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Research article

References

  1. Bianchi S, Vecchio AD, Vilariño ML, Romalde JL. 2011. Evaluation of different RNA-extraction kits for sensitive detection of hepatitis A virus in strawberry samples. Food Microbiol. 28: 38-42.
    Pubmed CrossRef
  2. Bower WA, Nainan OV, Han X, Margolis HS. 2000. Duration of viremia in hepatitis A virus infection. J. Infect. Dis. 182: 12-17.
    Pubmed CrossRef
  3. Butot S, Putallaz T, Sánchez G. 2007. Procedure for rapid concentration and detection of enteric viruses from berries and vegetables. Appl. Environ. Microbiol. 73: 186-192.
    Pubmed PMC CrossRef
  4. Cuthbert JA. 2001. Hepatitis A: old and new. Clin. Microbiol. Rev. 14: 38-58.
    Pubmed PMC CrossRef
  5. Di Pasquale S, Paniconi M, De Medici D, Suffredini E, Croci L. 2010. Duplex real-time PCR for the detection of hepatitis A virus in shellfish using feline calicivirus as a process control. J. Virol. Methods 163: 96-100.
    Pubmed CrossRef
  6. De Paula VS, Gaspar AMC, Villar LM. 2010. Optimization of methods for detecting hepatitis A virus in food. Food Environ. Virol. 2: 47-52.
    CrossRef
  7. Fuentes C, Guix S, Pérez-Rodriguez FJ, Fuster N, Carol M, Pintó RM, Bosch A. 2014. Standardized multiplex one-step qRT-PCR for hepatitis A virus, norovirus GI and GII quantification in bivalve mollusks and water. Food Microbiol. 40: 55-63.
    Pubmed CrossRef
  8. Hida K, Kulka M, Papafragkou E. 2013. Development of a rapid total nucleic acid extraction method for the isolation of hepatitis A virus from fresh produce. Int. J. Food Microbiol. 161: 143-150.
    Pubmed CrossRef
  9. Koopmans M, Duizer E. 2004. Foodborne viruses: an emerging problem. Int. J. Food Microbiol. 90: 23-41.
    CrossRef
  10. Lees D, CEN WG6 TAG4. 2010. International standardisation of a method for detection of human pathogenic viruses in molluscan shellfish. Food Environ. Virol. 2: 146-155.
    CrossRef
  11. Lees DN. 2000. Viruses and bivalve shellfish. Int. J. Food Microbiol. 59: 81-116.
    CrossRef
  12. Manso CF, Romalde JL. 2013. Detection and characterization of hepatitis A virus and norovirus in mussels from Galicia (NW Spain). Food Environ. Virol. 5: 110-118.
    Pubmed CrossRef
  13. Morales-Rayas R, Wolffs PFG, Griffiths MW. 2010. Simultaneous separation and detection of hepatitis A virus and norovirus in produce. Int. J. Food Microbiol. 139: 48-55.
    Pubmed CrossRef
  14. Nainan OV, Xia G, Vaughan G, Margolis HS. 2006. Diagnosis of hepatitis A virus infection: a molecular approach. Clin. Microbiol. Rev. 19: 63-79.
    Pubmed PMC CrossRef
  15. Papafragkou E, Plante M, Mattison K, Bidawid S, Karthikeyan K, Farber JM, Jaykus LA. 2008. Rapid and sensitive detection of hepatitis A virus in representative food matrices. J. Virol. Methods 147: 177-187.
    Pubmed CrossRef
  16. Robertson BH, Khanna B, Nainan OV, Margolis HS. 1991. Epidemiologic patterns of wild-type hepatitis A virus determined by genetic variation. J. Infect. Dis. 163: 286-292.
    Pubmed CrossRef
  17. Sánchez G, Elizaquível P, Aznar R. 2012. A single method for recovery and concentration of enteric viruses and bacteria from fresh-cut vegetables. Int. J. Food Microbiol. 152: 9-13.
    Pubmed CrossRef
  18. Stals A, Baert L, Van Coillie E, Uyttendaele M. 2012. Extraction of food-borne viruses from food samples: a review. Int. J. Food Microbiol. 153: 1-9.
    Pubmed CrossRef
  19. Summa M, von Bonsdorff CH, Maunula N. 2012. Evaluation of four virus recovery methods for detecting noroviruses on fresh lettuce, sliced ham, and frozen raspberries. J. Virol. Methods 183: 154-160.
    Pubmed CrossRef
  20. Terio V, Di Pinto A, Di Pinto P, Martella V, Tantillo G. 2010. RNA extraction method for the PCR detection of hepatitis A virus in shellfish. Int. J. Food Microbiol. 142: 198-201.
    Pubmed CrossRef
  21. Vaughan G, Xia G, Forbi JC, Purdy MA, Rossi LMG, Spradling PR, Khudyakov YE. 2013. Genetic relatedness among hepatitis A virus strains associated with food-borne outbreaks. PLoS One 8: e74546.
    Pubmed PMC CrossRef
  22. Woods JW, Escalona NG, Burkhardt III W. 2011. Direct sequencing of hepatitis A virus and norovirus RT-PCR products from environmentally contaminated oyster using M13-tailed primers. J. Virol. Methods 178: 253-257.
    Pubmed CrossRef

