Skip to main content
Log in

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

Food and Environmental Virology Aims and scope Submit manuscript

Cite this article


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.

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

Access this article

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


  • 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.

    Article  PubMed  CAS  Google Scholar 

  • 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.

    Article  PubMed  Google Scholar 

  • 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.

    PubMed  CAS  Google Scholar 

  • 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.

    PubMed  CAS  Google Scholar 

  • Chumakov, K. M. (1994). Reverse-transcriptase can inhibit PCR and stimulate primer-dimer formation. PCR Methods and Applications, 4(1), 62–64.

    Article  PubMed  CAS  Google Scholar 

  • 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.

    Article  Google Scholar 

  • Fehlmann, C., Krapf, R., & Solioz, M. (1993). Reverse-transcriptase can block polymerase chain-reaction. Clinical Chemistry, 39(2), 368–369.

    PubMed  CAS  Google Scholar 

  • 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.

    Article  PubMed  CAS  Google Scholar 

  • 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.

    Article  PubMed  CAS  Google Scholar 

  • 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 

  • 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.

    Article  PubMed  CAS  Google Scholar 

  • 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.

    Article  PubMed  Google Scholar 

  • 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.

    Article  PubMed  CAS  Google Scholar 

  • 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.

    Article  PubMed  Google Scholar 

  • 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.

    Article  PubMed  CAS  Google Scholar 

  • 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.

    Article  PubMed  CAS  Google Scholar 

  • 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.

    Article  PubMed  Google Scholar 

  • 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 

  • WHO. (2007). Combating waterborne disease at the household level. Geneva: WHO Press.

    Google Scholar 

  • Wilson, I. G. (1997). Inhibition and facilitation of nucleic acid amplification. Applied and Environmental Microbiology, 63(10), 3741–3751.

    PubMed  CAS  Google Scholar 

  • Wolin, C. D., & Franciskovich, P. P. (1995). mRNA purification. US Patent 5,459,253, 17 Oct 1995.

  • 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.

    Article  PubMed  CAS  Google Scholar 

  • 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.

    Article  PubMed  CAS  Google Scholar 

Download references


This research has been carried as part of research that was funded by the Korean Church of Brussels, Mangu Jeja Church, Seoul, Korea, and the Netherlands Ministry of Development Cooperation (DGIS) through the UNESCO-IHE Partnership Research Fund. It was carried out in the framework of the research project “Addressing the Sanitation Crisis in Unsewered Slum Areas of African Mega-cities” (SCUSA).

Author information

Authors and Affiliations


Corresponding author

Correspondence to J. W. Chung.

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Chung, J.W., Foppen, J.W. & Lens, P.N.L. Development of Low Cost Two-Step Reverse Transcription-Quantitative Polymerase Chain Reaction Assays for Rotavirus Detection in Foul Surface Water Drains. Food Environ Virol 5, 126–133 (2013).

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: