The Korean Journal of Malacology (한국패류학회지)

The Malacological Society of Korea (한국패류학회)
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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)
  • Received : 2011.12.14
  • Accepted : 2011.12.28
  • Published : 2011.12.31
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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.

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References

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