Establishment Of Sandwich Hybridization Integrated With Nuclease Protection Assay And Detection Of Several HAB Species With It

It is one of the research focuses for marine ecology that the emergence, development and vanishment of harmful algae blooms (HABs) at present. Among them, qualitative and quantitative detection for HAB species is the basic task. Besides traditional microscopy technology, more and more new technologies are introduced into this field. In this study, combining traditional S1 enzyme analysis with sandwich hybridization, sandwich hybridization integrated with nuclease protection assay (NPA-SH) was developed. The assay consists of three steps: firstly, cell-derived rRNA sample was hybridized with the nuclease-protection-assay probe (NPA probe) and subjected to digestion with S1 nuclease to clear all mismatched probes and preserve perfectly matched probes stoichiometrically; secondly, the remaining NPA probes were sandwich hybridized with the corresponding capture DNA probes immobilized on a microplate and then the corresponding signal probes were hybridized with the NPA probes; finally, the probe'sandwich'was quantified with enzyme-mediated color reaction. The signal intensity correlated to the abundance of the target species in the original samples. On the basis of feasibility, this method was optimized from cells splitting, nuclease protection assay and sandwich hybridization. Finally, twelve species-specific NPA probes were respectively designed, the standard curves for each alga, Gymnodinium sp., Akashiwo sanguinea, Prorocentrum micans, Prorocentrum minimum, Prorocentrum donghaiense, Alexandrium catenella, Scrippsiella trochoidea, Chaetoceros curvisetus, Skeletonema costatum, Pseudo-nitzschia pungens, Thalassiosira rotula and Phaeocystis globosa, were established and the equations were deducted. Good specificity and sensitivity had achieved. Moreover, there were good results when cultured samples and field ones were analyzed with this method.NPA-SH avoided complicated pre-treatments of purification of nucleic acid, transformed the targeted rRNA into the complemental DNA probes equally. This method solved the problem of RNA easy to degrade and upgraded the probe specificity from DNA probes hybridizing with RNA of microalgae to check reality of hybridization, using the ability of S1 nuclease, which degrades single-stranded nucleic acids to yield 5' phosphoryl mono or oligonucleotides to leave double stranded DNA, RNA and DNA/RNA hybrids intact (That is to say, the specific probes were kept and the others...