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Assessing Disinfection Efficacy of Hydrogen Peroxide Against Giardia Duodenalis Cysts: The PMA-QPCR Approach
Citation:
Kondo Nakada, L., J. Guimaraes, AND E. Villegas. Assessing Disinfection Efficacy of Hydrogen Peroxide Against Giardia Duodenalis Cysts: The PMA-QPCR Approach. Congress of the Sao Paolo State Society for Parasitology, Campinas, Sal Paolo, BRAZIL, December 07 - 09, 2018.
Impact/Purpose:
Presented at the Congress of the Sao Paolo State Society for Parasitology
Description:
Propidium Monoazide (PMA) is a photoactive dye that only penetrates permeabilized cells and, upon light exposure, covalently binds to DNA, resulting in permanently modified DNA, preventing PCR amplification [1]. The use of PMA coupled with real time quantitative polymerase chain reaction (qPCR) has already been reported to successfully differentiate viable from heat-treated Giardia cysts and Cryptosporidium oocysts [2]. In this study we have assessed the efficacy of hydrogen peroxide (10, 100 and 1000 mM / 30, 60 and 120 minutes) against G. duodenalis cysts using the PMA-qPCR approach. Two independent trials were performed for the disinfection experiments and each condition (disinfectant concentration x contact time) was analysed in triplicate. A heat treatment (80℃ / 30 minutes) was conducted to serve as a control experiment to each trial. All inactivation experiments were performed at room temperature using ≈ 2 x 106 freshly harvested G. duodenalis cysts. PMA was applied to a final concentration of 150 µM, the sample was incubated at room temperature in the dark for 5 minutes, and then PMA was photoactivated by the PMA-Lite™ LED Photolysis Device (Biotium, Inc.) for 15 minutes [2]. DNA extraction was carried out using the Power Soil DNA Isolation Kit (MO BIO), following the manufacturer’s protocol. The β-giardin gene of Giardia spp. was amplified using the primers P241 forward: 5’-CAT CCG CGA GGA GGT CAA-3’ and P241 reverse: 5’-GCA GCC ATG GTG TCG ATC T-3’, and the probe P241: 5’-FAM-AAG TCC GCC GAC AAC ATG TAC CTA ACG A-BHQ-3’ [3]. Our data show that PMA labeling prior to qPCR can increase up to 3-fold detection sensitivity of damaged G. duodenalis cysts, thus determining more accurately the disinfection efficacy. Our results demonstrated that water treatment with hydrogen peroxide (100 mM / 120 minutes) can achieve up to 3-log (99.9%) reduction of G. duodenalis cysts.