Rapid Detection and Quantitation of Microcystin-Producing Microcystis Using Real-Time PCR

  • Qiu Y
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Abstract

A system of analytical processes has been developed in order to serve as a cost-effective scheme for the monitoring of cyanobacterial toxins on a quantitative basis, in surface and drinking waters. Five cyclic peptide hepatotoxins, microcystin-LR, -RR, -YR, -LA and nodularin were chosen as the target compounds. Two different enzyme-linked immunosorbent assays (ELISA) were validated in order to serve as primary quantitative screening tools. Validation results showed that the ELISA methods are sufficiently specific and sensitive with limits of detection (LODs) around 0.1 ??g/L, however, matrix effects should be considered, especially with surface water samples or bacterial mass methanolic extracts. A??colorimetric protein phosphatase inhibition assay (PPIA) utilizing protein phosphatase 2A and p-nitrophenyl phosphate as substrate, was applied in microplate format in order to serve as a quantitative screening method for the detection of the toxic activity associated with cyclic peptide hepatotoxins, at concentration levels >0.2 ??g/L of MC-LR equivalents. A??fast HPLC/PDA method has been developed for the determination of microcystins, by using a short, 50 mm C18 column, with 1.8 ??m particle size. Using this method a 10-fold reduction of sample run time was achieved and sufficient separation of microcystins was accomplished in less than 3 min. Finally, the analytical system includes an LC/MS/MS method that was developed for the determination of the 5 target compounds after SPE extraction. The method achieves extremely low limits of detection (<0.02 ??g/L), in both surface and drinking waters and it is used for identification and verification purposes as well as for determinations at the ppt level. An analytical protocol that includes the above methods has been designed and validated through the analysis of a number of real samples. ?? 2009 Elsevier Ltd. All rights reserved.

Figures

  • Figure 1: Ct value obtained from real-time PCR for serial dilutions of mcyE DNA fragment.
  • Figure 2: mcyE copy numbers from 19 reference water samples of 2011. Microcystis mcyE copy numbers were determined by rt-QPCR, which was showed as mcyE copy numbers per millilitre. The sum of mcyE copy numbers (■) from centrifuge supernatant and centrifuge pellet were compared with mcyE copy numbers from direct extraction (♦).
  • Table 2: rt-QPCR and PPI results of 20 reference samples.
  • Figure 3: McyE copy numbers of Pine Lake (A) and Pigeon Lake (B) during the sampling period. Microcystis mcyE copy numbers were determined by rt-QPCR, which was showed as mcyE copy numbers per millilitre. Pine lake: Leisure Camp beach (♦) and Green Acre beach (■); Pigeon Lake: Mission beach (♦) and Zeiner Park beach (■).
  • Figure 4: 16S rRNA copy numbers of Pine Lake (A) and Pigeon Lake (B) during the sampling period. Cyanobacterial 16s rRNA copy numbers were determined by rt-QPCR, which was showed as 16S rRNA copy numbers per millilitre. Pine Lake: Leisure Camp beach (♦) and Green Acre beach (■); Pigeon Lake: Mission beach (♦) and Zeiner Park beach (■).

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APA

Qiu, Y. (2013). Rapid Detection and Quantitation of Microcystin-Producing Microcystis Using Real-Time PCR. Journal of Molecular Biomarkers & Diagnosis, S5. https://doi.org/10.4172/2155-9929.s5-006

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