Development Of A PCR-based Reverse Dot Hybridization Assay For Paralleldetection Of Several Harmful Algal Species

The identification and detection of harmful algae is the premise and foundation for redtide research and warning, so its methodology becomes a hot point. However, thetraditional identification method (light microscopy) based on morphologicalcharacteristics of red tide algae used to distinguishing causative species has disvantagesof low efficiency and accuracy, and especially not fit for parallel detection of severalalgae in the natural water samples. Therefore, this study aims to develop a moleculartechnique—PCR-based reverse dot blot hybridization for simultaneous detection ofseveral harmful algal species.Six common red tide algae, including Prorocentrum donghaiense Lu, Skeletonemacostatum (Greville) Cleve, Symbiodinium sp., Chaetoceros debilis, Heterosigmaakashiwo and Nitzschia closterium, were used as test algae in this paper. Firstly, thegenomic DNA from six algae were isolated to used as template to PCR-amplifythe largesubunit (LSU) D1–D2of rDNA sequences, and then the PCR products were purified wasand ligated with T-vector. The ligation product was used to transformedthe competentEscherich coli. Through blue–white spot screening, the positive colonies were used forcommercial sequences.According to the obtained sequences, we designed the universal primers using PrimerPremier5.0and the suitable probes with Array Designer4.20. Then the probes weevaluated with Oligo, and three probes with good specificity were screened. The selectedprobes were further tested using reverse dot blot hybridization, and finally got thespecific probes. Then the probes were labelled with digoxigenin. Six common red-tidealgae and some microalgae were choosen. The specificity of the probes were confirmedby dot blot hybridization assay. The probes were tailed and were used to preparelow-density membrane-based chip. This study preliminarily established a PCR-reversedot blot hybridization assay, which was based on the large subunit rDNA by labeling thePCR products with digoxigenin. Then the hybridization conditions, including probe dosage, the amount of digoxigenin-labeled PCR products, UV cross-linking time,hybridization temperature, hybridization time and washing time, were optimized. Theresults showed that the probes did have specificity and the final optimized conditionswere as follows: the probe sample volume is>1pmol, digoxigenin-labeled products>25ng, UV cross-linking time30s, hybridization42oC for2h, washing membranes at47oCfor2×5min.To further confirmed the practicability of the PCR-reverse dot blot hybridization assay,the simulated natural water samples and natural water samples of different concentrationand different fixed time were used as the samples, and tested with PCR-reverse dot blothybridization assay. The results showed that the simulated natural water samples coulddetect0.1cell/ml and six kinds of mixed algae that were fixed with Lugol’s solution for1d,3d,5d,10d,15d and30d can detected all the six kinds of algae. This technologycan detect the target algae within natural samples, and the results were identified with theresults under light microscopy.Finally, the ITS sequences of10algae were cloned, and used to design specific probe.The probe specificity was assessed by dot blot hybridization. Then, the low-density chipbased on the ITS sequences was designed, which provided a possibility for establishing aPCR-reverse dot blot hybridization assay based on ITS sequences.