Discrimination of infectious bacteriophage T4 virus by propidium monoazide real-time PCR.
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.
Fittipaldi
Mariana
M
Laboratori de Microbiologia Sanitària i Mediambiental (MSM-Lab) - Aquasost. UNESCO Chair in Sustainability, Universitat Politècnica de Catalunya, Violinista Vellsolá 37, Terrassa, Barcelona, Spain.
Rodriguez
Nancy J Pino
NJ
Codony
Francesc
F
Adrados
Bárbara
B
Peñuela
Gustavo A
GA
Morató
Jordi
J
D001386:Azides / Q000378:metabolism*; D017122:Bacteriophage T4 / Q000254:growth & development* / Q000302:isolation & purification; D006358:Hot Temperature; D006801:Humans; D007364:Intercalating Agents / Q000378:metabolism*; D050296:Microbial Viability; D016133:Polymerase Chain Reaction / Q000379:methods*; D011419:Propidium / Q000031:analogs & derivatives* / Q000378:metabolism; D012680:Sensitivity and Specificity; D013048:Specimen Handling / Q000379:methods*; D014773:Virology / Q000379:methods*
D001386:Azides; D007364:Intercalating Agents; C533957:propidium monoazide; D011419:Propidium