Sakai Yumiko, Kotoura Satoshi, Yano Takeo, Kurihara Takashi, Uchida Kouji, Miake Kiyotaka, Akiyama Hiroshi, Tanabe Soichi
Biochemical Production and Development Center, Nagahama Branch, Oriental Yeast Co., Ltd.
Biosci Biotechnol Biochem. 2011;75(9):1639-43. doi: 10.1271/bbb.110024. Epub 2011 Sep 7.
A standard plasmid was constructed as a novel reference molecule for use in real-time quantitative PCR assays to verify the identity of beef, pork, chicken, mutton, and horseflesh. The plasmid contained a target domain of the cytochrome b (cyt b) gene and an artificial DNA sequence. Primers CO-F and CO-R, and probe CO-P were specifically designed to detect the artificial sequence. The calculated R² values of the standard curves (10³-10⁷ copies per reaction) for the five species ranged between 0.998 and 0.999 in the quantification analysis. The constructed plasmid provides a universal method for measuring the copy number of cyt b DNA in minced meat. This method would be a useful procedure for verifying food labels.
构建了一种标准质粒，作为一种新型参考分子用于实时定量PCR检测，以验证牛肉、猪肉、鸡肉、羊肉和马肉的身份。该质粒包含细胞色素b（cyt b）基因的一个靶区域和一段人工DNA序列。特异性设计引物CO-F和CO-R以及探针CO-P来检测该人工序列。在定量分析中，这五个物种的标准曲线（每个反应10³-10⁷个拷贝）的计算R²值在0.998至0.999之间。构建的质粒为测量碎肉中cyt b DNA的拷贝数提供了一种通用方法。该方法将是验证食品标签的有用程序。
