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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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 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 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.
 Keywords
Perkinsus olseni;real-time PCR;TaqMan probe;quantification;
 Language
English
 Cited by
 References
1.
Abe, A., Inoue, K., Tanaka, T., Kato, J., Kajiyama, N., Kawaguchi, R., Tanaka, S., Yoshiba M. and Kohara, M. (1999) Quantitation of hepatitis B virus genomic DNA by real-time detection PCR. Journal of Clinical Microbiology, 37: 2899-2903.

2.
Audemard, C., Ragone Calvo, L. M., Paynter, K. T., Reece, K. S. and Burreson, E.M. (2006) Real-time PCR investigation of parasite ecology: In situ determination of oyster parasite Perkinsus marinus transmission dynamics in lower Chesapeake Bay. Parasitology, 132: 827-842. crossref(new window)

3.
Audemard, C., Reece, K.S. and Burreson, E.M., (2004) Real-time PCR for detection and quantification of the protistan parasite Perkinsus marinus in environmental waters. Applied Environmental Microbiology, 70: 6611-6618. crossref(new window)

4.
Bertrand, I., Gantzer, C., Chesnot, T. and Schwartzbrod, J. (2004) Improved specificity for Giardia lamblia cyst quantification in wastewater by development of a real-time PCR method. Journal of Microbiological Methods, 57: 41-53 crossref(new window)

5.
Blessmann, J., Buss, H., Nu, P.A., Dinh, B.T., Ngo, Q.T., Van, A.L., Alla, M.D., Jackson, T.F., Ravdin, J.I. and Tannich, E. (2002) Real-time PCR for detection and differentiation of Entamoeba histolytica and Entamoeba dispar in fecal samples. Journal of Clinical Microbiology, 40: 4413-4417. crossref(new window)

6.
Bushek, D., Dungan, C.F. and Lewitus, A. J. (2002) Serological affinities of the oyster pathogen Perkinsus marinus (Apicomplexa) with some dinoflagellates (Dinophyceae). Journal of Eukaryotic Microbiology, 49: 11-16. crossref(new window)

7.
Casas, S.M., Grau, A., Reece, K.S., Apakupakul, K., Azevedo, C. and Villalba, A. (2004) Perkinsus mediterraneus n. sp., a protistan parasite of the European flat oyster Ostrea edulis from the Balearic Islands, Mediterranean Sea. Disease of Aquatic Organisms, 58: 231-244. crossref(new window)

8.
Choi, K.S., Wilson, E.A., Lewis, D.H., Powell, E.N. and Ray, S.M. (1989). The energetic cost of Perkinsus marinus parasitism in oysters: quantification of the thioglycollate method. Journal of Shellfish Research, 8: 125-131.

9.
Coss, C.A., Robledo, J.A., Ruiz, G.M. and Vasta. G.R. (2001) Description of Perkinsus andrewsi n. sp. isolated from the Baltic clam (Macoma balthica) by characterization of the ribosomal RNA locus, and development of species-specific PCR-based diagnostic assay. Journal of Eukaryotic Microbiology, 48: 52-61. crossref(new window)

10.
Costa, C., Vidaud, D., Olivi, M.E., Bart-Delabesse, E., Vidaud, M. and Bretagne, S. (2001) Development of two real-time quantitative TaqMan PCR assays to detect circulating Aspergillus fumigatus DNA in serum. Journal of Microbiological Methods, 44: 263-269. crossref(new window)

11.
Dungan, C.F. and Reece, K.S. (2006) In vitro propagation of two Perkinsus spp. Parasites from Japanese Manila clams Venerupis philippinarum and description of Perkinsus honshuensis n. sp. Journal of Eukaryotic Microbiology, 53: 316-326. crossref(new window)

12.
Drago, L., Lombardi, A., De Vecchi, E. and Gismondo, M.R. (2002) Real- Time PCR assay for rapid detection of Bacillus anthracis spores in clinical samples. Journal of Clinical Microbiology, 40: 4399 crossref(new window)

13.
Elandalloussi, L.M., Leite, R., Afonso, R., Nunes, P., Robledo, J.A, Vasta, G.R. and Cancela, L. M. (2004) Development of a PCR-ELISA assay for diagnosis of Perkinsus marinus and Perkinsus atlanticus infection in bivalve molluscs. Molecular and Cellular Probes, 18: 89-96. crossref(new window)

14.
Espy, M.J., Uhl J.R., Sloan L.M., Buckwalter, S.P., Jones, M.F., Vetter, E.A., Yao, J.D.C., Wengewack, N.L., Rosenblatt, J.E., Cockerill, III. and Smith, T.F. (2006) Real-Time PCR in clinical microbiology: applications for routine laboratory testing. Clinical Microbiology Review, 19: 165-256. crossref(new window)

15.
Faveri, De.J., Somolowitz, R.M. and Roberts, S.B. (2009) Development and validation of a real-time quantitative PCR assay for the detection and quantification of Perkinsus marinus in the eastern oyster Crassostrea virginica. Journal of Shellfish Research, 28: 459-464. crossref(new window)

16.
Fong, D., Rodriguez, R., Koo, K., Sun, J., Sogin, M., Bushek, D., Littlewood, D.T. and Ford, S. (1993) Small subunit ribosomal RNA gene sequence of the oyster parasite Perkisnus marinus. Molecular Marine Biology and Biotechnology, 2: 346-350.

