Easy detection of karlodinium veneficum using PCR-based dot chromatography strip

In this study, a novel detection method by PCR-based dot chromatography strip (PDCS) is proposed. To investigate the application of PDCS in the detection of harmful microalgae, the internal transcribed spacer sequence of Karlodinium veneficum, one of the most common bloom-forming species, was cloned and sequenced to design and screen specific primers with tag sequences and probes, including gold nanoparticle probe, test probe, and control probe. The PDCS was prepared manually, and PCR amplicons prepared from the genomic DNA of K. veneficum using tagged specific primers were analyzed by PDCS for visual detection of the target species. The resulting test strip showed red spots at the predicted test and control points visible to the naked eyes, showing the successful development of PDCS. This detection technique is independent of expensive experimental equipment (except a DNA thermal cycler for PCR) but requires an aliquot of PCR amplicons mixed with development buffer to apply to the sample pad of PDCS for approximately 10 min to visualize the analytical results. Cross-reactivity test with 21 microalgae, including K. veneficum, showed that the established PDCS technique has excellent specificity. The detection limit of PDCS was 9.13 x 10(-2) ng mu L-1 for genomic DNA and 5.3 x 10(5) cells L-1 for crude DNA extracts of the target alga. In summary, the PDCS with high sensitivity and specificity can be prepared by hand, which is less expensive than traditional strip, thus providing a promising alternative to the detection of K. veneficum in natural samples.