Evaluation of Quantitative PCR Assays for Detection and Identification of Amoeba

Evaluation of Quantitative PCR assays for Detection and Identification of Amoebae

Tolu Awolusi*, Cassandra Karcs*, Marina E. Eremeeva

Jiann-Ping Hsu College of Public Health

Background : Free-living amoeba (FLA), particularly Acanthamoeba and Naegleria, are ubiquitous organisms distributed in soil, dust and air, and survive in brackish, fresh and sea water. Some FLAs are regarded as a significant cause of frequently fatal primary amoebic meningoencephalitis due to aquatic activities, and have become of significant public health concern in recent years. The purpose of this study is to evaluate the utility of previously published PCR-assays for detection of FLAs in environmental water samples. Methods : Specific fragments of the 18S rRNA gene of A. castellanii and N. fowleri , respectively, were amplified by PCR and cloned into the pCR2.1-TOPO plasmid by use of the TA cloning kit. The plasmids were transformed into Escherichia coli INVαF´ competent cells. Recombinant plasmids were isolated and their DNA was quantified. Multiplex TaqMan and EvaGreen PCR assays were performed according to published protocols. Results: A range of target specific primer and probe concentrations were tested to determine optimal amplification conditions for each reaction and multiplex assay. Amplification of 10-fold dilutions of standard plasmids exhibited linearity within the range of 10 ⁷ to 10 ² plasmid copies per sample. Amplification efficiency was 95%. TaqMan using either the Acanthamoeba or Naegleria primer pair could detect 10 to 100 copies of the homologous cloned plasmid. Limits of detection were determined to be equivalent to 1 trophozoite of A. castellanii when DNA of cultured amoebae was tested. Both assays could detect the presence of amoebae DNA when several environmental isolates were tested. Conclusions: Because amoebae present with a variety of morphologies that do not correlate consistently with genotype, our assays will allow us to determine the identity and quantity of amoebae in environmental samples in order to evaluate their relative prevalence and responses to environmental conditions and public health significance.

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