Parallel detection of harmful algae using reverse transcription polymerase chain reaction labeling coupled with membrane-based DNA array

Harmful algal blooms (HABs) are a global problem, which can cause economic loss to aquaculture industry's and pose a potential threat to human health. More attention must be made on the development of effective detection methods for the causative microalgae. The traditional microscopic examination has many disadvantages, such as low efficiency, inaccuracy, and requires specialized skill in identification and especially is incompetent for parallel analysis of several morphologically similar microalgae to species level at one time. This study aimed at exploring the feasibility of using membrane-based DNA array for parallel detection of several microalgae by selecting five microaglae, including Heterosigma akashiwo, Chaetoceros debilis, Skeletonema costatum, Prorocentrum donghaiense, and Nitzschia closterium as test species. Five species-specific (taxonomic) probes were designed from variable regions of the large subunit ribosomal DNA (LSU rDNA) by visualizing the alignment of LSU rDNA of related species. The specificity of the probes was confirmed by dot blot hybridization. The membrane-based DNA array was prepared by spotting the tailed taxonomic probes onto positively charged nylon membrane. Digoxigenin (Dig) labeling of target molecules was performed by multiple PCR/RT-PCR using RNA/DNA mixture of five microalgae as template. The Dig-labeled amplification products were hybridized with the membrane-based DNA array to produce visible hybridization signal indicating the presence of target algae. Detection sensitivity comparison showed that RT-PCR labeling (RPL) coupled with hybridization was tenfold more sensitive than DNA-PCR-labeling-coupled with hybridization. Finally, the effectiveness of RPL coupled with membrane-based DNA array was validated by testing with simulated and natural water samples, respectively. All of these results indicated that RPL coupled with membrane-based DNA array is specific, simple, and sensitive for parallel detection of microalgae which shows promise for monitoring natural samples in the future.