Development Of DNA Chip For Detecting Microcystin

In order to obtain probes for establishing a DNA chip to detect toxic microcystis, we carriedout a series of exploratory researches based on13laboratory strains of Microcystis sp.(Microcystis aeruginosa, Microcystis wesenbergii, Microcystis elabens, Microcystis viridis,Microcystis ichyoblabe, Microcystis flos-aquae). In this study, we chosed16S rDNA,16S-23SrDNA and microcystin biosynthesis (mcy) gene clusters of Microcystis as targets for screening ofspecific probes.The PCR amplification of the16S rDNA gene was done with universal primers27F and1492R. The assembled sequences were analyzed by using the BLAST program to ensure that thesequences generated were microcystis sp. in origin. Results showed that9strains named MA-1,MA-2, MA-3, ME-1, MF-2, MF-3, MW-1, MV-1and MI-1exhibited levels of sequence similarityranging from99%-100%. However,4strains named MF-4, MF-1, MW-2and MV-2showed nohomology with microcystis sp.. The alignment analysis of16S rDNA gene sequences indicatedthat the sequences between sepecies and subsepecies were very similar. It was difficult to designspecific probes.The internal transcribed spacer (ITS) region of9strains of Microcystis sp. cultured inlaboratory was sequenced. The identity of ITS sequence of the9strains and others retrieved fromGenBank were calculated. Based on the results, we designed18probes to identify species ofMicrocystis sp.. These probes were validated with DNA chip hybridization. Most of the18probeswere not specific. Finally, we selected3strains (MA-1, MW-1, MV-1) as representative strainsamong them. We designed9probes according to the difference of ITS sequences and validatedthese probes using DNA chip hybridzation. The results indicated that7of9porbes gave postivesignals, which could be use to developping the DNA chip in future.We designed6probes targeting at microcystin biosythesis (mcy) gene clusters and validatedeach probe with DNA chip hybridization. Results showed that all the expected signals were foundby the chip assay. In order to reduce the frequence of PCR, we developed multiplex PCR assay foramplification of microcystin biosythesis genes. The multiplex PCR reaction condition wasoptimized. The optimal conditions of PCR reaction were as followed: the concentration ratio offorward primer to reverse primers labeled with TAMRA was1:1. The volume of Mg2+(25mM),dNTP(2.5μM), Taq DNA polymerase(5U/μl) and each primer was3.5μl,4.5μl,0.4μl and1.5μlin a50μl reaction. The annealing temperature was51℃. Finally, we established a DNA chip for detecting microcystin-producing microcystisaccording to9probes of ITS and6mcy probes. We chosed3samples (Microcystis aeruginosa,Microcystis wesenbergii, Microcystis viridis) to test this DNA chip. We found perfectcorrespondence between the expected and actual DNA chip hybridization, except AMA probe.In conclusion, we demonstrated the feasibility of the DNA microarray for detectingmicrocystin-producing microcystis. The DNA chip we established was rapid, specific and high-throughout. This assay has wide potential for the real-time monitoring of microcystins in water.This method can be easily applied to the future study of other marker genes, one of the mostinsteresting of which one that could detect potentially toxic cyanobacteria.