Discrimination of viable Acanthamoeba castellani trophozoites and cysts by propidium monoazide real-time polymerase chain reaction.

Abstract

Even though the advent of quantitative polymerase chain reaction (PCR) has improved the detection of pathogen microorganisms in most of areas of microbiology, a serious limitation of this method may arise from the inability to discriminate between viable and nonviable pathogens. To overcome it, the use of real-time PCR and selective nucleic acid intercalating dyes like propidium monoazide (PMA) have been effectively evaluated for different microorganisms. To assess whether PMA pretreatment can inhibit PCR amplification of nonviable amoeba DNA, Acanthamoeba castellani survival was measured using cell culture and real-time PCR with and without PMA pretreatment. Autoclave and contact lens disinfecting solutions were used to inactivate amoebae. After these inactivation treatments, the results indicated that the PMA pretreatment approach is appropriate for differentiating viable A. castellani, both trophozoites and cysts. Therefore, the PMA-PCR approach could be useful as a rapid and sensitive analytical tool for monitoring treatment and disease control, assessing effective disinfection treatments, and for a more reliable understanding of the factors that contribute to the interaction amoeba-pathogenic bacteria.

• Authors: Mariana Fittipaldi, Nancy J Pino Rodriguez, Bárbara Adrados, Gemma Agustí, Gustavo Peñuela, Jordi Morató, Francesc Codony
• Journal: The Journal of eukaryotic microbiology (J Eukaryot Microbiol) 2011 Jul-Aug Vol. 58 Issue 4 Pg. 359-64 ISSN: 1550-7408 [Electronic] United States
• PMID: 21699621 (Publication Type: Journal Article, Research Support, Non-U.S. Gov't)
• Copyright: © 2011 The Author(s). Journal of Eukaryotic Microbiology © 2011 International Society of Protistologists.

Chemical References

• Azides
• Contact Lens Solutions
• DNA, Protozoan
• propidium monoazide
• Propidium

Topics

• Acanthamoeba castellanii (cytology, genetics, isolation & purification, physiology)
• Azides
• Contact Lens Solutions
• DNA, Protozoan (analysis, genetics)
• Polymerase Chain Reaction (methods)
• Propidium (analogs & derivatives)
• Staining and Labeling
• Trophozoites (cytology, physiology)