检测产毒微囊藻的特异性探针研究

通过对9株淡水微囊藻的ITS序列及其微囊藻毒素合成酶基因簇(mcy)的分析,以期获得用于快速检测产毒微囊藻的特异性探针.结果表明:9株淡水微囊藻的ITS序列差异百分比达1.0%~5.0%,平均差异度达3.0%,但序列比对得到的差异性片段与其是否产微囊藻毒素之间无明显相关性,无法用于检测产毒微囊藻特异性引物的设计.根据微囊藻毒素合成基因簇的保守结构域设计的6对引物(MCYAAAF\MCYAAAR,MCYAMAF\MCYAMAR,MCYBAF\CYBAR,MEAF\MCYEAR,MCYGAF\MCYGR,MCYKSF\MCYKSR),对9株微囊藻mcy基因簇的扩增进行了研究,结果与LC-MS的检测结果具有很好的一致性.同时,6对引物具有相同的退火温度,可进行PCR的同步扩增,在提高扩增效率的同时也大大增加了检测结果的准确性.此外,根据微囊藻毒素基因筛选设计了用于检测产毒微囊藻的特异性探针.

Research: Detection Probes for Microcystin-producing Microcystis

In order to obtain probes for detecting the microcystin producing Microcystis,9 strains belonging to the genus Microcysits are analyzed based on 16S to 23S ribosomal DNA intergenic spacer region(ITS) and microcystin biosythesis gene clusters.The results obtained using the similarity analysis of ITS sequences show that all 9 strains belong to Microcystis sp.Multiple sequences alignment show that different degree ranges within 1.0%-5.0%,and the average is 3.0%.The result indicates that the ITS sequences render no help in designing the probes for detecting microcystin producing Microcysits.Based on this finding,the analysis turns to the microcystin biosythesis(mcy) gene clusters.Six pair probes,respectively named as MCYAAAF\MCYAAAR,MCYAMAF\MCYAMAR,MCYBAF\MCYBAR,MCYEAF\MCYEAR,MCYGAF\MCYGR,MCYKSF\ MCYKSR are designed based on the domain of mcy gene.PCR-based analysis reveals that PCR and LC-MS methods give a similar result on toxicity of microcystis strains.Moreover,for sharing the same annealing temperature,it finds that six pair probes can be used to amplify simultaneously,which improves efficiency and accuracy.The probes introduced in this paper can also be used to detect the potential microcystin-producing Microcystis.