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Development of a quantitative PCR for detection and quantification of Prorocentrum donghaiense

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Abstract

Prorocentrum donghaiense is a dinoflagellate with a high frequency of bloom formation in the East China Sea. These blooms harm coastal ecosystems, marine fisheries, aquatic environments, and public health. Therefore, new and rapid methods that accurately process and specifically detect this alga are crucial to facilitate long-term monitoring or to provide timely warnings of P. donghaiense blooms. We report the development of a quantitative real-time PCR (qPCR) method to identify and detect P. donghaiense. The partial large subunit (LSU) rDNA D1–D2 was cloned and sequenced to design specific amplification primers. The specificity of the primers was tested using regular PCR and fluorescent PCR against a wide range of microalgae widely distributed along the Chinese coast. The qPCR detection protocol was based on two standard curves. Both curves were constructed from standard samples of tenfold serially diluted solutions of the recombinant plasmid containing the LSU D1–D2 fragment and crude DNA extracts with a known number of target cells. A quantitative relationship between the cell numbers and their corresponding plasmid copy numbers was established; this relationship can be used to determine the target cell number of unknown samples in combination with a standard curve that was generated from tenfold-diluted plasmid solutions and the determined C t value of target DNA. The effectiveness of the developed protocol was tested with a series of simulated and field samples. The developed qPCR had a detection sensitivity of up to 3.45 cells. The performance of qPCR was not affected by nontarget DNA. The detection test with a series of samples fixed for 40 days showed that qPCR is competent for long-term monitoring programs that require the quantitative analysis of fixative-preserved samples. qPCR can identify the target cells in the field samples within 3–4 h. No significant differences in the quantitative results of the target cells were observed between qPCR and light microscopy. Overall, the established qPCR method is specific, sensitive, rapid, accurate, and promising for the field detection of P. donghaiense in natural samples.

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References

  • Andrée KB, Fernandez-Tejedor M, Elandaloussi LM, Quijano-Scheggia S, Sampedro N, Garcés E, Camp J, Diogene J (2011) Quantitative PCR coupled with melt curve analysis for detection of selected Pseudo-nitzschia spp. (Bacillariophyceae) from the northwestern Mediterranean Sea. Appl Environ Microbiol 77:1651–1659

  • Bowers HA, Tengs T, Glasgow HB, Burkholder JM, Rublee PA, Oldach DW (2000) Development of real-time PCR assays for rapid detection of Pfiesteria piscicida and related dinoflagellates. Appl Environ Microbiol 66:4641–4648

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Cai Q, Li R, Zhen Y, Mi T, Yu Z (2006) Detection of two Prorocentrum species using sandwich hybridization integrated with nuclease protection assay. Harmful Algae 5:300–309

    Article  CAS  Google Scholar 

  • Cary SC, Coyne KJ, Rueckert A, Wood SA, Kelly S, Gemmill CEC, Vieglais C, Hicks BJ (2014) Development and validation of a quantitative PCR assay for the early detection and monitoring of the invasive diatom Didymosphenia geminata. Harmful Algae 36:63–70

    Article  CAS  Google Scholar 

  • Casper ET, Paul JH, Smith MC, Gray M (2004) Detection and quantification of the red tide dinoflagellate Karenia brevis by real-time nucleic acid sequence-based amplification. Appl Environ Microbiol 70:4727–4732

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Chen GF, Zhang CY, Zhang BY, Wang GC, Lu DD, Xu Z, Yan PS (2011) Development of a PNA probe for fluorescence in situ hybridization detection of Prorocentrum donghaiense. PLoS One 6:e25527

  • Chen GF, Liu Y, Zhang CY, Ma CS, Zhang BY, Wang GC, Xu Z, Lu DD (2013a) Development of rRNA-targeted probes for detection of Prorocentrum micans (Dinophyceae) using whole cell in situ hybridization. J Appl Phycol 25:1077–1089

    Article  CAS  Google Scholar 

  • Chen GF, Ma CS, Zhang CY, Zhou J, Wang YY, Wang GC, Zhang BY, Xu Z, Lu DD (2013b) A rapid and sensitive method for field detection of Prorocentrum donghaiense using reverse transcription-coupled loop-mediated isothermal amplification. Harmful Algae 29:31–39

