基于LAMP法快速检测羊肉及其制品中的猪、鸭和羊源性成分

Identification of porcin,duck and sheep-derived materials in mutton and its products by LAMP assay

目的建立肉制品中猪、鸭和羊源性成分的环介导等温扩增(loop-mediated isothermal amplification,LAMP)检测方法,对市售羊肉片、羊肉卷、羊肉串进行猪、鸭和羊源性成分检测分析。方法针对猪、鸭和羊线粒体Cytb基因分别设计2条外引物、2条内引物和2条环引物,优化LAMP反应条件,对猪、鸭和羊源性成分进行特异性和灵敏度试验,对羊肉制品进行上述动物源性成分监测。结果在0.2μmol/L外引物(F3和B3)、1.6μmol/L内引物(FIP和BIP)、0.8μmol/L环引物(FLP和BLP)、1 mol/L甜菜碱、6 mmol/L Mg SO4、1.6 mmol/L d NTP等参数的优化条件下,LAMP法对肉制品中猪、鸭和羊源性成分的检测灵敏度达0.5%。SYTO-9荧光染料的荧光实时检测结果与SYBR Green I染料肉眼观察结果一致。调查发现,该批市售羊肉及制品中存在掺假现象,掺假率为34.5%。结论 LAMP法具有操作简便、特异性强、灵敏度高、检测结果准确的特点,能有效地对肉制品中的猪、鸭和羊源性成分进行检测,可作为羊肉制品掺假的一种快速检测方法。

Objective Loop-mediated isothermal amplification （LAMP） method was developed to detect porcin, duck and sheep-derived materials in meat products （lamb chops, lamb meat roll, lamb shashlik）. Methods A set of four specific primers （F3, B3, FIP, BIP） and two loop primers （FLP and BLP） were designed, respectively. Optimization reaction was confirmed, and two different measurement platforms were compared. 29 samples of meat products were collected from local markets and the detection of porcin, duck and sheep-derived materials was carried out. Results The optimized condition was 0. 2 Ixmol/L each of F3 and B3, 1.6 Ixmol/L each of FIP and BIP, 0.8 I^mol/L each of FLP and BLP, 1 mol/L betaine, 6 mmol/L Mg2~, 1.6 mmol/L dNTP. The limit of detection was up to 0. 5% animal-derived materials in meat products under the platform of real time fluorescence and nake-eye inspection with SYBR Green I. The current results of LAMP obtained on the two measurement platforms were in good accordance. With the LAMP assay, meat products were successfully screened for porcin, duck and sheep-derived materials. Conclusion This method is a highly specific and sensitive detection system, and could be a very useful tool for animal components detection.