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Development of a real-time PCR method for detection and quantification of the parasitic protozoan Perkinsus olseni
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  • Journal title : The Korean Journal of Malacology
  • Volume 27, Issue 4,  2011, pp.387-393
  • Publisher : The Malacological Society of Korea
  • DOI : 10.9710/kjm.2011.27.4.387
 Title & Authors
Development of a real-time PCR method for detection and quantification of the parasitic protozoan Perkinsus olseni
Gajamange, Dinesh; Yoon, Jong-Man; Park, Kyung-Il;
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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 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 cells and 10-fold serial dilutions to the level of 5 cells were prepared. The standards and samples were analyzed in duplicate using an 96 real-time quantitative thermal block. For quantification, the threshold cycle () values of samples were compared with those obtained from standard dilutions. There was a strong linear relationship between the value and the log concentration of cells in the standard ( = 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.
Perkinsus olseni;real-time PCR;TaqMan probe;quantification;
 Cited by
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