ISSN : 1225-3480
The purpose of this study was to test the potential of digital PCR (dPCR) for diagnosing Perkinsus olseni. dPCR is currently one of the most effective molecular diagnosis techniques, as it is capable of quantifying the target sequence without a standard. Here, a primer pair and a probe were developed in the non-transcribed spacer region for diagnosis of P. olseni. In the sensitivity and specificity test, dPCR was able to detect as little as one P. olseni trophozoite, and could discriminate P. olseni from P. marinus. The number of P. olseni cells measured by dPCR was very similar to that determined by conventional cell counting. In conclusion, this study confirms that P. olseni can be quantified using dPCR. Additional validation is needed to access the efficiency of the technique in host tissues or environmental samples.
Adams, A. (2019) Rapid detection and identification of fish pathogens. http://www.fao.org/3/X4946E/x4946e0j.htm
Choi, K.S., Wilson, E.A., Lewis, D.H., Powell, E.N., Ray, S.M. (1989) The energetic cost of Perkinsus marinus parasitism in oysters: Quantication of the thioglycollate method. Journal of Shellfish Research, 8: 125-131.
Fisher, W.S., Oliver, L.M. (1996) A whole-oyster procedure for diagnosis of Perkinsus marinus disease using Ray's fluid thioglycollate culture medium. Journal of Shellfish Research, 15: 109-17.
Gevensleben, H., Garcia-Murillas, I., Graeser, M.K., Schiavon, G., Osin, P., Parton, M., Smith, I.E., Ashworth, A., Turner, N.C. (2013) Noninvasive detection of HER2 amplification with plasma DNA digital PCR. Clinical Cancer Research, 19(12):3276-284.
Hayden, R.T., Gu, Z., Ingersoll, J., Abdul-Ali, D., Shi, L., Pounds, S., Caliendo, A.M. (2013) Comparison of droplet digital PCR to real-time PCR for quantitative detection of cytomegalovirus. Journal of Clinical Microbiology, 51(2): 540-46.
Huggett, J.F., Cowen, S., Foy, C.A. (2014) Considerations for digital PCR as an accurate molecular diagnostic tool. Clinical Chemistry, 61(1): 79-88.
Hindson, B.J., Ness, K.D., Masquelier, D.A., Belgrader, P., Heredia, N.J., Makarewicz, A.J., Bright, I.J., Lucero, M.Y., Hiddessen, A.L., Legler, T.C., Kitano, T.K., Hoder, M.R., Pertersen, J.F., Wyatt, P.W., Steenblock, E.R., Shan, P.H., Bousse, L.J., Troup, C.B., Mellen, J.C., Wittmann, D.K., Erndt, N.G., Cauley, T.H., Koehler, R.T., So, A.P., Dube, S., Rose, K.A., Montesclaros, L., Wang, S., Stumbo, D.P., Hodges, S.P., Romine, S., Milanovich, F.P., White, H.E., Regan, J.F., Karlin-Neumann, G.A., Hindson, C.M., Saxonov, S., Colston, B.W. (2011)High-throughput droplet digital PCR system for absolute quantitation of DNA copy number. Analytical Chemistry, 83(22): 8604-8610.
Kahyo, T., Iwaizumi, M., Yamada, H., Tao, H., Kurachi, K., Sugimura, H. (2017) Application of digital PCR with chip-in-a-tube format to analyze Adenomatous polyposis coli (APC) somatic mosaicism. Clinica Chimica Acta, 475: 91-96.
Kiss, M.M., Ortoleva-Donnelly, L., Beer, N.R., Warner, J., Bailey, C.G., Colston, B.W., Rothberg, J.M., Link, D.R., Leamon, J.H. (2008) High-throughput quantitative polymerase chain reaction in picoliter droplets. Analytical Chemistry, 80(23): 8975-8981.
Laurent-Puig, P. Pekin, D., Normand, C., Kotsopoulos, S.K., Nizard, P., Perez-Toralla, K., Rowell, R., Olson, J., Srinivasan, P., Le Corre, D., Hor, T., El Harrak, Z., Li, X., Link, D.R., Bouché, O., Emile, J.F., Landi, B., Boige, V., Hutchison, J.B., Taly, V. (2015)Clinical relevance of KRAS-mutated subclones detected with picodroplet digital PCR in advanced colorectal cancer treated with anti-EGFR therapy. Clinical Cancer Research, 21(5): 1087-1097.
