Journal of Food Hygiene and Safety (한국식품위생안전성학회지)

The Korean Society of Food Hygiene and Safety (한국식품위생안전성학회)
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pH-Dependence of RNA Extraction for Norovirus by TRIzol Method

TRIzol을 이용한 노로바이러스 RNA 추출의 pH 의존성

  • Jhon, Deok-Young (Division of Food and Nutrition, Chonnam National University)
  • 전덕영 (전남대학교 식품영양과학부)
  • Received : 2017.12.02
  • Accepted : 2018.01.16
  • Published : 2018.02.28
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Abstract

Norovirus is a leading cause of sporadic pathogenic non-bacterial gastroenteritis worldwide. For the detection of norovirus, reverse transcription real-time PCR (RT qPCR) has quickly become a major tool due to its sensitivity and specificity. However, accurate viral RNA extraction methods are essential for RT qPCR analysis. TRIzol reagents are used to extract RNA from biological materials and are therefore widely used for norovirus RNA extraction. In this study, the yield of viral RNA extraction using TRIzol from genogroup II (GII) among the human norovirus genogroup I (GI) and GII, and murine norovirus (GV) depended on the pH of the virus sample solution. The yield of RNA extraction was higher at the alkaline pH than in the acidic region compared with the Ct (threshold cycle) value of the real-time PCR. From the results of this study, it was found that the pH condition is very important for the quantitative analysis of norovirus by extracting GII RNA using TRIzol.

노로바이러스는 전 세계적으로 산발적인 발병 관련 비세균성 위장염의 주요 원인 물질이다. 노로바이러스 검출을 위해 역전사 실시간 PCR (RT qPCR)이 그 민감도와 특이성으로 인해 주요 수단으로 빠르게 자리 잡았다. 그러나 RT qPCR 분석을 위해서는 정확한 바이러스 RNA 추출방법이 필수적이다. TRIzol 시약은 생물학적 물질로부터 RNA의 추출에 이용되고 따라서 노로바이러스 RNA 추출에도 널리 사용된다. 이 연구에서는 인체 노로바이러스 유전체 그룹 I (GI) 및 유전자 그룹 II (GII)와 생쥐 노로바이러스(GV) 중에서 GII로부터의 TRIzol 을 이용한 바이러스 RNA의 추출률이 바이러스 시료 용액의 pH에 의존했다는 내용이 다루어졌다. 실시간 PCR의 Ct값으로 비교한 RNA 추출 수율은 산성 영역보다 알칼리성 pH에서 높았다. 이 연구 결과로 부터 TRIzol을 이용하여 GII RNA를 추출하여 노로바이러스를 정량적으로 분석할 때 pH조건이 대단히 중요하다는 것을 알 수 있었다.

