manipulation. HAV RNA was isolated from HAV-spiked food samples and HAV-infected cell cultures. All reactions following HAV RNA isolation, including conventional reverse transcriptase PCR, nested-PCR, and RT-PCR were performed in a single tube. Our results demonstrated that all the samples tested positive by RT-PCR and nested-PCR were also positive by a single-tube NRT-PCR. The detection limits observed for HAV-infected cell cultures and HAV-spiked green onions were 0.1 and 1 PFU, respectively. This novel method retained the specificity and robustness of the original NRT-PCR method, while greatly reducing sample manipulation, turnaround time, and the risk of carry-over contamination. Single-tube NRT-PCR thus represents a promising new tool that can potentially facilitate the detection of HAV in foods thereby improving food safety and public health.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1

Similar content being viewed by others

Abbreviations

HAV:

Hepatitis A virus

NRT-PCR:

Nested real-time PCR

PFU:

Plaque-forming unit

TCID:

Tissue culture infective dose

References

  • Brooks, H. A., Gersberg, R. M., & Dhar, A. K. (2005). Detection and quantification of hepatitis A virus in seawater via real-time RT-PCR. Journal of Virological Methods, 127, 109–118.

    Article  CAS  PubMed  Google Scholar 

  • Butot, S., Putallaz, T., & Sanchez, G. (2007). Procedure for rapid concentration and detection of enteric viruses from berries and vegetables. Applied and Environment Microbiology, 73, 186–192.

    Article  CAS  Google Scholar 

  • Calder, L., Simmons, G., Thornley, C., Taylor, P., Pritchard, K., Greening, G., et al. (2003). An outbreak of hepatitis A associated with consumption of raw blueberries. Epidemiology and Infection, 131, 745–751.

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • CDC (Center for Disease Control and Prevention). (2011). Viral Hepatitis Statistics and Surveillance. Retrieved September 24, 2013 from http://www.cdc.gov/hepatitis/Statistics/index.html.

  • Costafreda, M. I., Bosch, A., & Pinto, R. M. (2006). Development, evaluation, and standardization of a real-time TaqMan ® reverse transcription-PCR assay for quantification of hepatitis A virus in clinical and shellfish samples. Applied and Environment Microbiology, 72, 3846–3855.

    Article  CAS  Google Scholar 

  • Coudray, C., Merle, G., Martin-Latil, S., Guillier, L., & Perelle, S. (2013). Comparison of two extraction methods for the detection of hepatitis A virus in lettuces using the murine norovirus as a process control. Journal of Virological Methods, 193, 96–102.

    Article  CAS  PubMed  Google Scholar 

  • Daemer, R. J., Feinstone, S. M., Gust, I. D., & Purcell, R. H. (1981). Propagation of human hepatitis A virus in African green monkey kidney cell culture: Primary isolation and serial passage. Infection and Immunity, 32, 388–393.

    CAS  PubMed Central  PubMed  Google Scholar 

  • Dey, K. K., Lin, H., Borth, W. B., Melzer, M. J., & Hu, J. S. (2012). A highly sensitive single-tube nested PCR assay for the detection of pineapple mealybug wilt associated virus-2 (PMWaV-2). Journal of Virological Methods, 183, 215–218.

    Article  CAS  PubMed  Google Scholar 

  • Dix, A. B., & Jaykus, L. A. (1998). Virion concentration method for the detection of human enteric viruses in extracts of hard-shelled clams. Journal of Food Protection, 61, 458–465.

    CAS  PubMed  Google Scholar 

  • D’Souza, D. H., & Jaykus, L. A. (2002). Zirconium hydroxide effectively immobilizes and concentrates human enteric viruses. Letters in Applied Microbiology, 35, 414–418.

    Article  PubMed  Google Scholar 

  • Dubois, E., Hennechart, C., Deboosere, N., Merle, G., Legeay, O., Burger, C., et al. (2006). Intra-laboratory validation of a concentration method adapted for the enumeration of infectious F-specific RNA coliphage, enterovirus, and hepatitis A virus from inoculated leaves of salad vegetables. International Journal of Food Microbiology, 108, 164–171.

    Article  CAS  PubMed  Google Scholar 

  • Fiore, A. E. (2004). Hepatitis A transmitted by food. Clinical Infectious Diseases, 38, 705–715.

    Article  PubMed  Google Scholar 

  • Goswami, B. B. (2004). Detection and quantitation of hepatitis A virus in shellfish by the polymerase chain reaction, chap 26 Bacteriological Analytical. Manual, Food and Drug Administration.

  • Guevremont, E., Brassard, J., Houde, A., Simard, C., & Trottier, Y. L. (2006). Development of an extraction and concentration procedure and comparison of RT-PCR primer systems for the detection of hepatitis A virus and norovirus GII in green onions. Journal of Virological Methods, 134, 130–135.

    Article  CAS  PubMed  Google Scholar 

  • Hartman, G., Lau, H., & Lin, A. (2005). A Rapid One-Step RT-PCR for the Detection and Screening of Hepatitis A Virus. FDA LIB, 4360, 1–10.

    Google Scholar 

  • Hewitt, J., & Greening, G. E. (2004). Survival and persistence of norovirus, hepatitis A virus, and feline calicivirus in marinated mussels. Journal of Food Protection, 67, 1743–1750.

    PubMed  Google Scholar 

  • Hu, Y., & Arsov, I. (2009). Nested real-time PCR for hepatitis A detection. Letters in Applied Microbiology, 49, 615–619.

    Article  CAS  PubMed  Google Scholar 

  • Jacobsen, K. H., & Wiersma, S. T. (2010). Hepatitis A virus seroprevalence by age and world region, 1990 and 2005. Vaccine, 28, 6653–6657.

