生鲜肉中牛源性和羊源性成分定量检测方法的建立

Establishment of quantitative methods for detecting bovine-derived and ovine-derived materials in fresh meat

中文关键词:  生鲜肉  定量检测  荧光PCR  细胞色素b

英文关键词:fresh meat  quantitative analysis  fluorescence quantitative PCR  cytochrome b

中文摘要:

      目的 建立生鲜肉中牛源性和羊源性成分荧光PCR定量检测方法。方法 以线粒体细胞色素b(Cytb)基因核苷酸序列为检测靶点, 设计并优化引物, 建立基于SYBR染料的荧光PCR定量检测方法, 并对该方法进行特异性和灵敏度验证。结果 在退火温度60 ℃的条件下, 荧光PCR检测体系中各条引物特异性良好, 目标成分检测信号Ct值均小于20, 非特异性检测信号Ct值均大于35, 该方法在模板浓度为0.016~10 ng/μL时线性关系良好; 以Ct30作为阳性或阴性结果的判定限, 目标源性成分荧光PCR定量检测的灵敏度可达1%。结论 本方法可为生鲜肉制品种源鉴定和掺假检测提供理论依据。

英文摘要:

      Objective To establish a method for detection of bovine-derived and ovine-derived materials in fresh meat by fluorescence quantitative PCR. Methods Fluorescence quantitative PCR method based on SYBR was established with cytochrome b nucleotide sequence from mammal’s plastochondria genome as target. Fluorescence quantitative PCR primers were designed and synthesized and the specificity and sensitivity of this method was respectively performed for validation. Results Primers for fluorescence quantitative PCR reaction had good specificity under the condition of annealing temperature 60 ℃, Ct values of target gene signals were less than 20, and Ct values of nonspecific signals were more than 35. This method had a good linearity in the range of 0.016~10 ng/μL. The sensitivity for quantitative assay of target gene was up to 1% when Ct30 was evaluated as positive or negative reaction. Conclusion This method can provide guarantee for fresh meat identification and adulteration analysis.