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.2019 Oct 10;9(1):14532.
doi: 10.1038/s41598-019-51074-3.

Evaluation of sxtA and rDNA qPCR assays through monitoring of an inshore bloom of Alexandrium catenella Group 1

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Evaluation of sxtA and rDNA qPCR assays through monitoring of an inshore bloom of Alexandrium catenella Group 1

Shauna A Murray et al. Sci Rep..

Abstract

Alexandrium catenella (formerly A. tamarense Group 1, or A. fundyense) is the leading cause of Paralytic Shellfish Poisoning in North and South America, Europe, Africa, Australia and Asia. The quantification of A.catenella via sxtA, a gene involved in Paralytic Shellfish Toxin synthesis, may be a promising approach, but has not been evaluated in situ on blooms of A. catenella, in which cell abundances may vary from not detectable to in the order of 106 cells L-1. In this study, we compared sxtA assay performance to a qPCR assay targeted to a species-specific region of ribosomal DNA (rDNA) and an established fluorescent in situ hybridization (FISH) microscopy method. Passing-Bablok regression analyses revealed the sxtA assay to overestimate abundances when <5 cell equivalents A. catenella DNA were analysed, but otherwise was closer to microscopy estimates than the rDNA assay, which overestimated abundance across the full range of concentrations analysed, indicative of a copy number difference between the bloom population and a culture used for assay calibration a priori. In contrast, the sxtA assay performed more consistently, indicating less copy number variation. The sxtA assay was generally reliable, fast and effective in quantifying A. catenella and was predictive of PST contamination of shellfish.

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Conflict of interest statement

The authors declare no competing financial interests. SM declares a non-financial potential competing interest as she has received research funding from Diagnostic Technology Pty Ltd, as they are an Industry Partner in several awarded grants through the Australian Research Council Linkage Grant scheme.

Figures

Figure 1
Figure 1
Map of the sampling location, Salt Pond, MA, USA. The inset box shows the Nauset Marsh system.
Figure 2
Figure 2
Standard curve ofsxtA (A) and LSU rRNA (B) using DNA extractions ofA. catenella, strain ATMP7E8 isolated from Salt Pond, showing efficiency (%), dilution series, (+/− error bars are standard deviations).
Figure 3
Figure 3
Mean of cell concentration estimates from samples collected at 5 m by survey day through the full course of the 2013A. catenella bloom in Salt Pond. LSUFC data are revised estimates of cell concentration based on comparison to microscopy counts as described in the text. Gray bars indicate PST levels measured by mouse bioassay inMytelis edulis collected from Salt Pond by the Massachusetts Department of Marine Fisheries (limit of detection: 40 µg STX eq./100 g shellfish meat). Linear correlations between PST levels and cell concentrations from 7 days previously as measured by microscopy andsxtA (n = 8) were highly significant (r2 = 0.93, 0.91, respectively, p < 0.0001 for both).
Figure 4
Figure 4
Bland Altman plot comparing log-transformedsxtA and field-calibrated LSU assay derived estimates ofA. catenella abundance. Points are means from triplicate qPCR assays of a single DNA extract. ThesxtA assay significantly overestimated cell concentration in samples with fewer than 200 cells, a threshold that corresponds to about 5 cell equivalents or approximately 103sxtA copies per reaction. Were assays equivalent points would be tightly clustered about 0 across the full range of measured concentrations (x-axis).
Figure 5
Figure 5
Passing Bablok regression and residual analyses. (A) Results from regression of microscopy and field-calibrated, LSU qPCR estimates of cell concentration. Solid line is the linear relationship described by parameters in Table 2, dotted line is 1:1, and dashed lines are upper and lower bounds from 95% confidence interval estimates of the regression. (B) Log-transformed residuals from the LSU-microscopy regression across the range of concentrations as estimated by microscopy. (C) The same log-transformed residuals but plotted by sampling day. The LSU assay tended to overestimate cell concentrations during early bloom development (March 4 and 11) and bloom termination (May 13 and 22). (D) Regression of microscopy and field-calibrated,sxtA qPCR estimates of cell concentration derived from the subset of samples ≥100 cells L−1 by microscopy (closed, black circles). Open circles are data points excluded from the regression (microscopy estimate <100 cells L−1). (E) Log-transformed residuals from thesxtA-microscopy regression across the range of observed cell concentrations. ThesxtA assay increasingly overestimated cell concentrations in samples below the 100 cells L−1 threshold. (F) Log-tranformed residuals fromsxtA-microscopy regression plotted by sampling day. Samples with cell concentrations >100 cells L−1 (closed, black circles) are more tightly clustered about 0 (no error) than samples with lower concentrations (open circles) and as estimated using the LSU assay (left column).
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