    Article  CAS  Google Scholar 

  • Chomerat N, Sellos DY, Zentz F, Nezan E (2010) Morphology and molecular phylogeny of Prorocentrum consutum sp. nov. (Dinophyceae), a new benthic dinoflagellate from South Brittany (northwestern France). J Phycol 46:183–194

    Article  CAS  Google Scholar 

  • Créach V, Ernst A, Sabbe K, Vanelslander B, Vyverman W, Stal LJ (2006) Using quantitative PCR to determine the distribution of a semicryptic benthic diatom, Navicula phyllepta (Bacillariophyceae). J Phycol 42:1142–1154

    Article  Google Scholar 

  • Diercks S, Medlin LK, Metfies K (2008) Colorimetric detection of the toxic dinoflagellate Alexandrium minutum using sandwich hybridization in a microtiter plate assay. Harmful Algae 7:137–145

    Article  CAS  Google Scholar 

  • Erdner DL, Percy L, Keafer B, Lewis J, Anderson DM (2010) A quantitative real-time PCR assay for the identification and enumeration of Alexandrium cysts in marine sediments. Deep-Sea Res II 57:279–287

    Article  CAS  Google Scholar 

  • Faust MA, Vandersea MW, Kibler SR, Tester PA, Litaker RW (2008) Prorocentrum levis, a new benthic species (Dinophyceae) from a mangrove island, Twin Cays, Belize. J Phycol 44:232–240

    Article  CAS  PubMed  Google Scholar 

  • Fitzpatrick E, Caron D, Schnetzer A (2010) Development and environmental application of a genus-specific quantitative PCR approach for Pseudo-nitzschia species. Mar Biol 157:1161–1169

    Article  CAS  Google Scholar 

  • Garneau M, Schnetzer A, Countway PD, Jones AC, Seubert EL, Caron DA (2011) Examination of the seasonal dynamics of the toxic dinoflagellate Alexandrium catenella at Redondo Beach, California, by quantitative PCR. Appl Environ Microbiol 77:7669–7680

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Guillard RRL (1975) Culture of phytoplankton for feeding marine invertebrates. In: Smith WL, Chanley MH (eds) Culture of marine invertebrate animals. Plenum Press, New York, pp 29–60

    Chapter  Google Scholar 

  • Hall TA (1999) BioEdit: a user-friendly biological sequence alignment editor and analysis program for Windows 95/98/NT. Nucleic Acids Symp Ser 41:95–98

    CAS  Google Scholar 

  • Hosoi-Tanabe S, Sako Y (2005) Species-specific detection and quantification of toxic marine dinoflagellates Alexandrium tamarense and A. catenella by real-time PCR assay. Mar Biotechnol 7:506–514

    Article  CAS  PubMed  Google Scholar 

  • Huang H, Wu MG, Lei HL, Lai HY, Hou JJ (2008) Quantitative detection of harmful algae species using lectin probes. J Fish Sci China 15:279–292

    CAS  Google Scholar 

  • Kamikawa R, Nagai S, Hosoi-Tanabe S, Itakura S, Yamaguchi M, Uchida Y, Baba T, Sako Y (2007) Application of real-time PCR assay for detection and quantification of Alexandrium tamarense and Alexandrium catenella cysts from marine sediments. Harmful Algae 6:413–420

    Article  CAS  Google Scholar 

  • Ki JS, Han MS (2006) A low-density oligonucleotide array study for parallel detection of harmful algal species using hybridisation of consensus PCR products of LSU rDNA D2 domain. Biosens Bioelectron 21:1812–1821

    Article  CAS  PubMed  Google Scholar 

  • Li J, Glibert PM, Zhou MJ, Lu SH, Lu DD (2009) Relationships between nitrogen and phosphorus forms and ratios and the development of dinoflagellate blooms in the East China Sea. Mar Ecol Prog Ser 383:11–26

    Article  CAS  Google Scholar 

  • Lin S, Zhang H, Dubois A (2006) Low abundance distribution of Pfiesteria piscicida in pacific and western Atlantic as detected by mtDNA-18S rDNA real-time polymerase chain reaction. J Plankton Res 28:667–681

