Bertolini Francesca, Ghionda Marco Ciro, D'Alessandro Enrico, Geraci Claudia, Chiofalo Vincenzo, Fontanesi Luca
Department of Agricultural and Food Sciences, Division of Animal Sciences, University of Bologna, Viale Fanin 46, 40127, Bologna, Italy.
Department of Agricultural and Food Sciences, Division of Animal Sciences, University of Bologna, Viale Fanin 46, 40127, Bologna, Italy; Department of Veterinary Sciences, Animal Production Unit, University of Messina, Polo Universitario dell'Annunziata, 98168, Messina, Italy.
PLoS One. 2015 Apr 29;10(4):e0121701. doi: 10.1371/journal.pone.0121701. eCollection 2015.
The identification of the species of origin of meat and meat products is an important issue to prevent and detect frauds that might have economic, ethical and health implications. In this paper we evaluated the potential of the next generation semiconductor based sequencing technology (Ion Torrent Personal Genome Machine) for the identification of DNA from meat species (pig, horse, cattle, sheep, rabbit, chicken, turkey, pheasant, duck, goose and pigeon) as well as from human and rat in DNA mixtures through the sequencing of PCR products obtained from different couples of universal primers that amplify 12S and 16S rRNA mitochondrial DNA genes. Six libraries were produced including PCR products obtained separately from 13 species or from DNA mixtures containing DNA from all species or only avian or only mammalian species at equimolar concentration or at 1:10 or 1:50 ratios for pig and horse DNA. Sequencing obtained a total of 33,294,511 called nucleotides of which 29,109,688 with Q20 (87.43%) in a total of 215,944 reads. Different alignment algorithms were used to assign the species based on sequence data. Error rate calculated after confirmation of the obtained sequences by Sanger sequencing ranged from 0.0003 to 0.02 for the different species. Correlation about the number of reads per species between different libraries was high for mammalian species (0.97) and lower for avian species (0.70). PCR competition limited the efficiency of amplification and sequencing for avian species for some primer pairs. Detection of low level of pig and horse DNA was possible with reads obtained from different primer pairs. The sequencing of the products obtained from different universal PCR primers could be a useful strategy to overcome potential problems of amplification. Based on these results, the Ion Torrent technology can be applied for the identification of meat species in DNA mixtures.
确定肉类及肉制品的物种来源是预防和检测可能具有经济、伦理和健康影响的欺诈行为的重要问题。在本文中，我们评估了基于下一代半导体的测序技术（Ion Torrent个人基因组测序仪）通过对从扩增12S和16S rRNA线粒体DNA基因的不同通用引物对获得的PCR产物进行测序，来鉴定DNA混合物中肉类物种（猪、马、牛、羊、兔、鸡、火鸡、野鸡、鸭、鹅和鸽子）以及人类和大鼠DNA的潜力。制备了六个文库，包括分别从13个物种获得的PCR产物，或从等摩尔浓度或以猪和马DNA的1:10或1:50比例包含所有物种或仅禽类或仅哺乳动物物种DNA的DNA混合物中获得的PCR产物。测序共获得33,294,511个已识别核苷酸，其中29,109,688个具有Q20（87.43%），共215,944条读数。使用了不同的比对算法根据序列数据来确定物种。通过桑格测序确认所得序列后计算的不同物种的错误率在0.0003至0.02之间。不同文库之间每个物种的读数数量相关性对于哺乳动物物种较高（0.97），对于禽类物种较低（0.70）。对于某些引物对，PCR竞争限制了禽类物种的扩增和测序效率。通过从不同引物对获得的读数可以检测到低水平的猪和马DNA。从不同通用PCR引物获得的产物测序可能是克服潜在扩增问题的有用策略。基于这些结果，Ion Torrent技术可应用于鉴定DNA混合物中的肉类物种。
