Identification of 11 Adulterated Meat Species in Cooked Meat Products Using DNA Mini-barcoding Methods Based on Cytochrome C Oxidase Subunit I Sequence(COI)
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摘要: 建立并优化了基于COI序列的DNA微条形码技术（mini-barcoding）检测熟肉制品中11种常见肉类掺假的方法。样品经超声与真空冷冻干燥处理，提取DNA模板和PCR扩增后，目标扩增物经切胶纯化后进行克隆测序，并将测序结果提交GenBank数据库Blast比对。筛选出适合猪、牛、羊、鸡、鸭、鸽子、马、驴、鹅、兔、鼠11种肉类的扩增的通用引物COI-A，对PCR扩增条件进行优化，并建立18个掺假模式，对低经济价值肉类（猪、鸡、鸭、马、驴、鼠）的掺入的最低比例进行考察。结果表明：11种熟肉的DNA经通用引物COI-A扩增后，其扩增效率均为100%，18个掺假模式中，牛肉和羊肉中掺入6种低经济价值肉类的最低检出比例为5%，而掺入鸡肉的最低检出比例为10%，而鹅肉、鸽子肉和兔肉的掺假模式中最低检出比例为10%；利用本方法检测30批次市售样品，发现有18批次的熟肉制品存在掺假情况。该方法前处理简单，灵敏度合适，重现性好，可作为高经济价值熟肉制品中掺假低经济价值肉类的有效检测方法。Abstract: A DNA min-barcoding method with cytochrome C oxidase subunit I sequence(COI)was developed to test 11 adulterated meat species in cooked meat products. DNA extraction and amplification of the mini-barcode region were carried out after samples were treated with ultrasonication and vacuum freeze drying. PCR products were purified with DNA purification kit and analyzed by clone sequencing. The sequencing results were submitted in GenBank using Blast. The primer set COI-A was found to be optimal,showing 100% amplification efficiency in 11 cooked meat species(including pork,beef,mutton,chicken,duck,pigeon meat,horsemeat,ass meat,goose,rabbit meat and rat meat)with optimized PCR procedure. 18 meat adulteration models were founded to test the minimum detected adulteration rate by cheap meat species including pork,chicken,duck,horsemeat,ass meat,and rat meat,and results showed that the minimum detected adulteration rates of beef and mutton added by the 6 cheap species were 5% with chicken was 10%,and the rates of pigeon,goose and rabbit meat were 10%. Using this method to test 30 cooked meat products,18 were found to be adulterated. The method was simple,sensitive and reproducible which would be suitable for the detection of 11 adulterated meat species in cooked meat products.
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Keywords:
- DNA mini-barcoding /
- meat /
- cytochromec coxidase subunit I /
- adulteration
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