Food and Environmental Virology

, Volume 5, Issue 2, pp 126–133 | Cite as

Development of Low Cost Two-Step Reverse Transcription-Quantitative Polymerase Chain Reaction Assays for Rotavirus Detection in Foul Surface Water Drains

Original Paper

Abstract

Commercial kits to determine RNA concentrations are expensive, and sometimes too expensive for laboratories working with tight budgets, especially those in developing countries. We developed, tested, and evaluated two home-made two-step reverse transcription-quantitative polymerase chain reaction assays aimed to detect rotavirus in surface water samples. A commercial one-step master kit was used for comparison. Our results indicated that the efficiency of the home-made assays was comparable to the commercial kit. Furthermore, the lowest detection limit of all assays corresponded to 10−0.2 TCID50 (50 % tissue Culture Infective Dose) per ml. The home-made assays were able to detect rotavirus concentrations in complex surface waters in a slum area in Kampala (Uganda) and their performance was comparable to the commercial kit. The total costs of the two home-made assays was 11 times less than the selected commercial kit. Although preparing home-made assays is more time consuming, the assays can be useful for cases in which consumable costs are more important than personnel costs.

Keywords

Rotavirus RT-qPCR Surface water Slums Kampala Uganda 

References

  1. Asano, K. M., de Souza, S. P., de Barros, I. N., Ayres, G. R., Silva, S. O. S., Richtzenhain, L. J., et al. (2010). Multiplex semi-nested RT-PCR with exogenous internal control for simultaneous detection of bovine coronavirus and group A rotavirus. Journal of Virological Methods, 169(2), 375–379. doi:10.1016/j.jviromet.2010.08.008.PubMedCrossRefGoogle Scholar
  2. Bengtsson, M., Hemberg, M., Rorsman, P., & Stahlberg, A. (2008). Quantification of mRNA in single cells and modelling of RT-qPCR induced noise. BMC Molecular Biology, 9, 63. doi:6310.1186/1471-2199-9-63.PubMedCrossRefGoogle Scholar
  3. Boom, R., Sol, C., Beld, M., Weel, J., Goudsmit, J., & Wertheim-van Dillen, P. (1999). Improved silica-guanidinium thiocyanate DNA isolation procedure based on selective binding of bovine alpha-casein to silica particles. Journal of Clinical Microbiology, 37(3), 615–619.PubMedGoogle Scholar
  4. Boom, R., Sol, C. J. A., Salimans, M. M. M., Jansen, C. L., Wertheimvandillen, P. M. E., & Vandernoordaa, J. (1990). Rapid and simple method for purification of nucleic-acids. Journal of Clinical Microbiology, 28(3), 495–503.PubMedGoogle Scholar
  5. Chumakov, K. M. (1994). Reverse-transcriptase can inhibit PCR and stimulate primer-dimer formation. PCR Methods and Applications, 4(1), 62–64.PubMedCrossRefGoogle Scholar
  6. Deprez, R. H. L., Fijnvandraat, A. C., Ruijter, J. M., & Moorman, A. F. M. (2002). Sensitivity and accuracy of quantitative real-time polymerase chain reaction using SYBR green I depends on cDNA synthesis conditions. Analytical Biochemistry, 307(1), 63–69.CrossRefGoogle Scholar
  7. Fehlmann, C., Krapf, R., & Solioz, M. (1993). Reverse-transcriptase can block polymerase chain-reaction. Clinical Chemistry, 39(2), 368–369.PubMedGoogle Scholar
  8. He, X. Q., Cheng, L., Zhang, D. Y., Xie, X. M., Wang, D. H., & Wang, Z. (2011). One-year monthly survey of rotavirus, astrovirus and norovirus in three sewage treatment plants in Beijing, China and associated health risk assessment. Water Science and Technology, 63(1), 191–198. doi:10.2166/wst.2011.032.PubMedCrossRefGoogle Scholar
  9. Ijzerman, M. M., Dahling, D. R., & Fout, G. S. (1997). A method to remove environmental inhibitors prior to the detection of waterborne enteric viruses by reverse transcription-polymerase chain reaction. Journal of Virological Methods, 63(1–2), 145–153. doi:10.1016/s0166-0934(96)02123-4.PubMedCrossRefGoogle Scholar
