Universal mitochondrial 16s rRNA biomarker for mini-barcode to identify fish species in Malaysian fish products

Mislabelling in fish products is a highly significant emerging issue in world fish trade in terms of health and economic concerns. DNA barcoding is an efficient sequencing-based tool for detecting fish species substitution but due to DNA degradation, it is in many cases difficult to amplify PCR products of the full-length barcode marker (~650 bp), especially in severely processed products. In the present study, a pair of universal primers targeting a 198 bp sequence of the mitochondrial 16s rRNA gene was designed for identification of fish species in the processed fish products commonly consumed in Malaysia. The specificity of the universal primers was tested by both in-silico studies using bioinformatics software and through cross-reaction assessment by practical PCR experiments against the DNA from 38 fish species and 22 other non-target species (animals and plants) and found to be specific for all the tested fish species. To eliminate the possibility of any false-negative detection, eukaryotic endogenous control was used during specificity evaluation. The developed primer set was validated with various heat-treated (boiled, autoclaved and microwaved) fish samples and was found to show high stability under all processing conditions. The newly developed marker successfully identified 92% of the tested commercial fish products with 96–100% sequence similarities. This study reveals a considerable degree of species mislabelling (20.8%); 5 out of 24 fish products were found to be mislabelled. The new marker developed in this work is a reliable tool to identify fish species even in highly processed products and might be useful in detecting fish species substitution thus protecting consumers’ health and economic interests.

水产制品的标签错误（mislabelling）是全球水产贸易中在健康与经济维度均备受关注的新兴重大议题。DNA条形码（DNA barcoding）是一种基于测序的高效工具，可用于检测水产物种替代行为，但由于DNA发生降解，多数情况下难以扩增出全长条形码标记（~650 bp）的聚合酶链式反应（PCR）产物，对于深度加工的水产制品而言尤为困难。本研究针对线粒体16S核糖体RNA（16S rRNA）基因的一段198 bp序列设计了一对通用引物，用于鉴定马来西亚民众日常食用的加工水产制品中的水产物种。本研究通过两种方式验证该通用引物的特异性：一是利用生物信息学软件开展的计算机模拟（in-silico）研究，二是以38种水产物种及22种非靶标物种（动物与植物）的DNA为模板进行的实际PCR交叉反应评估，结果显示该引物对所有受试水产物种均具有特异性。为排除假阴性检测结果的可能性，特异性评估过程中引入了真核生物内参对照。本研究针对煮沸、高压灭菌及微波处理等多种热处理水产样品，对所开发的引物套装进行了验证，结果显示该引物在所有加工条件下均表现出良好的稳定性。该新开发的分子标记成功鉴定出92%的受试市售水产制品，其序列相似度达96%~100%。本研究发现存在相当比例的物种标签错误现象，占比达20.8%：24份受试水产制品中有5份被检出标签错误。本研究开发的新型分子标记是一种可靠的水产物种鉴定工具，即便对于高度加工的水产制品也可适用，可用于检测水产物种替代行为，从而保障消费者的健康与经济权益。