Journal of Food Hygiene and Safety (한국식품위생안전성학회지)

The Korean Society of Food Hygiene and Safety (한국식품위생안전성학회)
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Quantitative Analysis of Feline Calicivirus Inactivation using Real-time RT-PCR

Real-time RT-PCR을 이용한 Feline Calicivirus 불활성화의 정량적 분석

  • Jeong, Hye Mi (Dept. of Food Science and Biotechnology, Chungbuk National University) ;
  • Kim, Kwang Yup (Dept. of Food Science and Biotechnology, Chungbuk National University)
  • 정혜미 (충북대학교 식품생명공학과) ;
  • 김광엽 (충북대학교 식품생명공학과)
  • Received : 2013.09.03
  • Accepted : 2014.02.25
  • Published : 2014.03.30
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Abstract

Norovirus causes acute gastroenteritis in all age groups and its food poisoning outbreaks are rapidly increasing in Korea. Reverse transcription-polymerase chain reaction (RT-PCR) is most widely used for the rapid detection of foodborne viruses due to high sensitivity. However, the false positive results of RT-PCR obtained against already inactivated viruses could be a serious drawbacks in food safety area. In this study, we investigated a method to yield true positive RT-PCR results only with alive viruses. To decompose the RNA genes from dead viruses, the enzymatic treatments composed of proteinse K and Ribonuclease A were applied to the sanitized and inactivated virus particles. Another aim of this study was to quantify the efficiencies of several major sanitizing treatments using real-time RT-PCR. Feline calicivirus (FCV) that belongs to the same Caliciviridae family with norovirus was used as a surrogate model for norovirus. The initial level of virus in control suspension was approximately $10^4$ PFU/mL. Most of inactivated viruses treated with the enzymatic treatment for 30 min at $37^{\circ}C$ were not detected in RT-PCR, Quantification results to verify the inactivation efficiencies of sanitizing treatments using real-time RT-PCR showed no false positive in most cases. We could successfully develope a numerical quantification process for the inactivated viruses after major sanitizing treatments using real-time RT-PCR. The results obtained in this study could provide a novel basis of rapid virus quantification in food safety area.

본 연구에서는 FCV 현탁액에 물리, 화학적 위생처리 후 복합효소처리라는 전처리과정을 적용한 뒤 real-time RT-PCR법을 이용하여 살균효능을 분석하였다. RT-PCR 이전에 $37^{\circ}C$에서 30분 동안 PK와 RNase A를 처리함으로써 UV, 열, 염소, 에탄올, 과초산계열 제품에 의해 불활성화 된 바이러스들은 음성 결과를 나타내었고, real-time RTP-CR법을 통해 살균 효능을 정량분석한 결과, 복합효소처리를 했을 경우 무처리구보다 더 높은 살균 효능을 보이는 것을 확인할 수 있었다. 이로써 Nuanualsuwan S. 등의 선행연구에서와 같이 PK와 RNase A로 전처리하는 단계를 통하여 물리, 화학적 위생처리에 의해 손상되지 않은 바이러스가 RT-PCR법에 의해 증폭되는 것을 방지함으로써 Real-time PCR법에 대한 검출 감도를 높일 수 있음을 확인하였다. 또한, FCV를 검출하기 위해 사용된 RT-PCR과 real-time RT-PCR 두 방법 중에서도 real-time RT-PCR법이 가장 신속하면서도 민감도 높은 결과로 도출되었다. 따라서, 유전자 분석 이전에 복합효소처리는 물리, 화학적 위생처리에 의해 불활성화 된 바이러스의 RNA가 transcription 또는 증폭되는 것을 방지하기 위한 수단으로 real-time RT-PCR법과 결합됨으로써 노로바이러스를 비롯한 식중독 바이러스를 검출하는데 효과적으로 적용될 것으로 판단된다. 또한 식품현장에서 전기영동 과정없이 신속하게 살아있는 바이러스만을 수치적으로 정량화함으로써 식품안전에도 기여할 것으로 사료된다.

