Depatrment of Infectious Diseases and Preventive Medicine, Veterinary Research Institute, v.v.i., Hudcova 296/70, 62100 Brno, Czech Republic.
Molecules. 2021 Feb 20;26(4):1137. doi: 10.3390/molecules26041137.
This study was conducted to develop systems for the identification of four tuna species (skipjack tuna , yellowfin tuna , bullet tuna sp. and Atlantic bonito sp). At first, raw samples of these species and a mix intended as internal control were prepared for the authentication of fish muscle tissue of the genus sp., sp. and sp. DNA from raw muscle tissue, the mix and samples was extracted with the DNeasy mericon Food Kit (Qiagen GmbH, Hilden, Germany). The concentration and purity of DNA in raw samples were evaluated using a spectrophotometer. Primers and probe sequences were specifically designed to identify the selected species. In addition, primers and a probe for the endogenous gene were designed to determine the presence of amplifiable fish (especially tuna) DNA in samples. Furthermore, the species specificity of the designed primers and probes was verified in DNA samples of various tuna and bonito species. Limit of detection for the selected species was calculated as well as the coefficient of determination and efficiency of real-time PCR testing was determined. To evaluate the developed real-time PCR methods, 70 commercial tuna products were analysed. The results show that mislabelling of fish products can still be encountered and, moreover, the presence of an additional species can be identified.
本研究旨在开发用于鉴定四种金枪鱼物种(鲣鱼、黄鳍金枪鱼、箭鱼和大西洋鲣鱼)的系统。首先,为了鉴定属 sp.、sp. 和 sp. 的鱼类肌肉组织,制备了这些物种的原始样本和用作内部对照的混合物。使用 DNeasy mericon Food Kit(Qiagen GmbH,德国希尔德恩)从原始肌肉组织、混合物和样本中提取 DNA。使用分光光度计评估原始样本中 DNA 的浓度和纯度。专门设计了引物和探针序列来鉴定所选物种。此外,还设计了用于内源性基因的引物和探针,以确定样品中可扩增鱼类(尤其是金枪鱼)DNA 的存在。此外,还验证了设计的引物和探针在各种金枪鱼和鲣鱼物种的 DNA 样本中的物种特异性。还计算了所选物种的检测限,以及实时 PCR 测试的决定系数和效率。为了评估开发的实时 PCR 方法,分析了 70 种商业金枪鱼产品。结果表明,仍然可以遇到鱼类产品的错误标签,而且可以识别出另一种物种的存在。