Rotavirus in drinking water : molecular methods for measurement of infectivity

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http://urn.fi/URN:NBN:fi:hulib-201507212272
Title: Rotavirus in drinking water : molecular methods for measurement of infectivity
Author: Lee, Hyejeong
Other contributor: Helsingin yliopisto, Maatalous-metsätieteellinen tiedekunta, Elintarvike- ja ympäristötieteiden laitos
University of Helsinki, Faculty of Agriculture and Forestry, Department of Food and Environmental Sciences
Helsingfors universitet, Agrikultur- och forstvetenskapliga fakulteten, Institutionen för livsmedels- och miljövetenskaper
Publisher: Helsingfors universitet
Date: 2014
Language: eng
URI: http://urn.fi/URN:NBN:fi:hulib-201507212272
http://hdl.handle.net/10138/135966
Thesis level: master's thesis
Discipline: Mikrobiologi
Microbiology (Food Safety)
Mikrobiologia
Abstract: Quantitative reverse transcription PCR (RT-qPCR) assay is widely used for the detection of RNA viruses in environmental water samples. However, a major limitation of using RT-qPCR assay to quantify virus titers is its inability to discriminate between infectious and non-infectious viruses, resulting in overestimation of viral infectivity. Thus, the aim of this study was to develop a reliable molecular method for rotavirus detection with information on viral infectivity, and which may contribute to the development of molecular detection methods for correct estimation of infectivity of non-cultivable viruses. In experimental work, the potential of using propidium monoazide (PMA) or RNase treatment prior to RT-qPCR assay was evaluated to measure the infectivity of human rotavirus. In brief, original human rotavirus (HRV) stock was produced by propagating viruses in MA-104 cells. The virus stocks (including HRV stock A and B) were thermally treated at 80 °C at different time points. The virus titer was measured by (1) cell culture-based infectivity assay, (2) RT-qPCR assay, and (3) RT-qPCR assay with PMA or RNase pretreatment. The result of cell culture-based infectivity assay showed that heat exposure for 5 min at 80 °C was sufficient to inactivate the HRV, while RT-qPCR assay alone overestimated the viral infectivity. The results of RT-qPCR assay with pre-treatments showed that, for thermally-inactivated HRV stock A, similar level of false-positive results was reduced with PMA treatment regardless of inactivation time (ranges from 1.04 to 1.18 log10 PCR-units), while higher reduction level was observed with RNase treatment (ranges from 2.64 to 2.89 log10 PCR-units). On the other hand, the effects of both pre-treatments on thermally-inactivated HRV stock B were negligible. In conclusion, both PMA and RNase pre-treatments eliminated the false-positive results of RT-qPCR assay to some extent in defined conditions, while the discrepancy between the infectivity assay and RT-qPCR assay even with PMA or RNase treatment was observed. In order to confirm the potential of using RT-qPCR assay combined with pre-treatments to measure the infectivity of rotavirus, further studies on optimization of PMA and RNase treatments and production of optimal virus stock would be necessary.
Subject: rotavirus
RT-qPCR
PMA
RNase
viral infectivity


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