应用SYBR Green I染料能选择性结合双链DNA的特点,可检测到沙门氏菌fimI基因特异性靶序列扩增所产生的荧光信号,通过熔解曲线可知其熔点值约为85.6℃,而对其他非沙门氏菌则检测不到荧光信号。建立了一种肉品中的沙门氏菌Real-time PCR...应用SYBR Green I染料能选择性结合双链DNA的特点,可检测到沙门氏菌fimI基因特异性靶序列扩增所产生的荧光信号,通过熔解曲线可知其熔点值约为85.6℃,而对其他非沙门氏菌则检测不到荧光信号。建立了一种肉品中的沙门氏菌Real-time PCR检测方法,用该方法检测市售牛肉、香肠中的沙门氏菌,其检测灵敏度分别为13,12 cfu/25 g,从样品的处理到得出检验结果可以在10 h内完成。该检测方法具有简便、快速、特异性强、敏感度高等特点。展开更多
Two real-time PCR methods for the relative quantitation of DNA from meat species in food samples are described: these methods are applicable for horse in processed beef meat products, and pork in raw/processed beef me...Two real-time PCR methods for the relative quantitation of DNA from meat species in food samples are described: these methods are applicable for horse in processed beef meat products, and pork in raw/processed beef meat products. Test samples were prepared using raw meat admixtures or processed horse/pork in beef food products made to an industry-standard recipe. The methods were subjected to single laboratory method validation, evaluating the performance characteristics of specificity, PCR efficiency and r-squared (r<sup>2</sup>), Limit of Detection (LOD), Limit of Quantitation (LOQ), and precision and trueness. A limited UK-based inter-laboratory trial of the two methods was completed involving four participating laboratories. Full statistical analysis of the data qualified the applicability of the methods for accurate and sensitive trace-level analysis. The methods were deemed fit for purpose for reproducibly distinguishing between adventitious contamination at 0.1% (w/w), the level for further enforcement action at 1% (w/w), and a level representative of deliberate economically motivated adulteration (10% (w/w)). The data provided evidence that the precision of the two methods was applicable for qualitative and quantitative detection at topically important levels of adulteration. This work has added significant value to the current state of the art in quantitative determination of topical meat species adulteration, allowing analysts to distinguish between adventitious contamination and deliberate adulteration. The resulting methods described in this paper can easily be deployed and used by analytical laboratories for controls and due-diligence testing based on standard laboratory equipment.展开更多
文摘Two real-time PCR methods for the relative quantitation of DNA from meat species in food samples are described: these methods are applicable for horse in processed beef meat products, and pork in raw/processed beef meat products. Test samples were prepared using raw meat admixtures or processed horse/pork in beef food products made to an industry-standard recipe. The methods were subjected to single laboratory method validation, evaluating the performance characteristics of specificity, PCR efficiency and r-squared (r<sup>2</sup>), Limit of Detection (LOD), Limit of Quantitation (LOQ), and precision and trueness. A limited UK-based inter-laboratory trial of the two methods was completed involving four participating laboratories. Full statistical analysis of the data qualified the applicability of the methods for accurate and sensitive trace-level analysis. The methods were deemed fit for purpose for reproducibly distinguishing between adventitious contamination at 0.1% (w/w), the level for further enforcement action at 1% (w/w), and a level representative of deliberate economically motivated adulteration (10% (w/w)). The data provided evidence that the precision of the two methods was applicable for qualitative and quantitative detection at topically important levels of adulteration. This work has added significant value to the current state of the art in quantitative determination of topical meat species adulteration, allowing analysts to distinguish between adventitious contamination and deliberate adulteration. The resulting methods described in this paper can easily be deployed and used by analytical laboratories for controls and due-diligence testing based on standard laboratory equipment.