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Article

Research article

J. Microbiol. Biotechnol. 2016; 26(8): 1398-1403

Published online August 28, 2016 https://doi.org/10.4014/jmb.1604.04047

Copyright © The Korean Society for Microbiology and Biotechnology.

Development of a One-Step Duplex RT-PCR Method for the Simultaneous Detection of VP3/VP1 and VP1/P2B Regions of the Hepatitis A Virus

Mi-Ju Kim 1, Shin-Young Lee 1, Hyun-Joong Kim 1, Jeong Su Lee 2, In Sun Joo 2, Hyo Sun Kwak 2 and Hae-Yeong Kim 1*

1Graduate School of Biotechnology and Institute of Life Sciences & Resources, Kyung Hee University, Yongin 17104, Republic of Korea, 2Division of Food Microbiology, National Institute of Food and Drug Safety Evaluation, Cheongju 28159, Republic of Korea

Received: April 18, 2016; Accepted: May 19, 2016

Abstract

The simultaneous detection and accurate identification of hepatitis A virus (HAV) is critical in
food safety and epidemiological studies to prevent the spread of HAV outbreaks. Towards this
goal, a one-step duplex reverse-transcription (RT)-PCR method was developed targeting the
VP1/P2B and VP3/VP1 regions of the HAV genome for the qualitative detection of HAV. An
HAV RT-qPCR standard curve was produced for the quantification of HAV RNA. The
detection limit of the duplex RT-PCR method was 2.8 × 101 copies of HAV. The PCR products
enabled HAV genotyping analysis through DNA sequencing, which can be applied for
epidemiological investigations. The ability of this duplex RT-PCR method to detect HAV was
evaluated with HAV-spiked samples of fresh lettuce, frozen strawberries, and oysters. The
limit of detection of the one-step duplex RT-PCR for each food model was 9.4 × 102 copies/20 g
fresh lettuce, 9.7 × 103 copies/20 g frozen strawberries, and 4.1 × 103 copies/1.5 g oysters. Use
of a one-step duplex RT-PCR method has advantages such as shorter time, decreased cost, and
decreased labor owing to the single amplification reaction instead of four amplifications
necessary for nested RT-PCR.

Keywords: Hepatitis A virus (HAV), simultaneous detection, epidemiological study, one-step duplex reverse transcription-polymerase chain reaction (RT-PCR), reverse transcription-quantitative polymerase chain reaction (RT-qPCR)