    Article  CAS  Google Scholar 

  • Long H, Zhou Y, Yu J, Hu YF, Fu GJ (2008) Analyses on harmful algal blooms in Zhejiang coastal waters from 2001 to 2007. Mar Environ Sci 27(suppl 1–4):11–18

    Google Scholar 

  • Lu DD, Goebel J (2001) Five red tide species in genus Prorocentrum including the description of Prorocentrum donghaiense Lu sp. nov. from the East China Sea. Chin J Oceanogr Limnol 19:337–344

    Article  Google Scholar 

  • Lu DD, Qi YZ, Goebel J, Zou JZ, Gao YH (2003) Redescription of Prorocentrum donghaiense Lu and comparison with relevant Prorocentrum species. Chin J Appl Ecol 14:1060–1064

    Google Scholar 

  • Lu DD, Goebel J, Qi YZ, Zou JZ, Han XT, Gao YH, Li RX (2005) Morphological and genetic study of Prorocentrum donghaiense Lu from the East China Sea, and comparison with some related Prorocentrum species. Harmful Algae 4:493–505

    Article  CAS  Google Scholar 

  • Mikulski CM, Park YT, Jones KL, Lee CK, Limb WA, Lee Y, Scholin CA, Doucette GJ (2008) Development and field application of rRNA-targeted probes for the detection of Cochlodinium polykrikoides Margalef in Korean coastal waters using whole cell and sandwich hybridization formats. Harmful Algae 7:347–359

    Article  CAS  Google Scholar 

  • Murray SA, Nagahama Y, Fukuyo Y (2007) Phylogenetic study of benthic, spine-bearing prorocentroids, including Prorocentrum fukuyoi sp. nov. Phycol Res 55:91–102

    Article  CAS  Google Scholar 

  • Murray SA, Wiese M, Stüken A, Brett S, Kellmann R, Hallegraeff G, Neilan BA (2011) STXA-based quantitative molecular assay to identify saxitoxin-producing harmful algal blooms in marine waters. Appl Environ Microbiol 77:7050–7057

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Penna A, Galluzzi L (2013) The quantitative real-time PCR applications in the monitoring of marine harmful algal bloom (HAB) species. Environ Sci Pollut Res 20:6851–6862

    Article  Google Scholar 

  • Percopo I, Siano R, Cerino F, Sarno D, Zingone A (2011) Phytoplankton diversity during the spring bloom in the northwestern Mediterranean Sea. Bot Mar 54:243–267

    Article  Google Scholar 

  • Popels LC, Cary SC, Hutchins DA, Forbes R, Pustizzi F, Gobler CJ, Coyne KJ (2003) The use of quantitative polymerase chain reaction for the detection and enumeration of the harmful alga Aureococcus anophagefferens in environmental samples along the United States East Coast. Limnol Oceanogr Methods 1:92–102

    Article  Google Scholar 

  • Qi YZ, Wang Y (2003) What the Prorocentrum species should be?—a review on identification of Prorocentrum species from the East China Sea. Chin J Appl Ecol 14:1188–1190

    Google Scholar 

  • Scholin CA, Herzog M, Sogin M, Anderson DM (1994) Identification of group- and strain-specific genetic markers for globally distributed Alexandrium (Dinophyceae): sequence analysis of a fragment of the LSU rRNA gene. J Phycol 30:999–1011

    Article  CAS  Google Scholar 

  • Scholin CA, Buck KR, Britschgi T, Cangelosi G, Chavez FP (1996) Identification of Pseudo-nitzschia australis (Bacillariophyceae) using rRNA-targeted probes in whole cell and sandwich hybridization formats. Phycologia 35:190–197

    Article  Google Scholar 

  • Shi YH, Zhang FY, Ma LB (2010) Development of a real-time PCR assay for rapid detection and quantification of Heterocapsa circularisquama. J Fish Sci China 17:267–273

