Rumiyati Rumiyati, Arini Rien Larasati, Purwanto Purwanto, Rohman Abdul
Faculty of Pharmacy, Universitas Gadjah Mada, Yogyakarta, Indonesia.
Center of Excellence, Institute of Halal Industry and Systems (IHIS), Universitas Gadjah Mada, Yogyakarta, Indonesia.
J Adv Vet Anim Res. 2024 Jun 4;11(2):247-253. doi: 10.5455/javar.2024.k770. eCollection 2024 Jun.
Meatballs are a popular meat-based food consumed widely in Indonesian society. However, the issue of unethical substitution of halal meatballs with non-halal meats, particularly pork and canine meat (CM), has emerged. The existence of non-halal meats, including CM, in food products is prohibited in Islam, necessitating the development of reliable analytical techniques for their identification. In this study, we designed species-specific primers (SSPs) targeting the D-loop region of mitochondrial DNA for CM meatball product identification.
The study was commenced by creating specific primers for canine DNA using Integrated DNA Technologies software and subsequently performing DNA isolation. The designed primers were then subjected to comprehensive evaluation using RT-PCR, including specification, linearity, limit of detection, efficiency, and repeatability.
The results indicated that the primer D-Loop 443 (forward: 5'-GGG ACA TCT CGA TGG ACTA ATG-3', reverse: 5'-GCG GTC ATA GAT GAG TGA TAG C-3') designed and validated in silico using primer-basic local alignment search tool nucleotide (BLAST) program from NCBI accurately identified canine DNA when the optimal annealing temperature was set at 57.5C. The real-time PCR technique utilizing the D-loop 443 primer exhibited the ability to amplify canine DNA down to a minimum quantity of 100 pg, with an efficiency value of 91.8%, a correlation coefficient (R) of 0.990, and a precision value (RSD) of 0.30%.
The SSP-based RT-PCR method developed is a versatile and efficient tool for detecting CM in meatballs. Its implementation helps maintain consumer trust and addresses concerns regarding the substitution of halal meats with non-halal alternatives.
肉丸是印度尼西亚社会广泛消费的一种受欢迎的肉类食品。然而，出现了用非清真肉类，特别是猪肉和狗肉（CM）不道德地替代清真肉丸的问题。伊斯兰教禁止食品中存在包括CM在内的非清真肉类，因此需要开发可靠的分析技术来识别它们。在本研究中，我们设计了针对线粒体DNA D环区域的物种特异性引物（SSP）用于CM肉丸产品鉴定。
该研究首先使用Integrated DNA Technologies软件创建犬DNA的特异性引物，随后进行DNA分离。然后使用RT-PCR对设计的引物进行全面评估，包括特异性、线性、检测限、效率和重复性。
结果表明，使用来自NCBI的引物基本局部比对搜索工具核苷酸（BLAST）程序在计算机上设计和验证的引物D-Loop 443（正向：5'-GGG ACA TCT CGA TGG ACTA ATG-3'，反向：5'-GCG GTC ATA GAT GAG TGA TAG C-3'）在最佳退火温度设定为57.5°C时能够准确鉴定犬DNA。利用D-loop 443引物的实时PCR技术能够将犬DNA扩增至最低量100 pg，效率值为91.8%，相关系数（R）为0.990，精密度值（RSD）为0.30%。
所开发的基于SSP的RT-PCR方法是检测肉丸中CM的一种通用且高效的工具。其应用有助于维护消费者信任并解决有关用非清真替代品替代清真肉类的问题。
