用双特异探针技术定性定量分析微型原甲藻

针对微型原甲藻核糖体大亚基和小亚基RNA分别设计了大亚基探针(LSU probe)和小亚基探针(SSU probe),发展了对微型原甲藻进行定性和定量分析的双特异探针技术.分析数据表明针对微型原甲藻核糖体大亚基RNA的LSU probe能够特异地将微型原甲藻和其他7种微藻分开,而且检测信号的强弱在一定的范围内与细胞数呈线性关系;由于针对微型原甲藻核糖体小亚基的SSU probe探针由于与具齿原甲藻存在核酸序列一致性,该探针与具齿原甲藻有严重的交叉反应,但未发现与海洋原甲藻和其他藻的交叉反应.另外,研究还优化了破碎微型原甲藻细胞所需的超声时间以及获得探针的特异性所需的S1酶反应温度.本研究为实现对微型原甲藻海水样品的快速准确监测奠定了基础.

Qualitative and quantitative detection of Prorocentrum minimum with double specific probes assay

Two groups of oligonucleotide probes targeted respectively large subunit ribosomal RNA and small subunit ribosomal RNA of Prorocentrum minimum,and the eight species of harmful algae were included to decide the specificity of the probes.In order to get better result,some experiment conditions such as the time of lysating the algae cells with ultrasonic and the time digesting the DNA/RNA hybrids with nuclease S1.The results showed that the probes LSU targeted large subunit ribosomal RNA were specific to Prorocentrum minimum,the color intensity is positively linear related to the cellular number,and the detection limit is low to 13 cells.Although the probes SSU targeted the small subunit ribosomal RNA were specific to most harmful algae,they seriously crossreacted with Prorocentrum dentatum due to the high homology between the small subunit ribosomal RNA of P.minimum and P.dentatum.Such,the technology is amenable to automation and can be used to monitor the low density of P.minimum in flied sample in long-term program.