长棘海星(Acanthaster planci)幼体特异性PCR检测技术与应用

Specific PCR detection for Acanthaster planci larvae and its application

长棘海星(Acanthaster planci)的暴发是导致我国南海乃至印度—太平洋海域珊瑚礁退化的主要原因之一。浮游幼体的密度是决定成体种群是否暴发的重要指标,但是由于幼体肉眼不可见且不易分辨,常规调查和显微镜观察均无法有效检测到自然海域的长棘海星幼体,因此迫切需要开发一种高灵敏性且特异性的长棘海星幼体检测技术。本研究针对长棘海星幼体线粒体细胞色素氧化酶亚基I(mt COI,mitochondrial cytochrome oxidase subunit I)基因序列,建立了基于聚合酶链式反应(PCR,polymerase chain reaction)的长棘海星幼体特异性检测技术,并对西沙七连屿珊瑚礁海域的长棘海星幼体进行了检测。结果表明,设计筛选的4对特异性引物均可以扩增长棘海星Apmt COI基因片段,且与蓝指海星(Linckia laevigata)、面包海星(Culcita novaeguineae)、粒皮海星(Choriaster granulatus)和吕宋棘海星(Echinaster luzonicus)没有交叉反应。在退火温度为58.5℃时,引物2aooni F/2aooni R的特异性最佳,具有较高的灵敏度,可检测到皮克级的长棘海星基因组DNA。利用该技术检测了西沙七连屿海域长棘海星幼体分布情况,发现10月底西沙七连屿珊瑚礁海域能检测到长棘海星幼体,且幼体分布并不均匀。因此,该检测技术可作为今后长棘海星幼体种群监测的有效方法。

The outbreak of crown-of-thorns seastar(CoTS, Acanthaster planci) is one of the main causes to coral reef degradation in the South China Sea and the Indo-Pacific region. The density of CoTS larvae is an important indicator to determine whether the outbreak of CoTS adult population occurs or not. However, as the larvae are not visible and thus difficult to be distinguished, conventional investigation and microscope observation cannot effectively detect the CoTS larvae in natural seawaters. Therefore, it is needed to develop a sensitive and specific method for detecting CoTS larvae. This study established specific polymerase chain reaction(PCR) method to detect the CoTS larvae based on CoTS mitochondrial cytochrome oxidase subunit I gene(Apmt COI), and this method was applied to detect the CoTS larvae in the coral reef around Qilianyu Island, Xisha. The results showed that the designed and screened four pairs of specific primers could succeed in amplifying Apmt COI gene, and had no cross-reaction with Linckia laevigata, Culcita novaeguineae, Choriaster granulatus and Echinaster luzonicus. Moreover, the primer 2aooniF/2anooiR had the best specificity and higher sensitivity when annealing temperature was 58.5°C, which could detect the CoTS genomic DNA in pictogram grade. Furthermore, this method had succeeded in detecting the CoTS larvae in the Qilianyu Island of Xisha at the end of October, and we found that the distribution of CoTS larvae was not uniform. Therefore, this detection technology can be used as an effective method for monitoring CoTS larvae population in the future.