Li, H., Bai, R., Zhao, Z., Tao, L., Ma, M., Ji, Z., Jian, M., Ding, Z., Dai, X., Bao, F., Liu, A. (2018)Application of droplet digital PCR to detect the pathogens of infectious diseases. Bioscience Report, 15: 38(6):BSR20181170. doi: 10.1042/BSR20181170. PMID: 30341241; PMCID: PMC6240714.
Li, X., Liu, Y., Shi, W., Xu, H., Hu, H., Dong, Z., Zhu, G., Sun, Y., Liu, B., Gao, H. Tang, C., Liu, X. (2017)Droplet digital PCR improved the EGFR mutation diagnosis with pleural fluid samples in non-small-cell lung cancer patients. Clinica Chimica Acta, 471:177-184.
Low, H., Chan, S.J., Soo, G.H., Ling, B., Tan, E.L. (2017) Clarity™ digital PCR system: a novel platform for absolute quantification of nucleic acids. Anal. Bioanal. Chemistry, 409(7): 1869-1875.
Montes, J.F., Durfort, M., Llado, A., Garcia Valero, J. (2002) Characterization and immunolocalization of a main proteinaceous component of the cell wall of the protozoan parasite Perkinsus atlanticus. Parasitology, 124: 477-484.
Moss, J.A., Burreson, E.M., Reece, K.S. (2006) Advanced Perkinus marinus infections in Crassostrea ariakensis maintained under laboratory conditions. Journal of Shellfish Research, 25: 65-72.
Moss, J.A. (2007) Characterization of exotic pathogens associated with the suminoe oyster, Crassostrea ariakensis. Ph.D. Dissertation. Virginia Institute of Marine Science, College of William and Mary, Gloucester Point, Virginia, USA, 230 pp.
Ottesen, E.A., Hong, J.W., Quake, S.R., Leadbetter, J.R. (2006) Microfluidic digital PCR enables multigene analysis of individual environmental bacteria. Science, 314(5804): 1464-1467.
Park, K.I., Choi, K.S. (1997) Report on the occurrence of Perkinsus sp. In the Manila clams, Ruditapes philippinarum in Korea. Aquaculture, 10: 227-237.
Park, K.I., Choi, K.S. (2001) Spatial distribution of the protozoan parasite Perkinsus sp., found in the Manila clams, Ruditapes philippinarum, in Korea. Aquaculture, 203: 9-22.
Park, K.I., Yang, H.S., Kang, H.S., Cho, M., Park, K.J., Choi, K.S. (2010) Isolation and identification of Perkinsus olseni from feces and marine sediment using immunological and molecular techniques. Journal of Invertebrate Pathology, 105: 261-269.
Powell, E.N., Wilson-Ormond, E.A., Choi, K.S. (1993)Gonadal analysis-Cassostrea virginica NOAA Tech. Memo. NOS ORCA, 71, pp. 11.55-11.62.
Ray, S.M. (1966) A review of the culture method of detecting Dermocystidium marinum with suggested modifications and precautions. Proc. National Shellfish Association, 54: 55-69.
Simoska, O., Stevenson, K.J. (2019) Electrochemical sensors for rapid diagnosis of pathogens in real time. Analyst, 144: 6461-6478.
Strain, M.C., Lada, S.M., Luong, T., Rought, S.E., Gianella, S., Terry, V.H., Spina, C.A., Woelk, C.H., Richman, D.D. (2013) Highly precise measurement of HIV DNA by droplet digital PCR. PLoS One, 8(4):e55943.
Sykes, P.J., Neoh, S.H., Brisco, M.J., Hughes, E., Condon, J., Morley, A.A. (1992) Quantitation of targets for PCR by use of limiting dilution. BioTechniques, 13(3): 444-449.
Villalba, A., Reece, K.S., Ordas, M.C., Casa, S.M., Figueras, A. (2004) Perkinsosis in molluscs: a review. Aquatic Living Resources, 17: 411-432.
Vogelstein, B., Kinzler, K.W. (1999) Digital PCR. PNAS, 96(16): 9236-9241. https://doi.org/10.1073/pnas.96.16.9236
Whale, A.S., Huggett, J.F., Tzonev, S. (2016)Fundamentals of multiplexing with digital PCR. Biomolecular Detection and Quantification, 10: 15-23.
Zonta, E., Garlan, F., Pécuchet, N., Perez-Toralla, K., Caen, O., Milbury, C., Didelot, A., Fabre, E., Blons, H., Laurent-Puig, P., Taly, V. (2016) Multiplex Detection of Rare Mutations by Picoliter Droplet Based Digital PCR: Sensitivity and Specificity Considerations. PLoS One, 11(7): e0159094.