应用实时荧光定量PCR技术研究九龙江口水域米氏凯伦藻的分布

Application of real-time PCR for detection of Karenia mikimotoi in Jiulongjiang Estuary waters

为快速精确监测九龙江口小型有毒赤潮藻的分布,本工作应用实时荧光定量PCR技术检测了2009年春(5月)、夏(8月)、秋(11月)3个季节九龙江口18个站位水样中米氏凯伦藻(Kareniamikimotoi)的密度.这3个季节分别对应九龙江口水域的丰水期、平水期、枯水期.结果表明,在九龙江口水中米氏凯伦藻的检出范围为未检出至2.3×104cells/dm3;其空间分布差异比较大,主要分布在厦门西港海域,其次是在高潮时的海门岛附近海域;海门岛以西水域几乎未监测到该藻的存在,仅5号站位有1个航次检出.米氏凯伦藻密度的季节分布差异也很明显,春、夏季的密度(最高检出值都达到了2.3×104cells/dm3)明显高于秋季的密度(最高检出值仅为5.4×103cells/dm3).本研究结果可为厦门西港以及九龙江口水域赤潮的研究与监测提供参考.同步进行的显微镜镜检技术没有观测到米氏凯伦藻的存在,可见在藻密度较低(低于103cells/dm3)的情况下,实时荧光定量PCR技术较传统镜检技术(检出限一般在103cells/dm3以上)可能更灵敏.该技术特异性好且操作简便,使对大样本的检测具有可行性,为实现沿岸海域赤潮的动态监测和深入研究奠定了基础.

This study aimed to rapid detecting the distribution of small harmful algae in Jiulongjiang Estuary waters. The density of Karenia mikimotoi in water samples at 18 stations of Jiulongjiang Estuary in spring（ May）, summer （August） and autumn （Novebmer）,2009 was investigated with realtime PCR. These seasons are of wet period, common period and dry period in Jiulongjiang Estuary, respectively. It was found that the K. mikimotoi densities were ranged from undetectable to 2.3 x 10^4 cells/dm^3 , the spatial distribution varied distinctively in the estuary. Higher densities were mostly detected in west waters of Xiamen Harbor ,then in Haimen Island during hightide period except for the waters in the north of Haimen Island where it was only detected once at station 5. The densities varied with seasons. In spring and summer the highest detectable density reached 2.3 x 10^4 cells/dm^3 ,which is obviously higher than that in autumn（ only 5.4 x 10^3 cells/dm^3 ）. The results can be taken as reference for red tide investigation in west waters of Xiamen Harbor and in Jiulongjiang Estuary. As none of K. mikimotoi was found under the light microscope, it suggested that realtime PCR technology may be much sensitive and accurate than traditional technology which normally works at cell density higher than 1 000 cells/dm^3. This technology has advantages of high specific and simple operation with a possible for large sample detections and thus it laid the foundation for further dynamic monitoring and red tide studying in coastal areas.