    CAS  Google Scholar 

  • Smith MW, Maier MA, Suciu D, Peterson TD, Bradstreet T, Nakayama J, Simon HM (2012) High resolution microarray assay for rapid taxonomic assessment of Pseudo-nitzschia spp. (Bacillariophyceae) in the field. Harmful Algae 19:169–180

    Article  Google Scholar 

  • Tang DL, Di BP, Wei GF, Ni IH, Oh IS, Wang SF (2006a) Spatial, seasonal and species variations of harmful algal blooms in the South Yellow Sea and East China Sea. Hydrobiologia 568:245–253

    Article  Google Scholar 

  • Tang JL, Mao HY, Guo MR (2006b) Analysis on the red tide of Prorocentrum dentatum in the north coastal water of Zhejiang. Mar Environ Sci 25:63–66

    CAS  Google Scholar 

  • Wang DZ, Huang XG, Chan LL, Hong HS (2007) Development of an immunofluorescence technique for detecting Prorocentrum donghaiense Lu. J Appl Phycol 19:325–332

    Article  Google Scholar 

  • Wang HX, Lu DD, He PX, Xia P, Dai XF (2012) Morphological characteristic and ITS sequence analysis of Prorocentrum triestinum isolated from the East China Sea. Acta Oceanol Sin 34:155–162

    CAS  Google Scholar 

  • Yuan J, Mi TZ, Zhen Y, Yu ZG (2012a) Development of a rapid detection and quantification method of Karenia mikimotoi by real-time quantitative PCR. Harmful Algae 17:83–91

    Article  CAS  Google Scholar 

  • Yuan J, Mi TZ, Zhen Y, Yu ZG (2012b) Development of a real-time PCR method (Taqman) for rapid identification and quantification of Prorocentrum donghaiense. J Ocean Univ China 11:366–374

    Article  CAS  Google Scholar 

  • Zhang BY, Wang GC, Qi YZ, Zou JZ, Tseng CK (2005) Identification of two species of dinoflagellate using fluorescence in situ hybridization. Chin High Technol Lett 15:101–105

    CAS  Google Scholar 

  • Zhen Y, Mi TZ, Yu ZG (2009) Detection of several harmful algal species by sandwich hybridization integrated with a nuclease protection assay. Harmful Algae 8:651–657

    Article  CAS  Google Scholar 

  • Zingone A, Percopo I, Sims PA, Sarno D (2005) Diversity in the genus Skeletonema (Bacillariophyceae) I. A reexamination of the type material of S. costatum with the description of S. grevillei sp. nov. J Phycol 41:140–150

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Acknowledgments

This work was supported by the National Scientific Foundation of China (41476086, 41106082, 41176141); the S & T Project of Shenzhen Science and Technology Innovation Committee (JSKF201505293000320); the Open Fund of Key Laboratory of Marine Ecology and Environmental Science, Institute of Oceanology, Chinese Academy of Sciences (KLMEES201303); and the Basic Research of Harbin Institute of Technology Outstanding Talents Cultivation Plan of Class III.

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Authors and Affiliations

  1. School of Marine Science and Technology, Harbin Institute of Technology (Weihai), Weihai, 264209, People’s Republic of China

    Chun Yun Zhang, Guo Fu Chen & Yuan Yuan Wang

  2. Key Laboratory of Marine Ecology and Environmental Science, Institute of Oceanology, Chinese Academy of Sciences, Qingdao, 266071, People’s Republic of China

    Chun Yun Zhang & Guo Fu Chen

  3. Division of Ocean Science and Technology, Graduate School at Shenzhen, Tsinghua University, Shenzhen, Guangdong, 518055, People’s Republic of China

    Jin Zhou

  4. The Second Institute of Oceanography, SOA, Hangzhou, 310012, People’s Republic of China

    Dou Ding Lu

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  1. Chun Yun Zhang
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  2. Guo Fu Chen
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Correspondence to Guo Fu Chen.

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Zhang, C.Y., Chen, G.F., Zhou, J. et al. Development of a quantitative PCR for detection and quantification of Prorocentrum donghaiense . J Appl Phycol 28, 1683–1693 (2016). https://doi.org/10.1007/s10811-015-0682-6

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  • DOI: https://doi.org/10.1007/s10811-015-0682-6

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