Keywords

References

  1. van Beek J., de Graaf M., Xia M., Jiang X., Vinje J., Beersma M., de Bruin E., van de Vijver D., Holwerda M., van Houten M., Buisman A.M., van Binnendijk R., Osterhaus A.D.M.E., van der Klis F., Vennema H., Koopmans M.P.G.: Comparison of norovirus genogroup i, ii and iv seroprevalence among children in the Netherlands, 1963, 1983 and 2006. J. Gen. Virol. 97, 2255-2264 (2016). https://doi.org/10.1099/jgv.0.000533
  2. Vinje J.: Advances in laboratory methods for detection and typing of norovirus. J. Clin. Microbiol. 53, 373-381 (2015). https://doi.org/10.1128/JCM.01535-14
  3. DiCaprio E.: Recent advances in human norovirus detection and cultivation methods. Curr. Opin. Food Sci. 14, 93-97 (2017). https://doi.org/10.1016/j.cofs.2017.02.007
  4. Ettayebi K., Crawford S.E., Murakami K., Broughman J.R., Karandikar U., Tenge V.R., Neill F.H., Blutt S.E., Zeng X.-L., Qu L., Kou B., Opekun A.R., Burrin D., Graham D.Y., Ramani S., Atmar R.L., Estes M.K.: Replication of human noroviruses in stem cell-derived human enteroids. Science (New York, N.Y.) 353, 1387 (2016). https://doi.org/10.1126/science.aaf5211
  5. Jones M.K., Watanabe M., Zhu S., Graves C.L., Keyes L.R., Grau K.R., Gonzalez-Hernandez M.B., Iovine N.M., Wobus C.E., Vinj J., Tibbetts S.A., Wallet S.M., Karst S.M.: Enteric bacteria promote human and mouse norovirus infection of B cells. Science (New York, N.Y.) 346, 755 (2014). https://doi.org/10.1126/science.1257147
  6. Kageyama T., Kojima S., Shinohara M., Uchida K., Fukushi S., Hoshino F.B., Takeda N., Katayama K.: Broadly reactive and highly sensitive assay for Norwalk-like viruses based on real-time quantitative reverse transcription-PCR. J. Clin. Microbiol. 41, 1548 (2003). https://doi.org/10.1128/JCM.41.4.1548-1557.2003
  7. Vinje J., Hamidjaja R.A., Sobsey M.D.: Development and application of a capsid VP1 (region D) based reverse transcription PCR assay for genotyping of genogroup I and II noroviruses. J. Virol. Methods. 116, 109-117 (2004). https://doi.org/10.1016/j.jviromet.2003.11.001
  8. Teunis P.F.M., Moe C.L., Liu P., Miller S.E., Lindesmith L., Baric R.S., Pendu J.L., Calderon R.L.: Norwalk virus: How infectious is it? J. Med. Virol. 80, 1468-1476 (2008). https://doi.org/10.1002/jmv.21237
  9. Patel P.K., Araujo-Castillo R.: Norovirus and infection control. Hosp. Med. Clin. 6, 28-37 (2017). https://doi.org/10.1016/j.ehmc.2016.07.003
  10. Prasad B.V.V., Hardy M.E., Dokland T., Bella J., Rossmann M.G., Estes M.K.: X-ray crystallographic structure of the norwalk virus capsid. Science. 286, 287-290 (1999). https://doi.org/10.1126/science.286.5438.287
  11. Karst S.M.: Pathogenesis of noroviruses, emerging rna viruses. Viruses. 2, 748 (2010). https://doi.org/10.3390/v2030748
  12. Li X., Chen H., Kingsley D.H.: The influence of temperature, pH, and water immersion on the high hydrostatic pressure inactivation of GI.1 and GII.4 human noroviruses. Int. J. Food Microbiol. 167, 138-143 (2013). https://doi.org/10.1016/j.ijfoodmicro.2013.08.020
  13. Shoemaker G.K., van Duijn E., Crawford S.E., Uetrecht C., Baclayon M., Roos W.H., Wuite G.J.L., Estes M.K., Prasad B.V.V., Heck A.J.R.: Norwalk virus assembly and stability monitored by mass spectrometry. Mol. Cell. Proteomics. 9, 1742-1751 (2010).
  14. Chomczynski P., Sacchi N.: Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal. Biochem. 162, 156-159 (1987).
  15. Xu Q., Liu H., Yuan P., Zhang X., Chen Q., Jiang X., Zhou Y.: Development of a simplified RT-PCR without RNA isolation for rapid detection of RNA viruses in a single small brown planthopper (Laodelphax striatellus Falln). Virol. J. 14, 90 (2017). https://doi.org/10.1186/s12985-017-0732-6