17.
Frostegard, A., Courtois, S., Ramisse, V., Clerc, S., Bernillon, D., Le Gall, F., Jeannin, P., Nesme, X. and Simonet, P. (1999) Quantification of bias to the extraction of DNA directly from soils. Applied and Environmental Microbiology, 65: 5409-5420.

18.
Gauthier, J.D., Miller, C.R. and Wilbur, A.E. (2006) TaqMan MGB Real-time PCR approach to quantification of Perkinsus marinus and Perkinsus spp. on oysters. Journal of Shellfish Research, 20: 619-624.

19.
Hamaguchi, M.N., Suzuki, N., Usuki, H. and Ishioka, H. (1998) Perkinsus protozoan infection in short-necked clam Tapes (=Ruditapes) philippinarum in Japan. Fish Pathology, 33: 473-480. crossref(new window)

20.
Holland, P.M., Abramson, R.D., Watson, R. and Gelfand, D.H. (1991) Detection of specific polymerase chain reaction product by utilizing the 5'$\rightarrow$3' exonuclease activity of Thermus aquaticus DNA polymerase. Proceedings of the National Academy of Science USA, 88: 7276-7280.

21.
Mackin, J.G. (1962) Oyster disease caused by Dermocystidiun marinum and other microorganisms in Louisiana. Publication of the Institute of Marine Science. University of Texas, 7: 132-229.

22.
Marsh, A., Gauthier, J.D. and Vasta, G.R. (1995) A semiquantitative PCR assay for assessing Perkinsus marinus infections in the eastern oyster, Crassostrea virginica. Jounal of Parasitology, 81: 577-583. crossref(new window)

23.
Nagle, L., Place, A.R., Schott, E.J., Jagus, R., Messick, G. and Pitula, J.S. (2009) Real-time PCR-based assay for quantitative detection of Hematodinium sp. in the blue crab Callinectes sapidus. Disease of Aquatic Organisms, 84: 79-87. crossref(new window)

24.
Park, K.I. and Choi, K.S. (2001) Spatial distribution and infection intensity of the protozoan parasite Perkinsus sp. in the Manila clam Ruditapes philippinarum in Korea. Aquaculture, 203: 9-22. crossref(new window)

25.
Park, K.I., Park, J.K., Lee, J. and Choi, K.S. (2005) Use of DNA sequences to determine the identity of Perkinsus sp. found in Manila clam, Ruditapes philippinarum in Korean water. Disease of Aquatic Organisms, 66: 255-263. crossref(new window)

26.
Park, K.I., Ngo, T.T.T., Choi, S.D., Cho, M. and Choi, K.S. (2006a) Occurrence of Perkinsus olseni in the Venus clam Protothaca jedoensis in Korean waters. Journal of Invertebrate Pathology, 93: 81-87. crossref(new window)

27.
Park, K.I., Figueras, A. and Choi, K.S. (2006b) Application of enzyme-linked immunosorbent assay (ELISA) for the study of reproduction in the Manila clam Ruditapes philippinarum: (Mollusca: Bivalvia): II. Impacts of Perkinsus olseni on clam reproduction. Aquaculture, 251: 182-191. crossref(new window)

28.
Ragone Calvo, L.M.., Dungan, C.F., Roberson, B.S. and Burreson, E.M. (2003) Systematic evaluation of factors controlling Perkinsus marinus transmission dynamics in lower Chesapeake Bay. Disease of Aquatic Organisms, 56: 75-86. crossref(new window)

29.
Ray, S.M. (1954) Experimental studies on the transmission and pathogenicity of Dermocytidium marinum, a fungus parasite of oyster. Journal of Parasitology, 40: 235 crossref(new window)

30.
Robledo, J.A.F., Gauthier, J.D, Coss, C.A., Wright, A.C. and Vasta, G.R. (1998) Species- specificity and sensitivity of a PCR- based Assay for Perkinsus marinus in the eastern oyster, Crassostrea virginica: A comparison with the fluid thioglycollate assay. Journal of Parasitology, 85: 1237-1244.

31.
Robledo, J.A.F., P. Nunes, P.A, Cancela, M.L. and Vasta G.R. (2002) Development of an in vitro clonal culture and characterization of the rRNA gene cluster of Perkinsus atlanticus, a protozoan parasite of the clam Tapes decussatus. Journal of Eukaryotic Microbiology, 49: 414-422. crossref(new window)

32.
Watson, R. J., and B. Blackwell. 2000. Purification and characterization of a common soil component which inhibits the polymerase chain reaction. Canadian Journal of Microbiology, 46: 633-642. crossref(new window)

33.
Wilson, I.G. (1997) Inhibition and facilitation of nucleic acid amplification. Applied and Environmental Microbiology, 63: 3741-3751.

34.
Xie, Z.X., Xie, L.J., Liu, J.B., Xie, Z.Q., Pang, Y.W. and Deng, X.W. (2009) Development and application of a real-time PCR assay for detection of Perkinsus olseni. Journal of Pathogen Biology, 4: 517-519.

35.
Yang, H.S., Kang, H.S, and Choi, K.S. (2011) Detection of Perkinsus olseni and Perkinsus honshuensis in Manila clam Ruditapes philippinarum in Korean waters using molecular markers. The 9th Asian Fisheries and Aquaculture Forum, Book of Abstracts. Shanghai Ocean University, China, p. 79.