基于荧光定量PCR技术构建水源地典型致嗅物质2-甲基异莰醇的评估方法及其应用

我国地表水源中由丝状蓝藻代谢产生2-甲基异莰醇(2-MIB)导致的嗅味问题十分普遍。由于传统显微镜检测方法无法鉴别产嗅藻种，不能满足水源水质管理要求，故有必要构建能特异性表征水体产嗅潜力的检测方法。由于丝状藻代谢产生2-MIB主要受其合成功能基因调控，设计了2-MIB功能基因引物，经引物特异性检验及PCR条件优化后，构建了基于2-MIB功能基因的定量PCR方法，并采用实际环境样品进行测试与验证。结果表明：引物特异性良好，定量PCR方法能有效检测2-MIB功能基因，并绘制了标准曲线，得出检测限为8.44×10² copies·L⁻¹；实际样品测试结果显示其2-MIB功能基因浓度在2.09×10⁷~1.94×10¹⁰ copies·L⁻¹范围内，与基于仪器分析方法测定的2-MIB浓度符合线性关系(R²=0.63，P<0.01)，表明定量方法可行。该方法具有灵敏性高、特异性好的特点，能特异性检测产嗅基因，可应用于水源地中产嗅潜力评估与预警。

Evaluation of typical odorant 2-methylisoborneol based on real time qPCR in source water and its application

Two-methylisoborneol (2-MIB) is a typical secondary metabolite released from cyanobacteria which causes off-flavor odor problem in surface water across China. Microscopy is the traditional method to identify such cyanobacteria. However, merely it is troublesome to distinguish between 2-MIB producers and non-producers by morphological structure and cannot meet the requirement of the source water quality management. It is necessary to build the test method which can specifically characterize the odor-producing capacity of waterbody. Due to the functional gene regulation for 2-MIB yield from cyanobacteria, the primers of 2-MIB function gene were designed and its specificity test was performed, then the PCR conditions were optimized. Real-time quantitative polymerase chain reaction (qPCR) can be an alternative method to evaluate 2-MIB syntheses which is controlled by 2-MIB functional gene. Here, 2-MIB identification primers (MIBF/MIBR) have been designed and tested with field samples. A good standard curve was established, R2=0.999 5, P<0.01, and the detection limit was 8.44×102 copies·L−1. The concentrations of 2-MIB gene of field samples were between 2.09×107 copies·L−1 to 1.94×1010 copies·L−1, which were significantly conformity with 2-MIB concentrations measured by GC-MS (R2=0.63, P<0.01). The high sensitivity and specificity of this qPCR-based method suggests that it can effectively evaluate the risk of 2-MIB occurrence and able to monitor source water quality management.