Yang Lixia, Fu Shujun, Peng Xinkai, Li Le, Song Taoping, Li Lin
Changsha Center of Supervision & Inspection on Food Quality Safety , Yinshuang Road, Yuelu District, Hunan, Changsha 410013 , People's Republic of China.
Biotechnol Biotechnol Equip. 2014 Sep 3;28(5):882-888. doi: 10.1080/13102818.2014.963789. Epub 2014 Oct 22.
In this study, a one-step, real-time, loop-mediated isothermal amplification (RealAmp) assay was developed, for the highly specific detection of pork DNA. For the assay, the mtDNA of () gene was amplified at 63 °C using SYBR Green I for 45 min with a Real-Time Polymerase Chain Reaction (PCR) System that measured the fluorescent signal at one-minute intervals. As little as 1 pg of template DNA could be detected, without any cross-reactivity with non-target species. Meat mixtures, heat-treated at 100 °C for 15 min, prepared by mixing pork meat with beef at different ratios (0.01%-10%) were tested, and the RealAmp assays allowed the detection of as little as 0.01% pork in the meat mixtures. Thus, this work showed that RealAmp could be used for specific identification and sensitive quantification of meat species, even for heat-treated meat products.
在本研究中，开发了一种一步式实时环介导等温扩增（RealAmp）检测方法，用于高特异性检测猪肉DNA。对于该检测方法，使用SYBR Green I在63°C下对（）基因的线粒体DNA进行扩增45分钟，采用实时聚合酶链反应（PCR）系统，每隔一分钟测量荧光信号。可检测到低至1 pg的模板DNA，且与非目标物种无任何交叉反应。对通过将猪肉与牛肉按不同比例（0.01%-10%）混合制备并在100°C下热处理15分钟的肉混合物进行了测试，RealAmp检测方法能够检测到肉混合物中低至0.01%的猪肉。因此，这项工作表明，RealAmp可用于肉类物种的特异性鉴定和灵敏定量，即使对于热处理的肉类产品也是如此。
