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J Virol Methods. 2010 Sep;168(1-2):228-32. doi: 10.1016/j.jviromet.2010.06.011. Epub 2010 Jun 25.

Discrimination of infectious bacteriophage T4 virus by propidium monoazide real-time PCR.

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Laboratori de Microbiologia Sanitària i Mediambiental (MSM-Lab) - Aquasost. UNESCO Chair in Sustainability, Universitat Politècnica de Catalunya, Violinista Vellsolá 37, Terrassa, Barcelona, Spain.


The advent of quantitative PCR has improved the detection of human viral pathogens in the environment. However, a serious limitation of this method may arise from the inability to discriminate between viruses that are infectious and viruses that have been inactivated and do not represent a human health hazard. To assess whether propidium monoazide (PMA) pre-treatment is a good approach to inhibiting DNA amplification from non-infectious viruses, bacteriophage T4 survival was measured using cell culture titration and real-time PCR with and without PMA pre-treatment. Heat (85 degrees C) and proteolysis methods were carried out. After these inactivation treatments, the results indicated that the PMA pre-treatment approach is not appropriate for differentiating infectious viruses. However, when a heat treatment at 110 degrees C was undertaken, PMA pre-treatment did allow differentiation of non-infectious from infectious viruses. In this case, effective binding of PMA to bacteriophage T4 DNA could be taken to indicate capsid damage. Therefore, PMA pre-treatment may be appropriate for assessing effective disinfection treatments and for a more reliable understanding of the factors that contribute to viral inactivation through capsid damage monitoring. The PMA-PCR approach could be useful as a rapid and inexpensive analytical tool for screening and evaluation of the efficacy of disinfectants.

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