Determination of the viability of Toxoplasma gondii oocysts by PCR real-time after treatment with propidium monoazide

• Published in: Revista do Instituto de Medicina Tropical de São Paulo
• Main Authors: Maria Aparecida Moraes Marciano, Rafaela Aparecida Silva, Maria Luisa Barbosa, Antônio Roberto Souza Ferreira, Vera Lucia Pereira-Chioccola
• Format: Article in Journal/Newspaper
• Language: English
• Published: Universidade de São Paulo (USP) 2020
• Subjects: Viability; Toxoplasma gondii; Oocysts; Vital dyes; Propidium monoazide; Real time PCR; Arctic medicine. Tropical medicine; RC955-962; Infectious and parasitic diseases; RC109-216; Arctic
• Online Access: https://doi.org/10.1590/s1678-9946202062084; https://doaj.org/article/321489f0b78641338f2f4a4317db5701

ABSTRACT This study aimed to investigate a methodology for discriminating viable and non-viable T. gondii oocysts in water. Analyses included two steps: (i) microscopic investigation with vital dyes; (ii) molecular investigation, using a real time PCR (qPCR), after parasite treatment (or not) with propidium monoazide (PMA). The method was called qPCR-PMA. Oocyst aliquots were incubated (15 min) at 25 ºC or 100 ºC and analyzed by microscopy, after trypan blue and neutral red staining. Microscopic investigation determined viable and non-viable oocysts. For the molecular investigation, both aliquots of oocysts were treated with PMA. Non-viable oocysts, after PMA treatment, exhibited an inhibition of DNA amplification by qPCR. Although analyses were carried out with oocysts treated experimentally, these results suggest that qPCR-PMA can be a useful strategy to distinguish viable and non-viable T. gondii oocysts in water safety testing, showing if water is safe to drink.