Rapid Detection And Enrichment Of Risk Microorganisms In Aquatic Environment

Rapid diagnosis of microorganisms is of great significance in water quality monitoring. In this study,achieved fluorescence array corresponds to the test microorganism specificly based on fluorescence array-based microbial detection using a new way, in which the fluorescent signals were generated through the interaction between fluorophores and quenchers labeled double strands oligonucleotide DNA and target microorganisms. Besides, "magnet beads-aptamer" was functionalized with polyethylene glycol(PEG) to improve the specificity against target microorganisms.Specially designed nucleic acid oligonucleotides and its complementary strand was labeled with fluorephore and quencher respectively. Through the interaction of DNA oligonucleotide chain and microorganisms achieved fluorescence array corresponds to the test microorganism specificly. The basic parameters was optimized for detecting microorganisms: DNA oligonucleotide chain l ength are 13 to 15 bp; the GC content from40% to 53%;the range of oligo-stranded DNA concentration is 0.1-0.5 μM; the reaction buffer p H is 6.5 and the reaction temperature is 22-25 ℃.When 6 strains including3 genus and 6 species(Escherichia coli, Staphylococcus aureus, Bacillus cereus, Peanibacillus alvei, Fusiform lysine bacillus, Vogesella perlucida) were selected as the test strains, PCo A(Principle Coordinate Analysis) of the fluorescence arrays indicates: fluorescence array and target microorganisms have characteristic correspondence; increase of oligonucleotide chain type(three kinds up to five kinds) can significantly improve the sensitivity of the method; the method is not affected by cell concentration of 108-109 CFU/m L; and activity has no significant effect on the result of discrimination.PEG was functionalized onto "magnet bead-aptamer" to enhance the specificity against target microorganisms. When "magnet bead-aptamer" was modified with PEG-1000 for the enrichment of Staphylococcus aureus in a mixed system containing both Staphylococcus aureus and Escherichia coli, the selectivity against Staphylococcus aureus was significantly higher under the PEG: aptamer=4:1(mol/mol)conditions, comparing with PEG: aptamer =2:1. While under both conditions, the achieved selectivity was much higher than unmodified beads. "Magnet bead-aptamer" modified with PEG-400 and PEG-1000 both can increase the adsorption ratio of Staphylococcus aureus.