Keywords

References

  1. Dean O Cliver and Hans P Riemann: Foodborne Deseases. 2nd Ed. Academin press. San Diego. Califronia. USA. pp. 161-175 (2002).
  2. Woo, G.J., Hwang, I.G., Kwak, H.S., Kim, M.G., Park, J.S., Lee, G.Y. and Koh, Y.H.: Apply of detection method and evaluation for foodborne virus. The Annual Report of KFDA, 8-1, 569-575 (2004).
  3. Jung, W.Y., Eom, J.H., Kim, B.J., Yun, M.H., Ju, I.S., Kim, C.S., Kim, M.R., Byun, J.A., Park, Y.G., Son, S.H., Lee, E.M., Jung, R.S., Na, M.A., Yuk, D.Y., Gang, J.Y. and H대, O.S.: Investigation of norovirus occurrence and influence of environmental factors in food service institiutions of chungcheong area. J. Fd Hyg. Safety, 25, 153-161 (2010).
  4. Sarbelio M, Espinosa M, Farkas T, Jiang X.: Human caliciviruses and pediatric gastoenteritis. Semin. Pediatr. Infect. Dis, 15, 237-245 (2004). https://doi.org/10.1053/j.spid.2004.07.004
  5. Koopmans M, Durzer E.: Foodborne viruses: An emerging problem. Int. J. Food Microbiol. 90, 23-41 (2004). https://doi.org/10.1016/S0168-1605(03)00169-7
  6. Blackburn, B. G., G. F. Craun, J. S. Yoder, V. Hill, R. L. Calderon, N. Chen, S. H. Lee, D. A. Levy, and M. J. Beach.: Surveillance for waterborne-disease outbreaks associated with drinking water-United States. MMWR Surveillance Summaries, 53, 23-45 (2004).
  7. Centers for Disease Control and Prevention. Available at: http://www.cdc.gov
  8. Ministry of Food and Drug Safety. Present Condition of Foodborne. Available at: http://www.mfds.go.kr
  9. Lee, S.Y., Jang, K.I., Woo, G.J., Kwak, H.S. and Kim, K.Y.: Development of protocol for the effective detection of feline calicivirus as norovirus surrogate in oyster and lettuce. KOREAN J. FOOD SCI. TECHNOL, 39, 71-76 (2007).
  10. Cho, M.G., Jeong, H.M., Ahn, J.B. and Kim, K.Y.: Detection of feline calicivirus as norovirus surrogate in food and water sources using filtration and real-time RT-PCR. Food Sci. Biotechnol, 20, 1475-1480 (2011). https://doi.org/10.1007/s10068-011-0204-5
  11. Nuanualsuwan S., Mariam T., Himathongkham S., and Cliver D. O.:. Ultraviolet inactivation of feline calicivirus, human enteric viruses and coliphages. Photochemistry and phtobiology, 76, 406-410 (2002). https://doi.org/10.1562/0031-8655(2002)0760406UIOFCH2.0.CO2
  12. Hewitt J., Rivera-Aban M., and Greening G. E.: Evalution of murine norovirus as a surrogate human norovirus and hepatitis A virus in heat inactivation studies. Journal of Applied microbiology. 107, 65-71 (2009). https://doi.org/10.1111/j.1365-2672.2009.04179.x
  13. Shin, G.A. and Mark D. Sobsey.: Inactivation of norovirus by chlorine disinfection of water. water research 42, 4562-4568 (2008). https://doi.org/10.1016/j.watres.2008.08.001
  14. Malik, Maherchandani, and Goyal.: Comparative efficacy of ethanol and isopropanol against feline calicivirus, a norovirus surrogate. Association for professionals in infection control and epidemiology. 34, 31-35 (2006).
  15. Lee J. H.: Real-time PCR in the viral hepatitis. Korean Society of Gastroenterolohy. 2, 198-206 (2003).
  16. Yang C, Jinag Y, Huang K, Zhu C, Yin Y.: Application of real-time PCR for quantitative detection of Campylobacter jejuni in poultry, milk, and environmental water. FEMS Immunol Med Mic. 38, 265-271(2003). https://doi.org/10.1016/S0928-8244(03)00168-8
  17. Manish M. Patel, Aron J. Halla, Jan Vinje, and Umesh D.: Parashara. Noroviruses: A comprehensive review. Journal of Clinical Virology 44, 1-8 (2009). https://doi.org/10.1016/j.jcv.2008.10.009