References

  1. Bianchi S, Vecchio AD, Vilariño ML, Romalde JL. 2011. Evaluation of different RNA-extraction kits for sensitive detection of hepatitis A virus in strawberry samples. Food Microbiol. 28: 38-42.
    Pubmed CrossRef
  2. Bower WA, Nainan OV, Han X, Margolis HS. 2000. Duration of viremia in hepatitis A virus infection. J. Infect. Dis. 182: 12-17.
    Pubmed CrossRef
  3. Butot S, Putallaz T, Sánchez G. 2007. Procedure for rapid concentration and detection of enteric viruses from berries and vegetables. Appl. Environ. Microbiol. 73: 186-192.
    Pubmed KoreaMed CrossRef
  4. Cuthbert JA. 2001. Hepatitis A: old and new. Clin. Microbiol. Rev. 14: 38-58.
    Pubmed KoreaMed CrossRef
  5. Di Pasquale S, Paniconi M, De Medici D, Suffredini E, Croci L. 2010. Duplex real-time PCR for the detection of hepatitis A virus in shellfish using feline calicivirus as a process control. J. Virol. Methods 163: 96-100.
    Pubmed CrossRef
  6. De Paula VS, Gaspar AMC, Villar LM. 2010. Optimization of methods for detecting hepatitis A virus in food. Food Environ. Virol. 2: 47-52.
    CrossRef
  7. Fuentes C, Guix S, Pérez-Rodriguez FJ, Fuster N, Carol M, Pintó RM, Bosch A. 2014. Standardized multiplex one-step qRT-PCR for hepatitis A virus, norovirus GI and GII quantification in bivalve mollusks and water. Food Microbiol. 40: 55-63.
    Pubmed CrossRef
  8. Hida K, Kulka M, Papafragkou E. 2013. Development of a rapid total nucleic acid extraction method for the isolation of hepatitis A virus from fresh produce. Int. J. Food Microbiol. 161: 143-150.
    Pubmed CrossRef
  9. Koopmans M, Duizer E. 2004. Foodborne viruses: an emerging problem. Int. J. Food Microbiol. 90: 23-41.
    CrossRef
  10. Lees D, CEN WG6 TAG4. 2010. International standardisation of a method for detection of human pathogenic viruses in molluscan shellfish. Food Environ. Virol. 2: 146-155.
    CrossRef
  11. Lees DN. 2000. Viruses and bivalve shellfish. Int. J. Food Microbiol. 59: 81-116.
    CrossRef
  12. Manso CF, Romalde JL. 2013. Detection and characterization of hepatitis A virus and norovirus in mussels from Galicia (NW Spain). Food Environ. Virol. 5: 110-118.
    Pubmed CrossRef
  13. Morales-Rayas R, Wolffs PFG, Griffiths MW. 2010. Simultaneous separation and detection of hepatitis A virus and norovirus in produce. Int. J. Food Microbiol. 139: 48-55.
    Pubmed CrossRef
  14. Nainan OV, Xia G, Vaughan G, Margolis HS. 2006. Diagnosis of hepatitis A virus infection: a molecular approach. Clin. Microbiol. Rev. 19: 63-79.
    Pubmed KoreaMed CrossRef
  15. Papafragkou E, Plante M, Mattison K, Bidawid S, Karthikeyan K, Farber JM, Jaykus LA. 2008. Rapid and sensitive detection of hepatitis A virus in representative food matrices. J. Virol. Methods 147: 177-187.
    Pubmed CrossRef
  16. Robertson BH, Khanna B, Nainan OV, Margolis HS. 1991. Epidemiologic patterns of wild-type hepatitis A virus determined by genetic variation. J. Infect. Dis. 163: 286-292.
    Pubmed CrossRef
  17. Sánchez G, Elizaquível P, Aznar R. 2012. A single method for recovery and concentration of enteric viruses and bacteria from fresh-cut vegetables. Int. J. Food Microbiol. 152: 9-13.
    Pubmed CrossRef
  18. Stals A, Baert L, Van Coillie E, Uyttendaele M. 2012. Extraction of food-borne viruses from food samples: a review. Int. J. Food Microbiol. 153: 1-9.
    Pubmed CrossRef
  19. Summa M, von Bonsdorff CH, Maunula N. 2012. Evaluation of four virus recovery methods for detecting noroviruses on fresh lettuce, sliced ham, and frozen raspberries. J. Virol. Methods 183: 154-160.
    Pubmed CrossRef
  20. Terio V, Di Pinto A, Di Pinto P, Martella V, Tantillo G. 2010. RNA extraction method for the PCR detection of hepatitis A virus in shellfish. Int. J. Food Microbiol. 142: 198-201.
    Pubmed CrossRef
  21. Vaughan G, Xia G, Forbi JC, Purdy MA, Rossi LMG, Spradling PR, Khudyakov YE. 2013. Genetic relatedness among hepatitis A virus strains associated with food-borne outbreaks. PLoS One 8: e74546.
    Pubmed KoreaMed CrossRef
  22. Woods JW, Escalona NG, Burkhardt III W. 2011. Direct sequencing of hepatitis A virus and norovirus RT-PCR products from environmentally contaminated oyster using M13-tailed primers. J. Virol. Methods 178: 253-257.
    Pubmed CrossRef