    Article  PubMed  Google Scholar 

  • Jothikumar, N., Cromeans, T. L., Sobsey, M. D., & Robertson, B. H. (2005). Development and evaluation of a broadly reactive TaqMan ® assay for rapid detection of hepatitis A virus. Applied and Environment Microbiology, 71, 3359–3363.

    Article  CAS  Google Scholar 

  • Kingsley, D. H., & Richards, G. P. (2001). Rapid and efficient extraction method for reverse transcription-PCR detection of hepatitis A and Norwalk-like viruses in shellfish. Applied and Environment Microbiology, 67, 4152–4157.

    Article  CAS  Google Scholar 

  • Llop, P., Bonaterra, A., Penalver, J., & Lopez, M. M. (2000). Development of a highly sensitive nested-PCR procedure using a single closed tube for detection of Erwinia amylovora in asymptomatic plant material. Applied and Environment Microbiology, 66, 2071–2078.

    Article  CAS  Google Scholar 

  • Love, D. C., Casteel, M. J., Meschke, J. S., & Sobsey, M. D. (2008). Methods for recovery of hepatitis A virus (HAV) and other viruses from processed foods and detection of HAV by nested RT-PCR and TaqMan ® RT-PCR. International Journal of Food Microbiology, 126, 221–226.

    Article  CAS  PubMed  Google Scholar 

  • Moser, D. A., Neuberger, E. W., & Simon, P. (2012). A quick one-tube nested PCR-protocol for EPO transgene detection. Drug Testing and Analysis, 4, 870–875.

    Article  CAS  PubMed  Google Scholar 

  • Ott, J. J., Irving, G., & Wiersma, S. T. (2012). Long-term protective effects of hepatitis A vaccines. A systematic review. Vaccine, 31, 3–11.

    CAS  Google Scholar 

  • Rutjes, S. A., Lodder-Verschoor, F., van der Poel, W. H., van Duijnhoven, Y. T., & de Roda-Husman, A. M. (2006). Detection of noroviruses in foods: a study on virus extraction procedures in foods implicated in outbreaks of human gastroenteritis. Journal of Food Protection, 69, 1949–1956.

    PubMed  Google Scholar 

  • Sair, A. I., D’Souza, D. H., Moe, C. L., & Jaykus, L. A. (2002). Improved detection of human enteric viruses in foods by RT-PCR. Journal of Virological Methods, 100, 57–69.

    Article  CAS  PubMed  Google Scholar 

  • Sanchez, G., Bosch, A., & Pinto, R. M. (2007). Hepatitis A virus detection in food: current and future prospects. Letters in Applied Microbiology, 45, 1–5.

    Article  CAS  PubMed  Google Scholar 

  • Sanchez, G., Pinto, R. M., Vanaclocha, H., & Bosch, A. (2002). Molecular characterization of hepatitis a virus isolates from a transcontinental shellfish-borne outbreak. Journal of Clinical Microbiology, 40, 4148–4155.

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Scallan, E., Hoekstra, R. M., Angulo, F. J., Tauxe, R. V., Widdowson, M. A., Roy, S. L., et al. (2011). Foodborne illness acquired in the United States–major pathogens. Emerging Infectious Diseases, 17, 7–15.

    Article  PubMed Central  PubMed  Google Scholar 

  • Shan, X. C., Wolffs, P., & Griffiths, M. W. (2005). Rapid and quantitative detection of hepatitis A virus from green onion and strawberry rinses by use of real-time reverse transcription-PCR. Applied and Environment Microbiology, 71, 5624–5626.

    Article  CAS  Google Scholar 

  • Shieh, Y. C., Calci, K. R., & Baric, R. S. (1999). A method to detect low levels of enteric viruses in contaminated oysters. Applied and Environment Microbiology, 65, 4709–4714.

    CAS  Google Scholar 

  • Tanzer, L. R., Hu, Y., Cripe, L., & Moore, R. E. (1999). A hot-start reverse transcription-polymerase chain reaction protocol that initiates multiple analyses simultaneously. Analytical Biochemistry, 273, 307–310.

    Article  CAS  PubMed  Google Scholar 

  • Vega, E., Smith, J., Garland, J., Matos, A., & Pillaii, S. D. (2005). Variability of virus attachment patterns to butterhead lettuce. Journal of Food Protection, 68, 2112–2117.

    PubMed  Google Scholar 

  • Villar, L. M., de Paula, V. S., Diniz-Mendes, L., Lampe, E., & Gaspar, A. M. (2006). Evaluation of methods used to concentrate and detect hepatitis A virus in water samples. Journal of Virological Methods, 137, 169–176.

    Article  CAS  PubMed  Google Scholar 

  • Wheeler, C., Vogt, T. M., Armstrong, G. L., Vaughan, G., Weltman, A., Nainan, O. V., et al. (2005). An outbreak of hepatitis A associated with green onions. New England Journal of Medicine, 353, 890–897.

    Article  CAS  PubMed  Google Scholar 

Download references

Acknowledgment

The authors would like to thank Dr. Paul Morin (FDA Northeast Regional Laboratory) for critically reading the manuscript.

Author information

Authors and Affiliations

  1. FDA, Northeast Regional Laboratory, 158-15 Liberty Avenue, Jamaica, NY, USA

    Yuan Hu

  2. Department of Biology, York College, 94-20 Brewer Blvd, Jamaica, NY, USA

    Ivica Arsov

Authors
  1. Yuan Hu
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Ivica Arsov
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Yuan Hu.

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Hu, Y., Arsov, I. A Rapid Single-Tube Protocol for HAV Detection by Nested Real-Time PCR. Food Environ Virol 6, 189–195 (2014). https://doi.org/10.1007/s12560-014-9152-6

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s12560-014-9152-6

Keywords

Search

Navigation