Evaluation of loop-mediated isothermal amplification for detection of Toxoplasma gondii in water samples and comparative findings by polymerase chain reaction and immunofluorescence test (IFT).
The development and evaluation of a 1-step single-tube accelerated loop-mediated isothermal amplification (LAMP) assay for the rapid detection of Toxoplasma in water samples is described. The method has been evaluated based on the amplification of B1 and TgOWP Toxoplasma genes, and it demonstrated a sensitivity detection limit of 0.1 tachyzoites' DNA for both genes. LAMP detection was evaluated and compared with nested polymerase chain reaction (PCR) in 26 water sample pellets spiked with known numbers of Toxoplasma oocysts. After DNA extraction, the detection sensitivity in spiked pellets was 100% by LAMP and 53.8% by PCR. Subsequently, 52 natural water samples of different origin were directly investigated by 3 assays: LAMP, PCR, and immunofluorescence test (IFT). Twenty-five (48%) of 52 have been found positive for Toxoplasma DNA by LAMP, whereas nested PCR products were generated in 7 of 52 (13.5%) water samples. All 52 water samples were negative for Toxoplasma by IFT. These data clearly indicate LAMP as a rapid, specific, and sensitive tool for the detection of Toxoplasma contamination in water samples.
Sotiriadou
Isaia
I
National Research Center for Protozoan Diseases, Obihiro University of Agriculture and Veterinary Medicine, Obihiro, Hokkaido 080-8555, Japan.
Karanis
Panagiotis
P
D000818:Animals; D016054:DNA, Protozoan; D004784:Environmental Monitoring / Q000379:methods; D005455:Fluorescent Antibody Technique; D021141:Nucleic Acid Amplification Techniques / Q000379:methods*; D016133:Polymerase Chain Reaction; D012680:Sensitivity and Specificity; D014122:Toxoplasma / Q000302:isolation & purification*; D014867:Water / Q000469:parasitology*
D016054:DNA, Protozoan; D014867:Water