Detection of Prorocentrum minimum by hyperbranched rolling circle amplification coupled with lateral flow dipstick

Area of Science:

• Marine Biology
• Microbiology
• Biotechnology

Background:

• Prorocentrum minimum is a harmful algal bloom (HAB) species.
• Accurate and rapid detection of P. minimum is crucial for environmental monitoring.
• Traditional methods like microscopic examination can be time-consuming and require expertise.

Purpose of the Study:

• To develop a novel, specific, sensitive, and rapid detection method for P. minimum.
• To establish hyperbranched rolling circle amplification coupled with lateral flow dipstick (HRCA-LFD) for P. minimum detection.
• To evaluate the performance of HRCA-LFD compared to conventional methods.

Main Methods:

• Development of a padlock probe (PLP) targeting species-specific sites on the LSU rDNA D1-D2 region of P. minimum.
• Optimization of hyperbranched rolling circle amplification (HRCA) parameters including probe concentration, ligation, digestion, and amplification times and temperatures.
• Coupling HRCA with lateral flow dipstick (LFD) for visual detection.

Main Results:

• The HRCA-LFD method demonstrated high specificity, distinguishing P. minimum from other microalgae.
• Achieved a low detection limit: 1.42 × 10⁻⁷ ng μL⁻¹ for genomic DNA and 0.17 cells for crude DNA extract.
• The method showed 100-fold higher sensitivity than regular PCR and stable performance against interfering species.
• Field sample results correlated well with microscopic examination.

Conclusions:

• The developed HRCA-LFD is a specific, sensitive, and rapid method for P. minimum detection.
• HRCA-LFD offers a reliable and efficient alternative to traditional microscopic methods for field sample analysis.
• This technique has significant potential for HAB monitoring and management.