Recombinase Polymerase Amplification Combined with Lateral Flow Dipstick Assay for the Rapid and Sensitive Detection of Pseudo-nitzschia multiseries

Abstract

The harmful algal bloom (HAB) species Pseudo-nitzschia multiseries is widely distributed worldwide and is known to produce the neurotoxin domoic acid, which harms marine wildlife and humans. Early detection and preventative measures are more critical than late management. However, the major challenge related to early detection is the accurate and sensitive detection of microalgae present in low abundance. Therefore, developing a sensitive and specific method that can rapidly detect P. multiseries is critical for expediting the monitoring and prediction of HABs. In this study, a novel assay method, recombinase polymerase amplification combined with lateral flow dipstick (RPA-LFD), is first developed for the detection of P. multiseries. To obtain the best test results, several important factors that affected the amplification effect were optimized. The internal transcribed spacer sequence of the nuclear ribosomal DNA from P. multiseries was selected as the target region. The results showed that the optimal amplification temperature and time for the recombinase polymerase amplification (RPA) of P. multiseries were 37 °C and 15 min. The RPA products could be visualized directly using the lateral flow dipstick after only 3 min. The RPA-LFD assay sensitivity for detection of recombinant plasmid DNA (1.9 × 100 pg/μL) was 100 times more sensitive than that of RPA, and the RPA-LFD assay sensitivity for detection of genomic DNA (2.0 × 102 pg/μL) was 10 times more sensitive than that of RPA. Its feasibility in the detection of environmental samples was also verified. In conclusion, these results indicated that the RPA-LFD detection of P. multiseries that was established in this study has high efficiency, sensitivity, specificity, and practicability. Management measures made based on information gained from early detection methods may be able to prevent certain blooms. The use of a highly sensitive approach for early warning detection of P. multiseries is essential to alleviate the harmful impacts of HABs on the environment, aquaculture, and human health.