一种新型双链 SYBR Green 实时荧光 PCR 熔解曲线分析方法用于检测牛肉掺假。
A novel duplex SYBR Green real-time PCR with melting curve analysis method for beef adulteration detection.
机构信息
China Meat Research Center, 100068 Beijing, China; Beijing Key Laboratory of Meat Processing Technology, 100068 Beijing, China.
China Meat Research Center, 100068 Beijing, China; Beijing Key Laboratory of Meat Processing Technology, 100068 Beijing, China.
出版信息
Food Chem. 2021 Feb 15;338:127932. doi: 10.1016/j.foodchem.2020.127932. Epub 2020 Aug 26.
An efficient and reliable duplex SYBR Green real-time quantitative PCR (qPCR) method for beef products adulteration detection was developed based on bovine specific and vertebrate universal primers. By analyzing the numbers, positions (Tm value) of melting curve peaks of the duplex PCR products, we simultaneously identified bovine and preliminary screened non-bovine in samples, and also semi-quantified the bovine percentage according to the area ratios of peaks. All of these were necessary for adulteration determination. The specific and universal primers were designed based on mitochondrial genes ND4 and 16S rRNA respectively, their amplicons Tm values were 72.6 ± 0.5 °C and 79-81 °C. There might be some other peaks at 74-78 °C and above 81 °C if non-bovine components existed. Thelimit of detectionwas 1 pgforbovineDNA, and1 - 30 pg fornon-bovineDNAbasedon differentspecies.
建立了一种基于牛特异性和脊椎动物通用引物的高效可靠的牛肉制品掺假检测双 SYBR Green 实时荧光定量 PCR(qPCR)方法。通过分析双 PCR 产物的熔解曲线峰的数量和位置（Tm 值），我们可以同时鉴定样品中的牛源性和初步筛选的非牛源性，并根据峰面积比进行牛源性含量的半定量分析。这些都是掺假鉴定所必需的。特异性和通用引物分别基于线粒体基因 ND4 和 16S rRNA 设计，其扩增产物的 Tm 值分别为 72.6±0.5°C 和 79-81°C。如果存在非牛成分，可能会在 74-78°C 和 81°C 以上出现其他一些峰。基于不同物种，牛 DNA 的检测限为 1 pg，非牛 DNA 的检测限为 1-30 pg。
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