  16. O'Donnell T.B., Hyde J.L., Mintern J.D., Mackenzie J.M.: Mouse norovirus infection promotes autophagy induction to facilitate replication but prevents final autophagosome maturation. Virology. 492, 130-139 (2016). https://doi.org/10.1016/j.virol.2016.02.018
  17. Cuevas J.M., Combe M., Torres-Puente M., Garijo R., Guix S., Buesa J., Rodriguez-Diaz J., Sanjuan R.: Human norovirus hyper-mutation revealed by ultra-deep sequencing. Infect. Genet. Evol. 41, 233-239 (2016). https://doi.org/10.1016/j.meegid.2016.04.017
  18. Kim H.-Y., Kwak I.-S., Hwang I.-G., Ko G.: Optimization of methods for detecting norovirus on various fruit. J. Virol. Methods. 153, 104-110 (2008). https://doi.org/10.1016/j.jviromet.2008.07.022
  19. Somura Y., Kimoto K., Oda M., Nagano M., Okutsu Y., Mori K., Akiba T., Sadamasu K.: Detection of norovirus in swab specimens of restrooms and kitchens collected for investigation of suspected food poisoning outbreaks in tokyo. Shokuhin Eiseigaku Zasshi. 58, 201-204 (2017). https://doi.org/10.3358/shokueishi.58.201
  20. Tian P., Yang D., Shan L., Wang D., Li Q., Gorski L., Lee B.G., Quinones B., Cooley M.B.: Concurrent detection of human norovirus and bacterial pathogens in water samples from an agricultural region in central California coast. Front. Microbiol. 8, 1560 (2017).
  21. Morillo S.G., Luchs A., Cilli A., do Carmo Sampaio Tavares Timenetsky M.: Rapid detection of norovirus in naturally contaminated food: Foodborne gastroenteritis outbreak on a cruise ship in Brazil, 2010. Food Environ. Virol. 4, 124-129 (2012). https://doi.org/10.1007/s12560-012-9085-x
  22. Gentry-Shields J., Jaykus L.-A.: Comparison of process control viruses for use in extraction and detection of human norovirus from food matrices. Food Res. Int. 77, 320-325 (2015).
  23. Jothikumar N., Lowther J.A., Henshilwood K., Lees D.N., Hill V.R., Vinje J.: Rapid and sensitive detection of noroviruses by using taqman-based one-step reverse transcription-PCR assays and application to naturally contaminated shellfish samples. Appl. Environ. Microbiol. 71, 1870-1875 (2005). https://doi.org/10.1128/AEM.71.4.1870-1875.2005
  24. Tung G., Macinga D., Arbogast J., Jaykus L.A.: Efficacy of commonly used disinfectants for inactivation of human noroviruses and their surrogates. J. Food Prot. 76, 1210-1217 (2013). https://doi.org/10.4315/0362-028X.JFP-12-532
  25. Ausar S.F., Foubert T.R., Hudson M.H., Vedvick T.S., Middaugh C.R.: Conformational stability and disassembly of norwalk virus-like particles: Effect of pH and temperature. J. Biol. Chem. 281, 19478-19488 (2006). https://doi.org/10.1074/jbc.M603313200
  26. Lin Y., Fengling L., Lianzhu W., Yuxiu Z., Yanhua J.: Function of VP2 protein in the stability of the secondary structure of virus-like particles of genogroup II norovirus at different pH levels: Function of VP2 protein in the stability of Nov VLPs. J. Microbiol. 52, 970-975 (2014). https://doi.org/10.1007/s12275-014-4323-6
  27. Cuellar J.L., Meinhoevel F., Hoehne M., Donath E.: Size and mechanical stability of norovirus capsids depend on pH: A nanoindentation study. J. Gen. Virol. 91, 2449-2456 (2010). https://doi.org/10.1099/vir.0.021212-0
  28. Baclayon M., Shoemaker G.K., Uetrecht C., Crawford S.E., Estes M.K., Prasad B.V.V., Heck A.J.R., Wuite G.J.L., Roos W.H.: Prestress strengthens the shell of norwalk virus nanoparticles. Nano Lett. 11, 4865-4869 (2011). https://doi.org/10.1021/nl202699r