Rapid detection of Prorocentrum donghaiense using nuclease protection assay integrated with dot nucleic acid chromatography strip

Abstract

Harmful algal bloom (HAB), caused by harmful microalgae, is a global marine ecological disaster, and its frequency and damage have increased significantly in recent years. For effective monitoring of HAB, there is an urgent need to establish rapid and easy detection techniques for harmful microalgae. For this purpose, here a nuclease protection assay integrated with dot nucleic acid chromatography strip (NPA-dNACS) detection technology was proposed for the first time. Using Prorocentrum donghaiense that is one of the most typical HAB-forming species in the East China Sea as a target, the application of NPA-dNACS in the detection of harmful microalgae was explored in this study. NPA-dNACS does not require nucleic acid extraction and amplification. Instead, it directly targets high-copy rRNA in cells and employs the hand-made dNACS to automatically determine the detection results, making the detection process rather simple and fast. First, specific probes targeting the D1-D2 region of ribosomal large subunit DNA of P. donghaiense were designed, and the specificity of the probes for the target species was confirmed by the cross-test with 19 other microalgae. Next, all of the main parameters or conditions associated with NPA-dNACS were optimized separately. In addition, the sensitivity of NPA-dNACS was also evaluated, and the developed NPA-dNACS displayed a detection limit of 7 × 10−1 ng of target RNA. The test with spiked samples showed that NPA-dNACS can meet the demand for early warning of P. donghaiense-forming HAB. In conclusion, the proposed NPA-dNACS are specific, rapid, simple, and visualizable, providing a new alternative method for the rapid detection of harmful microalgae like P. donghaiense.