暗色刺参的实时荧光PCR鉴定

Accurate Identification of an Endangered Sea Cucumber Isostichopus fuscus with Real-Time Quantitative PCR

暗色刺参(Isostichopus fuscus)已被列入《濒危野生动植物种国际贸易公约》(CITES)目录。为精确辨别和有效保护该物种,作者进行了精准鉴定暗色刺参的实时荧光PCR方法的研究。在GenBank中收集暗色刺参及近似种海参的线粒体序列,使用海参的线粒体基因中细胞色素C氧化酶亚基Ⅰ(Cytochrome oxidase subunitⅠgene,COⅠ)进行实时荧光PCR中引物和探针的设计。根据收集到的17种海参的25条COⅠ序列,设计了2组具有暗色刺参特异性的引物探针组合I-fu-1和I-fu-2;同时根据暗色刺参实物测序得到的COⅠ序列,设计了引物探针I-fu-3~I-fu-6。实时荧光PCR检测结果显示:I-fu-1、I-fu-3、I-fu-4、I-fu-6仅在目标物种中检测到阳性,I-fu-2和I-fu-5则在墨西哥刺参(Isostichopus badionotus)中也检测到阳性;对纯暗色刺参样本的灵敏度(指在一次荧光PCR反应中能够检测到暗色刺参的最低浓度)检测显示,I-fu-4优于I-fu-1、I-fu-3和I-fu-6,可达到0.0008 ng/μL,因此认为I-fu-4是最佳的引物探针组合。利用I-fu-4对混合物中暗色刺参进行检测,在同属近似种墨西哥刺参含量高达99.9%,暗色刺参含量低至0.05%的混合样品中,暗色刺参的实时荧光PCR检测也显示了强阳性结果。本研究建立的鉴定方法具有高特异性和灵敏度的特性,有助于快速、精准地鉴定暗色刺参。

Isostichopus fuscus is a species in family Stichopodidae(Aspidochirotida:Holothuroidea)and has been listed as an endangered species in the AppendixⅢof the CITES.To accurately identify the species,a real-time quantitative PCR(qPCR)method was developed in this study.Cytochrome oxidase subunitⅠ(COⅠ)genes of I.fuscus and its close relatives were retrieved from GenBank,and primers and probes were designed.Two I.fuscus specific primer-probe sets,I-fu-1 and I-fu-2,were designed according to 25 COⅠgene sequences of 17 sea cucumber species,and other four sets,I-fu-3 through I-fu-6,were also designed according to the sequences isolated from I.fuscus samples.By comparing the 6 primer-probe sets,we found that I-fu-1,I-fu-3,I-fu-4 and I-fu-6 were highly specific,and positive results were obtained only in target species.I-fu-2 and I-fu-5 were not specific;they also targeted I.badionotus,a species in the same genus.I-fu-4 demonstrated the highest sensitivity among all sets,0.0008 ng/μL.As the optimum primer-probe set,I-fu-4 was also applicable for the detection of target species in mixed samples.Even in the deeply processed mixed samples in which the content of I.badionotus is as high as 99.9%and the target species is as low as 0.05%,the detection of I.fuscus showed a strong positive result.The identification method established in this study is highly specific and sensitive,which is conducive to the rapid and accurate identification of I.fuscus,and beneficial for the conservation of the species.