Detection of Prorocentrum minimum (Pavillard) Schiller with an Electrochemiluminescence–Molecular Probe Assay

• Published in: Marine biotechnology (New York, N.Y.) N.Y.), 2012-08, Vol.14 (4), p.502-511
• Main Authors: Zhu, Xia, Zhen, Yu, Mi, Tiezhu, Yu, Zhigang
• Format: Article
• Language: English
• Subjects: Algae; Algal blooms; Analysis; Biological Assay - instrumentation; Biomedical and Life Sciences; Biosensing Techniques - instrumentation; Biotechnology; Conductometry - instrumentation; Dinoflagellida - isolation & purification; Electrodes; Electrons; Engineering; Equipment Design; Equipment Failure Analysis; Eutrophication; Freshwater & Marine Ecology; Harmful Algal Bloom; Hybridization; Laboratories; Life Sciences; Luminescence; Luminescent Measurements - instrumentation; Marine; Marine environment; Microbiology; Molecular Probe Techniques - instrumentation; nucleic acid hybridization; Original Article; Oxidation; Prorocentrum; Prorocentrum minimum; Shellfish; Student's t-test; Studies; Toxins; Zoology
• ISSN: 1436-2228; 1436-2236
• DOI: 10.1007/s10126-012-9431-x

The occurrence of harmful algal blooms (HABs) caused by Prorocentrum minimum (Pavillard) Schiller is a crucial subject in the study of HABs. An electrochemiluminescence–molecular probe assay (ECL–MP) was developed to qualitatively and quantitatively detect P. minimum. It was based on the sandwich hybridization integrated with a nuclease protection assay (NPA–SH) and improved by electrochemiluminescence (ECL). An ECL analyzer was established in this study, and it was shown that this analyzer was stable and highly sensitive, with a detection range of 0.4 pmol to 4 nmol Ru(bpy)3Cl2·6H2O under optimal reaction conditions of 1.0 V, 1.0 mA, 1.5 mol·L−1 TPrA, and pH 7.4. The optimal amount of magnetic beads for separation of labeled NPA probes in a 20-μL hybridization mixture was 4 μg. The ECL counts per second was linear with the number of P. minimum cells in a range of 6.25 × 102 to 4 × 104, and there was no significant difference between ECL–MP and microscopy, with a 95% confidence level (t test) when individual, mixed cultures and field samples were treated. This study provides a convenient method for fast and accurate detection of P. minimum in the marine environment.