梯型熔解温度等温扩增检测食品中植物源成分的内标方法构建

Establishment of internal standard method for detection of plant-derived components in food with ladder-type melting temperature isothermal amplification

中文关键词:  梯型熔解温度等温扩增  植物源成分  特异性基因  内标方法  DNA提取方法

英文关键词:ladder-type melting temperature isothermal amplification  plant-derived components  specific gene  internal standard method  DNA extraction method

中文摘要:

      目的 为梯型熔解温度核酸等温扩增(ladder-type melting temperature isothermal amplification, LMTIA)检测食品中植物源特异性基因建立内标方法, 并用于检测肉制品中植物成分。方法 针对植物转录间隔区(internal transcribed spacer, ITS)的共有序列, 设计LMTIA引物, 优化反应温度, 考查方法灵敏度, 并评估淀粉制品、蜂蜜、食用油DNA提取方法与LMTIA检测方法的适应性, 以H2O、鸭肉、鸡肉、羊肉、猪肉和牛肉基因组为阴性对照测试LMTIA方法的假阳性。结果 所建立的LMTIA方法在最优温度57 ℃时, 灵敏度为10 pg/μL玉米基因组与10 pg/μL红枣基因组, 可检出肉制品掺入0.1%的玉米淀粉, 所选淀粉制品、蜂蜜、食用油DNA提取方法均适用于LMTIA检测, 并且所有阴性对照样品在测试中均未出现假阳性。结论 本研究成功建立了LMTIA检测食品中植物源特异性基因的内标方法, 排除了DNA提取引起的LMTIA检测的假阴性, 可用于肉制品中植物成分定性检测。

英文摘要:

      Objective To establish an internal standard method for detection of specific plant-derived gene in food with ladder-type melting temperature isothermal amplification (LMTIA), and detect the plant-derived components in meat products by the LMTIA method. Methods The LMTIA primers were designed with the common sequence on the internal transcribed spacer (ITS) of plants as the target, the reaction temperature was to optimized, the sensitivity was determined, the applicability of DNA extraction methods for starch products, honey and edible oil to the LMTIA method were evaluated, and the false positive rates of the LMTIA method were determined with H2O and genomic DNAs of duck meet, chicken, mutton, pork and beef as negative controls. Results At the optimized temperature of 57 ℃, the sensitivities of the LMTIA method were 10 pg/μL genomic DNA of Zea mays and 10 pg/μL genomic DNA of Ziziphus jujuba, the LMTIA method could detect 0.1% corn starch in pork products, the selected DNA extraction methods for starch products, honey and edible oil were highly suited the LMTIA method, and all negative controls were negative in the false positive rate determination of LMTIA method. Conclusion The internal standard method for the detection of specific plant-derived gene in food with LMTIA had been established in this study, it had excluded the false negative of the LMTIA method caused by the improper DNA extraction methods, and it can also be used for quantitative determination of plant-derived components in meat products.