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目的建立肉类的PCR-CE-LIF检测方法,并用以检测市售肉类的掺伪。方法采用DNA Ladder Marker(100 bp)优化CE的分离检测条件,包括分离电压、筛分介质浓度和电泳缓冲液浓度。采用试剂盒法提取肉的基因组DNA,并用特异性引物对其特异保守序列进行PCR扩增,经HindⅢ限制性核酸内切酶酶切,产物用优化的CE-LIF条件检测,测定结果与琼脂糖凝胶电泳法进行比对。结果在优化的CE条件下,同一天连续6次测定DNA Ladder Marker,迁移时间的日内相对标准差(RSD)为0.21%~0.71%;连续6天测定,迁移时间的日间RSD为1.02%~2.09%。毛细管电泳法能特异、灵敏地鉴定不同肉类,而琼脂糖凝胶电泳则无法检出目的条带。结论 CE-LIF方法具有高灵敏度、高分辨率、高特异性、绿色环保等优势,能够用于肉类掺伪测定。
Objective To establish a PCR-CE-LIF detection method for meat and to detect adulteration of commercially available meat. Methods The separation conditions of CE were optimized by using DNA Ladder Marker (100 bp), including the separation voltage, the concentration of screening medium and the concentration of electrophoresis buffer. The genomic DNA of the meat was extracted by the kit method and the specific conserved sequence of the genomic DNA was amplified by PCR using specific primers. After restriction endonuclease digestion with Hind III, the product was detected by the optimized CE-LIF conditions. The results were compared with agarose Gel electrophoresis method for comparison. Results Under the optimized conditions, the relative standard deviations (RSDs) were 0.21% -0.71% for the determination of DNA Ladder Marker on the same day for 6 consecutive times. The daytime RSD of migration time was 1.02% ~ 2.09%. Capillary electrophoresis method can identify different meat sensitively and sensitively, while agarose gel electrophoresis can not detect the target band. Conclusion The CE-LIF method has the advantages of high sensitivity, high resolution, high specificity and environmental protection. It can be used in the determination of meat adulteration.