| 虞惠贞,赵相鹏,尹文秀,等.海马特异性实时荧光PCR鉴定方法的建立及应用[J].中国海洋药物,2026,(4):-. |
| 海马特异性实时荧光PCR鉴定方法的建立及应用 |
| Establishment and application of real-time fluorescence PCR method for Seahorse specific identification |
| 投稿时间：2025-03-04 修订日期：2025-04-25 |
| DOI： |
| 中文关键词: 海马属 驼背海马 三斑海马 克氏海马 实时荧光聚合酶链式反应 |
| English Keywords:Hippocampus Hippocampus camelopardalis Hippocampus trimaculatus Hippocampus kelloggi real-time polymerase chain reaction |
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| 中文摘要: |
| 目的采用TaqMan探针实时荧光聚合酶链式反应（real-time polymerase chain reaction, real-time PCR）技术，建立海马属（Hippocampus）和驼背海马（Hippocampus camelopardalis）、三斑海马（Hippocampus trimaculatus）以及克氏海马（Hippocampus kelloggi）的特异性鉴定检测方法。方法 基于海马属的线粒体全基因，以及3种海马的线粒体细胞色素c氧化酶亚基Ⅰ（cytochrome c oxidase subunitⅠ，CO Ⅰ）基因序列，分别设计了属和种特异性引物和探针。通过特异性、灵敏度以及混合样本检测等对4组引物探针性能进行评估。结果 对32个物种（包括13种海马，4种海龙和15种其他常见物种）进行实时荧光PCR检测，仅在目标物种中检测到阳性。海马属和驼背海马的检测灵敏度达0.005 ng/μL，三斑海马和克氏海马达0.001 2 ng/μL。此外，建立了四重real-time PCR方法，优化后当引物探针比例为2∶2∶3∶3，检测灵敏度最佳（0.02 ng/μL），即使在同属近似种海马含量高达99.5%，目标海马低至0.5%的混合样中，目标海马的实时荧光PCR检测也呈现强阳结果。结论 该方法为海马物种的快速真伪鉴别和资源保护提供了有效的技术支撑。 |
| English Summary: |
| Objective Using TaqMan probe-based real-time fluorescence polymerase chain reaction (real-time PCR) technology, the authors established specific identification methods for the genus Hippocampus, as well as for Hippocampus camelopardalis, Hippocampus trimaculatus, and Hippocampus kelloggi. Methods Based on the complete mitochondrial genome of the genus Hippocampus and the mitochondrial cytochrome c oxidase subunit Ⅰ(COⅠ) gene sequences of the three seahorse species, genus- and species-specific primers and probes were designed. The performance of the four primer-probe sets was evaluated based on specificity, sensitivity, and mixed-sample testing. Results The results showed that real-time PCR testing of 32 species (including 13 seahorse species, 4 pipefish species, and 15 other common species) yielded positive results only for the target species. The detection sensitivity for the genus Hippocampus and H.camelopardalis reached 0.005 ng/μL, while for H.trimaculatus and H.kelloggi, it reached 0.001 2 ng/μL. Additionally, a quadruplex real-time PCR method was established and optimized. When the primer-to-probe ratio for the genus Hippocampus, H. camelopardalis, H. trimaculatus, and H.kelloggi was 2:2:3:3, the detection sensitivity was optimal (0.02 ng/μL). Even in mixed samples where the target seahorse content was as low as 0.5% and the content of closely related species was as high as 99.5%, the real-time PCR detection of the target seahorse still yielded strong positive results. Conclusion This method provides effective technical support for the rapid authenticity identification and conservation of seahorse species. |
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