Real-time PCR for detection and quantification of the protistan parasite Perkinsus marinus in environmental waters

Abstract

The protistan parasite Perkinsus marinus is a severe pathogen of the oyster Crassostrea virginica along the east coast of the United States. Very few data have been collected, however, on the abundance of the parasite in environmental waters, limiting our understanding of P. marinus transmission dynamics. Real-time PCR assays with SybrGreen I as a label for detection were developed in this study for quantification of P. marinus in environmental waters with P. marinus species-specific primers and of Perkinsus spp. with Perkinsus genus-specific primers. Detection of DNA concentrations as low as the equivalent of 3.3 x 10(-2) cell per 10-microl reaction mixture was obtained by targeting the multicopy internal transcribed spacer region of the genome. To obtain reliable target quantification from environmental water samples, removal of PCR inhibitors and efficient DNA recovery were two major concerns. A DNA extraction kit designed for tissues and another designed for stool samples were tested on environmental and artificial seawater (ASW) samples spiked with P. marinus cultured cells. The stool kit was significantly more efficient than the tissue kit at removing inhibitors from environmental water samples. With the stool kit, no significant difference in the quantified target concentrations was observed between the environmental and ASW samples. However, with the spiked ASW samples, the tissue kit demonstrated more efficient DNA recovery. Finally, by performing three elutions of DNA from the spin columns, which were combined prior to target quantification, variability of DNA recovery from different samples was minimized and more reliable real-time PCR quantification was accomplished.

FIG. 1.

Standard PCR results. (a) Specificity of the PerkITS and PmarITS primers tested on no DNA (lane N) and P. marinus (lane 1), P. chesapeaki (lane 2), P. andrewsi (lane 3), P. atlanticus (lane 4), P. mediterraneus (lane 5), H. perezi (lane 6), A. occelatum (lane 7), P. piscicida (lane 8), P. schumwayae (lane 9), A. carterae (lane 10), C. cohnii (lane 11), K. micrum (lane 12), P. foliaceum (lane 13), and P. micans (lane 14) DNAs. (b) Sensitivity of PerkITS and PmarITS primers tested on DNA extracted with either the tissue or the stool kit and no DNA (N) and serial dilutions of P. marinus DNA corresponding to 8 × 10⁰ (lane 1), 8 × 10⁻¹ (lane 2), 8 × 10⁻² (lane 3), and 8 × 10⁻³ cell per reaction mixture (lane 4).

FIG. 2.

Standard curves to quantify Perkinsus spp. and P. marinus in environmental samples by real-time PCR with PerkITS and PmarITS primers, respectively. The standard curves correspond to the C_T versus the logarithm of the estimated cell concentration in the sample. The DNA concentrations tested were obtained by performing 10-fold serial dilution of DNA extracted with either the tissue or the stool kit after spiking ASW with cultured P. marinus cells. Each value corresponds to the mean of three replicate DNA samples for each dilution and the respective standard deviation. After the dilution of P. marinus DNA, the corresponding concentrations of cells per reaction mixture ranged from 33 × 10⁰ to 3 × 10⁻² cell per real-time PCR, with each cell containing multiple ITS copies.

FIG. 3.

Recovery of DNA from the QIAGEN columns used with the tissue kit and the stool kit. Three replicates of each type of water sample (ASW, PTS, or DWS) were spiked with two P. marinus cell concentrations, 100 (a) and 20 (b) cells/ml of water. After each of the five elutions was performed, the mean cell concentration per reaction mixture and the standard deviation were calculated on the basis of the P. marinus DNA concentration, the C_T values, and the regression formulas for the PmarITS primers.

FIG. 4.

Quantification of P. marinus cell concentrations (cells per reaction mixture) after combining the first three elutions from DNA samples extracted with the tissue kit or the stool kit. Samples were obtained by spiking ASW or two environmental waters samples (PTS and DWS) with cultured P. marinus cells with a final concentration of 100 cells/ml of water. For each type of water and each cell concentration, three replicates were analyzed and the means and standard deviations are presented.