Development of a real-time PCR probe for quantification of the heterotrophic dinoflagellate Cryptoperidiniopsis brodyi (Dinophyceae) in environmental samples

Abstract

A TaqMan format real-time PCR probe was developed against the internal transcribed spacer 2 ribosomal DNA region for the specific detection and quantification of Cryptoperidiniopsis brodyi in environmental samples. The assay specificity was confirmed by testing against related dinoflagellates and verified by sequencing PCR amplicons from natural water samples. Phylogenetic analysis of the sequenced environmental samples also showed that this assay is specific to C. brodyi. The C. brodyi-specific assay was used in conjunction with Pfiesteria piscicida- and Pfiesteria shumwayae-specific real-time PCR assays to investigate the temporal variations of C. brodyi, P. piscicida, and P. shumwayae abundance in the Derwent estuary, Tasmania. The 18-month field survey from November 2004 to April 2006 revealed that C. brodyi occurred in all seasons at very low densities, mostly below 25 cells liter(-1), with higher abundance (maximum, 112 cells liter(-1)) in April and May. P. piscicida was detected only once, in May 2005 at 60 cells liter(-1). P. shumwayae was not detected during the survey.

FIG. 1.

Locations where water samples were collected for this study.

FIG. 2.

Agarose gel analysis showing C. brodyi-selective real-time PCR products. The real-time PCR was carried out using a SYBR green-based system with the primers CBITSF and CBITSR. The arrow indicates a positive real-time PCR product of 128 bp. Lanes: 1, 2-kbp-ladder molecular size marker; 2, C. brodyi; 3, P. piscicida; 4, P. shumwayae; 5, Karlodinium veneficum (=micrum); 6, Karenia mikimotoi; 7, Prorocentrum minimum; 8, no-template control.

FIG. 3.

Alignment of partial ITS2 rDNA sequences from environmental sample and C. brodyi (CBWA12). The field sample DE160505 was sampled from the Derwent River on 16 May 2005 and was detected by C. brodyi-specific real-time PCR. Dots indicate nucleotides identical to those of C. brodyi.

FIG. 4.

Phylogram based on Bayesian analysis of partial ITS2 rDNA sequences (128 bp) of environmental samples and closely related organisms. The environmental samples DE281104 to DE160505 were sampled from the Derwent River from 28 November 2004 to 16 May 2005. These samples were detected by C. brodyi-specific real-time PCR and were sequenced. The sample DE250505 was collected on 25 May 2005 and amplified with P. piscicida-specific real-time PCR. The numbers adjacent to each node represent probabilities which were derived from 150,000 generations using a general time-reversible evolution model with gamma-distributed (0.248) among-site rate variation. The strains sequenced in the present study are shown in bold. Consistency index, 0.837; rescaled consistency index, 0.696; retention index, 0.831; homoplasy index, 0.162.

FIG. 5.

Linear relationship between the C_T values and the cell numbers for C. brodyi (r² = 0.997) (A) and for P. piscicida (r² = 0.998) (B). The standard errors from two measurements are shown as error bars.

FIG. 6.

Temporal variations in C. brodyi, P. piscicida, and P. shumwayae abundances in the Derwent River, Tasmania, during 2004 to 2006, quantified by species-specific real-time PCR. The values are the means of triplicate wells.