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Evaluation of propidium monoazide-based qPCR to detect viable oocysts of Toxoplasma gondii
- Source :
- Parasitology Research, Parasitology Research, Springer Verlag (Germany), 2019, 118 (3), pp.999-1010. ⟨10.1007/s00436-019-06220-1⟩, Parasitology Research, 2019, 118 (3), pp.999-1010. ⟨10.1007/s00436-019-06220-1⟩
- Publication Year :
- 2018
-
Abstract
- Information on the viability of Toxoplasma gondii oocysts is crucial to establish the public health significance of this environmental transmission stage that can contaminate water and foods. Interest for molecular-based methods to assess viability is growing and the aim of our study was to assess, for the first time, a propidium monoazide (PMA)–qPCR approach to determine the viability of T. gondii oocysts. Untreated and heat-killed (99 °C, 5 min) oocysts were incubated with PMA, a photoreactive DNA binding dye, and analyzed by confocal microscopy and flow cytometry to characterize oocysts’ dye permeability. Different PMA concentrations (50 to 150 μM), incubation temperatures (22, 37, and 45 °C), amplicon length, selected targeted gene, and dyes (PMA, PMAxx™) were evaluated to define optimal conditions to discriminate specifically viable oocysts by PMA–qPCR. In theory, PMA binding to DNA would inhibit PCR amplification in dead but not in viable oocysts. Incubation at 22 °C with 100 μM PMA coupled to qPCR targeting a 123-bp sequence of the 529-bp repeat element allowed the distinction between viable and heated oocysts. However, the reduction of viability following heating of oocysts at high temperature was slight and, contrarily to reverse transcriptase-qPCR, the qPCR signal was not totally suppressed in heated suspensions. Therefore, PMA–qPCR is able to assess the impact of heating on T. gondii oocysts’ viability but underestimates the efficacy of this treatment. The relevance of this technique to evaluate the efficacy of other inactivation processes and assess exposure of humans to this pathogen requires further investigations.
- Subjects :
- Azides
030231 tropical medicine
Polymerase Chain Reaction
030308 mycology & parasitology
law.invention
Microbiology
Flow cytometry
03 medical and health sciences
0302 clinical medicine
[SDV.MHEP.CSC]Life Sciences [q-bio]/Human health and pathology/Cardiology and cardiovascular system
law
Propidium monoazide
[SDV.MHEP.MI]Life Sciences [q-bio]/Human health and pathology/Infectious diseases
parasitic diseases
medicine
Humans
[SDV.MP.PAR]Life Sciences [q-bio]/Microbiology and Parasitology/Parasitology
Coloring Agents
Incubation
Pathogen
Polymerase chain reaction
ComputingMilieux_MISCELLANEOUS
0303 health sciences
[SDV.MHEP.ME]Life Sciences [q-bio]/Human health and pathology/Emerging diseases
Microbial Viability
General Veterinary
biology
medicine.diagnostic_test
Staining and Labeling
Oocysts
Toxoplasma gondii
General Medicine
Amplicon
biology.organism_classification
[SDV.MP.BAC]Life Sciences [q-bio]/Microbiology and Parasitology/Bacteriology
Heat inactivation
Infectious Diseases
Insect Science
[SDV.MP.VIR]Life Sciences [q-bio]/Microbiology and Parasitology/Virology
Parasitology
Toxoplasma
Propidium
Subjects
Details
- ISSN :
- 14321955 and 09320113
- Volume :
- 118
- Issue :
- 3
- Database :
- OpenAIRE
- Journal :
- Parasitology research
- Accession number :
- edsair.doi.dedup.....2d96e20631e1b70a8f0d0f1cd8a5a456