  18. Suphachai Nuanualsuwan and Dean O. Cliver.: Pretreatment to avoid positive RT-PCR result with inactivatied viruses. Journal of Virological Methods. 104, 217-225 (2002). https://doi.org/10.1016/S0166-0934(02)00089-7
  19. Kim, K.R., Kim, Y.M., Lee, E.W., Lee, D.S., Lee, M.S.: Screening of antiviral activity from natural plants against feline calicivirus. Journal of Life Science, 19, 928-933 (2009). https://doi.org/10.5352/JLS.2009.19.7.928
  20. Duizer, E., K. J. Schwab, F. H. Neill, R. L. Atmar, M. P. G. Koopman, and M. K. Estes.: Laboratory efforts to cultivate noroviruses. J. Gen. Virol. 85, 79-87 (2004). https://doi.org/10.1099/vir.0.19478-0
  21. Clarke I. N., Lambden P. R., and Caul E. O.: Human enteric RNA viruses: caliciviruses and astroviruses. In: Mahy BWJ, Collier L, eds. Topley and Wilson's microbiology and microbial infections. London: Arnold, 511-535 (1998).
  22. Koopmans M. and Durzer E.: Foodborne viruses: an emerging problem. Int. J. Food Microbiol. 90, 23-41 (2004). https://doi.org/10.1016/S0168-1605(03)00169-7
  23. Park, K.J., Ha, H.C., Kim, H.S., Chiba, K., Yeo, I.H. and Lee, S.Y.: The neuroprotective and neurotrophic effects of korean gardenia(Gardenia jasminoides ellis) in PC12h cells. Food Sci. Biotechnol. 15, 735-738 (2006).
  24. Chi, H.Y., Lee, C.H., Kim, K.H., Kim, S.I. and Chung, I.M.: Induction of apoptotic cell death by red pericarp rice(Jakwangchalbyeo) extracts. Food Sci. Biotechnol. 15, 534-542 (2006).
  25. Kang, J.O.: Quantitation of virus. Korean J Clin Microbiol, 4, 1-4 (2001).
  26. Bidawid, S., Malika, N., Adegbunrinb, O., Sattarb, S.A. and Farber, J.M.: A feline kidney cell line-based plaque assay for feline calicivirus, a surrogate for norwalk virus. J Virol. Method. 107, 163-167 (2003). https://doi.org/10.1016/S0166-0934(02)00214-8
  27. Wutzler P. and Sauerbrei A.: Virucidal efficacy of a combination of 0.2% peracetic acid and 80% (v/v) ethanol (PAAethanol) as a potential hand disinfectant. Journal of Hospital infection. 46, 304-308 (2000). https://doi.org/10.1053/jhin.2000.0850
  28. Guerreiro-Tanomaru J. M., Morqental R. D., Faria-Junior N. B., Berbert F. L., and Tanomaru-Filho M.: Antibacterial effectiveness of peracetic acid and conventional endodontic irrigants. Braz Dent J. 22, 285-287 (2011). https://doi.org/10.1590/S0103-64402011000400004
  29. Suphachai Nuanualsuwan and Dean O. Cliver.: Capsid Functions of Inactivated Human Picornaviruses and Feline Calicivirus. Applied and environmental micobiology. 69, 350-357 (2003). https://doi.org/10.1128/AEM.69.1.350-357.2003
  30. Bidavid S., Farber J.M., Sattar S.A.: Rapid concentration and detection of hepatitis A virus from lettuce and strawberry. J. Virol. Methods. 88, 175-185 (2000). https://doi.org/10.1016/S0166-0934(00)00186-5
  31. Sykes J.E., Allen J.L., Studdert P., and Browning G.F.: Detection of feline calicivirus, feline herpesvirus I and Chlamydia psittaci mucosal swabs by multiplex RT-PCR/PCR. Vet. Microbiol. 81, 95-108 (2001). https://doi.org/10.1016/S0378-1135(01)00340-6
  32. Abd-Eldaim M. M., Wilkes R. P., Thomas K. V., and Kennedy M. A.: Development and validation of a Taqman real-time reverse transcription-PCR for rapid eetection of feline calicivirus. Arch. Virol. 154, 555-560 (2009). https://doi.org/10.1007/s00705-009-0337-5
  33. Kim, H.I., Jeon, D.H., Yoon, H.J., Choi, H.C., Eom, M.O., Sung, J.H., Park, N.Y., Won, S.A., Lim, N.Y. and Lee, Y.J.: Evaluation of the efficacy of sanitizers on food contact surface using a surface test method. J Fd Hyg Safety. 23, 291-296 (2008).