Development of real-time PCR assays for rapid detection of Pfiesteria piscicida and related dinoflagellates

Abstract

Pfiesteria complex species are heterotrophic and mixotrophic dinoflagellates that have been recognized as harmful algal bloom species associated with adverse fish and human health effects along the East Coast of North America, particularly in its largest (Chesapeake Bay in Maryland) and second largest (Albermarle-Pamlico Sound in North Carolina) estuaries. In response to impacts on human health and the economy, monitoring programs to detect the organism have been implemented in affected areas. However, until recently, specific identification of the two toxic species known thus far, Pfiesteria piscicida and P. shumwayae (sp. nov.), required scanning electron microscopy (SEM). SEM is a labor-intensive process in which a small number of cells can be analyzed, posing limitations when the method is applied to environmental estuarine water samples. To overcome these problems, we developed a real-time PCR-based assay that permits rapid and specific identification of these organisms in culture and heterogeneous environmental water samples. Various factors likely to be encountered when assessing environmental samples were addressed, and assay specificity was validated through screening of a comprehensive panel of cultures, including the two recognized Pfiesteria species, morphologically similar species, and a wide range of other estuarine dinoflagellates. Assay sensitivity and sample stability were established for both unpreserved and fixative (acidic Lugol's solution)-preserved samples. The effects of background DNA on organism detection and enumeration were also explored, and based on these results, we conclude that the assay may be utilized to derive quantitative data. This real-time PCR-based method will be useful for many other applications, including adaptation for field-based technology.

FIG. 1

Specificity of P. piscicida (A) and P. shumwayae sp. nov. (B) real-time PCR assays. DNA was extracted from five cultures (A, B, C, D, and E) determined to be P. piscicida by either SEM or LM (coupled with 18S rDNA sequence analysis) and analyzed with the real-time PCR assay specific for P. piscicida. DNA was extracted from three cultures (F, G, and H) determined to be P. shumwayae sp. nov. by SEM and analyzed with the real-time PCR assay specific for P. shumwayae sp. nov. Negative results in both graphs (below the noise band) represent morphologically close relatives. The negative (no-DNA) controls were negative. The corresponding results obtained are presented in Table 2.

FIG. 2

Real-time P. piscicida PCR assay on the Lightcycler to detect the organism in 10-fold serial dilutions of unpreserved and fixative (acidic Lugol's solution)-preserved culture material. A 10-ml volume of each dilution was filtered through a 5-μm-pore-size filter, and DNA was extracted from the retained organism. In graphs A and C (unpreserved and fixative preserved, respectively), fluorescence acquired from dilutions detected with the probe is plotted against the cycle number. The numbers indicate the equivalent numbers of cells (genomes) aliquoted into the PCR (i.e., extracted DNA was eluted in 100 μl, and 1 μl was assayed). In graphs B and D (unpreserved and fixative preserved, respectively), the log of the number of cells in the starting material is plotted against the cycle number at which the signal exceeded the threshold (set at 10% of the total fluorescence for the data set). In the unpreserved dilution, fewer than one cell per reaction could be detected, while in the fixative-preserved sample, the lower limit of detection was six cells per reaction.

FIG. 3

Single-cell specificity and sensitivity of P. piscicida real-time PCR-based assay. (A) Results of PCR performed on eight replicates of single P. piscicida cells (all detectable). (B) Results of PCR performed on G. galatheanum (seven replicates), a close morphological relative, to test assay specificity. The positive control was total DNA isolated from a P. piscicida culture. In both graphs, the values for the negative control are below the noise band.

FIG. 4

Detection of P. piscicida over time in unpreserved (A) and fixative (acidic Lugol's solution)-preserved (B) environmental water spiked with a known number of organisms. Spiked samples were stored on the benchtop, and DNA was extracted from 40-ml aliquots on the days indicated.

FIG. 5

Detection of P. piscicida to 120 days in a fixative (acidic Lugol's solution)-preserved culture. At time point indicated, DNA was extracted from a 2-ml aliquot of the culture. DNA from all time points was assayed with the P. piscicida probe assay in the same Lighcycler run. The inset is a graph depicting fluorescence versus cycle number for each time point.

FIG. 6

Effects of background DNA on detection of P. piscicida. Three 10-fold serial dilutions were prepared from a pure P. piscicida strain MDFDEPMR23 culture. Aliquots from one dilution set were spiked postfiltration with 12.8 ng of organism DNA extracted from a heterogeneous environmental water sample (Choptank River in Maryland). The third dilution set was spiked with 1,860,000 cells of Rhodomonas sp. prefiltration.