Abstract
Since 2009, Aureococcus anophagefferens has caused brown tide to occur recurrently in Qinhuangdao coastal area, China. Because the algal cells of A. anophagefferens are so tiny (~3 µm) that it is very hard to identify exactly under a microscope for natural water samples, it is very urgent to develop a method for efficient and continuous monitoring. Here specific primers and Taqman probe are designed to develop a real-time quantitative PCR (qPCR) method for identification and quantification continually. The algal community and cell abundance of A. anophagefferens in the study area (E 119°20′–119°50′ and N 39°30′–39°50′) from April to October in 2013 are detected by pyrosequencing, and are used to validate the specification and precision of qPCR method for natural samples. Both pyrosequencing and qPCR shows that the targeted cells are present only in May, June and July, and the cell abundance are July > June > May. Although there are various algal species including dinoflagellata, diatom, Cryptomonadales, Chrysophyceae and Chlorophyta living in the natural seawater simultaneously, no disturbance happens to qPCR method. This qPCR method could detect as few as 10 targeted cells, indicating it is able to detect the algal cells at pre-bloom levels. Therefore, qPCR with Taqman probe provides a powerful and sensitive method to monitor the brown tide continually in Qinhuangdao coastal area, China. The results provide a necessary technology support for forecasting the brown tide initiation, in China.
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References
Zhang QC, Qiu LM, Yu RC, Kong FZ, Wang YF, Yan T, Gobler CJ, Zhou MJ (2012) Emergence of brown tides caused by Aureococcus anophagefferens Hargraves et Sieburth in China. Harmful Algae 19:117–124. doi:10.1016/j.hal.2012.06.007
Qiu J (2012) China third country to be hit by ‘brown tide’. Nature. doi:10.1038/nature.2012.11015
Novais RC, Thorstenson YR (2011) The evolution of pyrosequencing for microbiology: from genes to genomes. J Microbiol Methods 86:1–7. doi:10.1016/j.mimet.2011.04.006
Kim TS, Kim HS, Kwon S, Park HD (2011) Nitrifying bacterial community structure of a full-scale integrated fixed-film activated sludge process as investigated by pyrosequencing. J Microbiol Biotechnol 21:293–298. doi:10.4014/jmb.1009.09042
Li YF, Wu B, Zhu GB, Liu Y, Ng WJ, Appan A, Tan SK (2016) High-throughput pyrosequencing analysis of bacteria relevant to cometabolic and metabolic degradation of ibuprofen in horizontal subsurface flow constructed wetlands. Sci Total Environ 562:604–613. doi:10.1016/j.scitotenv.2016.04.020
Maspolim Y, Zhou Y, Guo CH, Xiao KK, Ng WJ (2015) Determination of the archaeal and bacterial communities in two-phase and single-stage anaerobic systems by 454 pyrosequencing. J Environ Sci 36:121–129. doi:10.1016/j.jes.2015.02.017
Wang LP, Zheng BH, Nan BX, Hu PL (2014) Diversity of bacterial community and detection of nirS- and nirK-encoding denitrifying bacteria in sandy intertidal sediments along Laizhou Bay of Bohai Sea, China. Mar Pollut Bull 88:215–223. doi:10.1016/j.marpolbul.2014.09.002
Eckford-Soper LK, Daugbjerg N (2015) Development of a multiplex real-time qPCR assay for simultaneous enumeration of up to four marine toxic bloom-forming microalgal species. Harmful Algae 48:37–43. doi:10.1016/j.hal.2015.06.009
Nishimura T, Hariganeya N, Tawong W, Sakanari H, Yamaguchi H, Adachi M (2016) Quantitative PCR assay for detection and enumeration of ciguatera-causing dinoflagellate Gambierdiscus spp. (Gonyaulacales) in coastal areas of Japan. Harmful Algae 52:11–22. doi:10.1016/j.hal.2015.11.018
Yuan J, Mi TZ, Zhen Y, Yu ZG (2012) Development of a rapid detection and quantification method of Karenia mikimotoi by real-time quantitative PCR. Harmful Algae 17:83–91. doi:10.1016/j.hal.2012.03.004
Zheng BH, Wang LP, Liu LS (2014) Bacterial community structure and its regulating factors in the intertidal sediment along the Liaodong Bay of Bohai Sea, China. Microbiol Res 169:585–592. doi:10.1016/j.micres.2013.09.019
Schloss PD, Westcott SL, Ryabin T, Hall JR, Hartmann M, Hollister EB, Lesnniewski RA, Oakley BB, Parks DH, Robinson CJ, Sahl JW, Stres B, Thallinger GG, Van Jorn DJ, Weber CF (2009) Introducing mothur: open-source, platform-independent, community-supported software for describing and comparing microbial communities. Appl Environ Microbiol 75:7537–7541. doi:10.1128/AEM.01541-09
Otten TG, Crosswell JR, Mackey S, Dreher TW (2015) Application of molecular tools for microbial source tracking and public health risk assessment of a microcystis bloom traversing 300 km of the Klamath River. Harmful Algae 46:71–81. doi:10.1016/j.hal.2015.05.007
Johansson KSL, Lührig K, Klaminder J, Rengefors K (2016) Development of a quantitative PCR method to explore the historical occurrence of a nuisance microagla under expansion. Harmful Algae 56:67–76. doi:10.1016/j.hal.2016.04.012
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This research was financially supported by the central basic scientific research project in the Public Welfare for the scientific research institutes (gyk5091301) and the basic special project of science and technology (2013FY111100).
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Wang, Lp., Lei, K. Rapid Identification and Quantification of Aureococcus anophagefferens by qPCR Method (Taqman) in the Qinhuangdao Coastal Area: A Region for Recurrent Brown Tide Breakout in China. Indian J Microbiol 56, 491–497 (2016). https://doi.org/10.1007/s12088-016-0619-z
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DOI: https://doi.org/10.1007/s12088-016-0619-z