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.
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References
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.
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.
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.
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.
Chumakov, K. M. (1994). Reverse-transcriptase can inhibit PCR and stimulate primer-dimer formation. PCR Methods and Applications, 4(1), 62–64.
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.
Fehlmann, C., Krapf, R., & Solioz, M. (1993). Reverse-transcriptase can block polymerase chain-reaction. Clinical Chemistry, 39(2), 368–369.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
WHO. (2007). Combating waterborne disease at the household level. Geneva: WHO Press.
Wilson, I. G. (1997). Inhibition and facilitation of nucleic acid amplification. Applied and Environmental Microbiology, 63(10), 3741–3751.
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.
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.
Acknowledgments
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).
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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). https://doi.org/10.1007/s12560-013-9111-7
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DOI: https://doi.org/10.1007/s12560-013-9111-7