Rapid detection of Chattonella marina by PCR combined with dot lateral flow strip

Abstract

In this study a novel technique referred to as PCR combined with dot lateral flow strip (PCDS) is proposed and its application to the detection of harmful microalgae was explored. For this purpose, using Chattonella marina as a test algal species, PCR targeting the D1-D2 region of large subunit ribosomal gene of this alga was performed with the tagged specific primers. The amplicons were then analyzed with the manually prepared dot lateral flow strip, and the strip could produce a test dot and a control dot that are naked-eye detectable, indicating the successful establishment of PCDS. The established PCDS assay does not require expensive instruments for the detection, and the results can be observed visually after adding 7.5 μL of PCR amplicons in combination with 92.5 μL of chromatography buffer to the sample pad of the strip for about 10 min. The PCR conditions were optimized to enhance the effectiveness of detection. The cross-reactivity test with 23 microalgae species, including Chattonella marina, showed good specificity of the PCDS. The detection limit of PCDS was 1.25 × 10 −2 ng µL −1 for genomic DNA and 10 1 cells mL −1 for crude cell extracts, which can meet the detection needs. In summary, the PCDS proposed in this study has low cost, clear, and intuitive detection results and good specificity and sensitivity, providing a novel detection method for C. marina.