전자저널 > 전자저널 > Development of a real-time PCR method for detection and quantification of the parasitic protozoan Perkinsus olseni

Development of a real-time PCR method for detection and quantification of the parasitic protozoan Perkinsus olseni

Dinesh Gajamange, Jong-Man Yoon and Kyung-Il Park

Department of Aquatic Life Medicine, Kunsan National University

The objective of this study was to develop a real-time PCR method for the rapid detection and quantification of the protozoan pathogen Perkinsus olseni using a TaqMan probe. For the standard, genomic DNA was extracted from 105 in vitro-cultured P. olseni trophozoites, and then 10-fold serial dilutions to the level of a single cell were prepared. To test the reliability of the technique, triplicates of genomic DNA were extracted from 5 × 104 cells and 10-fold serial dilutions to the level of 5 cells were prepared. The standards and samples were analyzed in duplicate using an ExicyclerTM 96 real-time quantitative thermal block. For quantification, the threshold cycle (CT) values of samples were compared with those obtained from standard dilutions. There was a strong linear relationship between the CT value and the log concentration of cells in the standard (r2 = 0.996). Detection of DNA at a concentration as low as the equivalent of a single cell showed that the assay was sensitive enough to detect a single cell of P. olseni. The estimated number of P. olseni cells was similar to the original cell concentrations, indicating the reliability of P. olseni quantification by real-time PCR. Accordingly, the designed primers and probe may be used for the rapid detection and quantification of P. olseni from clam tissue, environmental water, and sediment samples.

v27_4_387-393.pdf (514.2K), Down : 215, 2012-03-17 17:13:30