  10. Katukiza, A. Y., Temanu, H., Chung, J. W., Foppen, J. W. A., Lens, P. N. L. (2013). Genomic copy concentrations of selected waterborne viruses in a slum environment in Kampala, Uganda. Journal of Water and Health. doi:10.2166/wh.2013.184.Google Scholar
  11. Kottaridi, C., Spathis, A. T., Ntova, C. K., Papaevangelou, V., & Karakitsos, P. (2012). Evaluation of a multiplex real time reverse transcription PCR assay for the detection and quantitation of the most common human rotavirus genotypes. Journal of Virological Methods, 180(1–2), 49–53. doi:10.1016/j.jviromet.2011.12.009.PubMedCrossRefGoogle Scholar
  12. Liss, B. (2002). Improved quantitative real-time RT-PCR for expression profiling of individual cells. Nucleic Acids Research, 30(17), e89. doi:e8910.1093/nar/gnf088.PubMedCrossRefGoogle Scholar
  13. Pang, X. L. L., Lee, B., Boroumand, N., Leblanc, B., Preiksaitis, J. K., & Ip, C. C. Y. (2004). Increased detection of rotavirus using a real time reverse transcription-polymerase chain reaction (RT-PCR) assay in stool specimens from children with diarrhea. Journal of Medical Virology, 72(3), 496–501. doi:10.1002/jmv.20009.PubMedCrossRefGoogle Scholar
  14. Parashar, U. D., Burton, A., Lanata, C., Boschi-Pinto, C., Shibuya, K., Steele, D., et al. (2009). Global mortality associated with rotavirus disease among children in 2004. Journal of Infectious Diseases, 200, S9–S15. doi:10.1086/605025.PubMedCrossRefGoogle Scholar
  15. Sellner, L. N., Coelen, R. J., & Mackenzie, J. S. (1992). Reverse-transcriptase inhibits Taq polymerase activity. Nucleic Acids Research, 20(7), 1487–1490. doi:10.1093/nar/20.7.1487.PubMedCrossRefGoogle Scholar
  16. Shaw, A. E., Reid, S. M., Ebert, K., Hutchings, G. H., Ferris, N. P., & King, D. P. (2007). Implementation of a one-step real-time RT-PCR protocol for diagnosis of foot-and-mouth disease. Journal of Virological Methods, 143(1), 81–85. doi:10.1016/j.jviromet.2007.02.009.PubMedCrossRefGoogle Scholar
  17. Suslov, O., & Steindler, D. A. (2005). PCR inhibition by reverse transcriptase leads to an overestimation of amplification efficiency. Nucleic Acids Research, 33(20), e181. doi:e18110.1093/nar/gni176.PubMedCrossRefGoogle Scholar
  18. Vilagines, P., Sarrette, B., Husson, G., & Vilagines, R. (1993). Glass wool for virus concentration at ambient water pH level. Water Science and Technology, 27(3–4), 299–306.Google Scholar
  19. WHO. (2007). Combating waterborne disease at the household level. Geneva: WHO Press.Google Scholar
  20. Wilson, I. G. (1997). Inhibition and facilitation of nucleic acid amplification. Applied and Environmental Microbiology, 63(10), 3741–3751.PubMedGoogle Scholar
  21. Wolin, C. D., & Franciskovich, P. P. (1995). mRNA purification. US Patent 5,459,253, 17 Oct 1995.Google Scholar
  22. Wyn-Jones, A. P., Carducci, A., Cook, N., D’Agostino, M., Divizia, M., Fleischer, J., et al. (2011). Surveillance of adenoviruses and noroviruses in European recreational waters. Water Research, 45(3), 1025–1038. doi:10.1016/j.watres.2010.10.015.PubMedCrossRefGoogle Scholar
  23. Yang, W., Gu, A. Z., Zeng, S. Y., Li, D., He, M. A., & Shi, H. C. (2011). Development of a combined immunomagnetic separation and quantitative reverse transcription-PCR assay for sensitive detection of infectious rotavirus in water samples. Journal of Microbiological Methods, 84(3), 447–453. doi:10.1016/j.mimet.2011.01.011.PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2013

Authors and Affiliations

  1. 1.Department of Environmental Engineering and Water TechnologyUNESCO-IHE Institute for Water EducationDelftThe Netherlands
  2. 2.Department of Water Science and EngineeringUNESCO-IHE Institute for Water EducationDelftThe Netherlands
  3. 3.DelftThe Netherlands

Personalised recommendations