Development of a PNA Probe for Fluorescence In Situ Hybridization Detection of Prorocentrum donghaienseReport as inadecuate

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Prorocentrum donghaiense is a common but dominant harmful algal bloom HAB species, which is widely distributed along the China Sea coast. Development of methods for rapid and precise identification and quantification is prerequisite for early-stage warning and monitoring of blooms due to P. donghaiense. In this study, sequences representing the partial large subunit rDNA D1–D2, small subunit rDNA and internal transcribed spacer region ITS-1, 5.8S rDNA and ITS-2 of P. donghaiense were firstly obtained, and then seven candidate DNA probes were designed for performing fluorescence in situ hybridization FISH tests on P. donghaiense. Based on the fluorescent intensity of P. donghaiense cells labeled by the DNA probes, the probe DP0443A displayed the best hybridization performance. Therefore, a PNA probe PP0443A analogous to DP0443A was used in the further study. The cells labeled with the PNA probe displayed more intensive green fluorescence than that labeled with its DNA analog. The PNA probe was used to hybridize with thirteen microalgae belonging to five families, i.e., Dinophyceae, Prymnesiophyceae, Raphidophyceae, Chlorophyceae and Bacillariophyceae, and showed no visible cross-reaction. Finally, FISH with the probes PP0443A and DP0443A and light microscopy LM analysis aiming at enumerating P. donghaiense cells were performed on the field samples. Statistical comparisons of the cell densities cells-L of P. donghaiense in the natural samples determined by FISH and LM were performed using one-way ANOVA and Duncan-s multiple comparisons of the means. The P. donghaiense cell densities determined by LM and the PNA probe are remarkably higher than p<0.05 that determined by the DNA probe, while no significant difference is observed between LM and the PNA probe. All results suggest that the PNA probe is more sensitive that its DNA analog, and therefore is promising for the monitoring of harmful algal blooms of P. donghaiense in the future.

Author: Guofu Chen, Chunyu Zhang , Baoyu Zhang , Guangce Wang , Douding Lu, Zhong Xu, Peishen Yan



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