Watanabe Shun, Minegishi Yuki, Yoshinaga Tatsuki, Aoyama Jun, Tsukamoto Katsumi
Ocean Research Institute, The University of Tokyo, 1-15-1 Minamidai, Nakano-ku, Tokyo, 164-8639, Japan.
Mar Biotechnol (NY). 2004 Nov-Dec;6(6):566-74. doi: 10.1007/s10126-004-1000-5. Epub 2005 Feb 2.
To compensate for the limited number of morphological characteristics of fish eggs and larvae, we established a convenient and robust method of species identification for eggs of the Japanese eel (Anguilla japonica) using a real-time polymerase chain reaction (PCR) that can be performed onboard research ships at sea. A total of about 1.2 kbp of the mitochondrial 16S ribosomal RNA gene sequences from all species of Anguilla and 3 other anguilliform species were compared to design specific primer pairs and a probe for A. japonica. This real-time PCR amplification was conducted for a total of 44 specimens including A. japonica, A. marmorata, A. bicolor pacifica, and 6 other anguilliform species. Immediate PCR amplification was only observed in A. japonica. We then tested this method under onboard conditions and obtained the same result as had been produced in the laboratory. These results suggest that real-time PCR can be a powerful tool for detecting Japanese eel eggs and newly hatched larvae immediately after onboard sampling during research cruises and will allow targeted sampling efforts to occur rapidly in response to any positive onboard identification of the eggs and larvae of this species.
为弥补鱼卵和仔鱼形态特征数量有限的不足，我们建立了一种便捷且可靠的日本鳗鲡（Anguilla japonica）鱼卵物种鉴定方法，该方法采用实时聚合酶链反应（PCR），可在海上研究船上进行。比较了鳗鲡属所有物种以及其他3种鳗鲡目物种的线粒体16S核糖体RNA基因序列，共计约1.2 kbp，以设计日本鳗鲡的特异性引物对和探针。对包括日本鳗鲡、花鳗鲡、太平洋双色鳗鲡以及其他6种鳗鲡目物种在内的44个样本进行了实时PCR扩增。仅在日本鳗鲡中观察到即时PCR扩增。然后我们在船上条件下测试了该方法，得到了与实验室相同的结果。这些结果表明，实时PCR可成为在研究航次中进行船上采样后立即检测日本鳗鲡鱼卵和新孵化仔鱼的有力工具，并将使针对该物种鱼卵和仔鱼的船上阳性鉴定迅速开展有针对性的采样工作。
