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ISSN : 1225-3480

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27권 4호

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

Gajamange, Dinesh (Department of Aquatic Life Medicine, Kunsan National University)
Yoon, Jong-Man (Department of Aquatic Life Medicine, Kunsan National University)
Park, Kyung-Il (Department of Aquatic Life Medicine, Kunsan National University)
한국패류학회지 / The Korean Journal of Malacology, (P)1225-3480;
2011, v.27 no.4, pp.387-393
https://doi.org/10.9710/kjm.2011.27.4.387
Gajamange, Dinesh, Yoon, Jong-Man, & Park, Kyung-Il. (2011). Development of a real-time PCR method for detection and quantification of the parasitic protozoan Perkinsus olseni. , 27(4), 387-393, https://doi.org/10.9710/kjm.2011.27.4.387

Abstract

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 $10^5$ 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 $5{\times}10^4$ cells and 10-fold serial dilutions to the level of 5 cells were prepared. The standards and samples were analyzed in duplicate using an $Exicycler^{TM}$ 96 real-time quantitative thermal block. For quantification, the threshold cycle ($C_T$) values of samples were compared with those obtained from standard dilutions. There was a strong linear relationship between the $C_T$ value and the log concentration of cells in the standard ($r^2$ = 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.

keywords
Perkinsus olseni, real-time PCR, TaqMan probe, quantification

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