Development of a multiplex PCR-nucleic acid chromatographic strip for rapid detection of representative harmful...

Harmful algal blooms (HABs) are often triggered by the combined effects of multiple phytoplankton species. Therefore, developing an efficient method for simultaneously detecting microalgae is key to providing early warning of HABs. This study developed a technique based on multiplex PCR combined with the nucleic acid chromatographic strip (mPCR-NACS), which can simultaneously detect three representative harmful microalgae in the East China Sea, i.e., Heterosigma akashiwo, Prorocentrum donghaiense, and Karenia mikimotoi. Here, three pairs of specific primers (Ha-FP/Ha-RP, Pd-FP/Pd-RP, and Km-FP/Km-RP) containing specific tags, spacer, and primer sequences were designed based on the ribosomal large subunit rDNA D1-D2 of the target species. The mPCR amplification system was initially established using these specific primers, followed by the optimization of key parameters to increase efficiency, including hybridization temperature (40°C) and detection probe concentration (5.0 μM). Next, a cross-reactivity test with representative control algal species revealed the specificity of the developed mPCR-NACS. A further stability test showed that non-target microalgal species did not interfere with the detection of target microalgae by mPCR-NACS, demonstrating excellent anti-interference ability. In addition, a final evaluation with simulated natural water samples showed that the detection limit of mPCR-NACS could reach 10 cells mL^-1. In summary, the established mPCR-NACS is characterized by simplicity, rapidity, and visualized results, which may provide reliable technical support for the early warning and monitoring of HABs.