基于双重聚合酶链式反应技术检测掺假驴乳中的牛乳

Detection of cow milk in adulterated donkey milk based on double polymerase chain reaction

中文关键词:  驴乳  牛乳  双重聚合酶链式反应技术  乳掺假  真实性鉴别

英文关键词:donkey milk  cow milk  double polymerase chain reaction  milk adulteration  verification of authenticity

中文摘要:

      目的 建立双重聚合酶链式反应(polymerase chain reaction, PCR)技术检测驴乳中掺假的牛乳的鉴别方法。方法 将牛乳按比例掺入驴乳制备掺假乳, 经热处理提取DNA, 以驴和牛的12S-RNA、16S-RNA为靶标设计特异性引物, 进行相应的PCR扩增, 优化双重PCR退火温度, 同时考察该方法在巴氏杀菌、高温灭菌和冷冻干燥条件下的适用性及灵敏度。结果 牛和驴的引物特异性均良好, 与非目标物种DNA不发生交叉反应, DNA检出限最低为10 ng; 优化后的双重PCR最佳退火温度为55.7℃; 单重PCR由于干扰因素少, 对驴乳中牛乳的掺假检出限均为0.1%, 而双重PCR也表现出较好的适用性及灵敏性, 在原料乳中检出限为0.5%, 巴氏杀菌乳、高温灭菌乳和冷冻干燥驴乳中检出限均为2.0%。相较于单重PCR, 双重PCR大大缩短了检测时间成本。结论 本研究为驴乳中牛乳的掺假检测提供了一种准确、灵敏和经济的方法, 具有实际参考价值。

英文摘要:

      Objective To establish a method for determination of adulterated cow milk in donkey milk by double polymerase chain reaction (PCR). Methods The adulterated milk was prepared by mixing cow milk into donkey milk in proportion, and DNA was extracted by heat treatment. The specific primers were designed with 12S-RNA and 16S-RNA of donkey and cow as targets, and the corresponding PCR amplification was carried out, and the annealing temperature of dual PCR was optimized. At the same time, the applicability and sensitivity of the method under the conditions of pasteurization, high-temperature sterilization and freeze-drying were investigated. Results The primers of cattle and donkeys had good specificity and did not cross react with the DNA of non target species, the lowest limit of detection of DNA was 10 ng; the optimal annealing temperature of the optimized dual PCR was 55.7℃; the limit of detection of single PCR for cow milk adulteration in donkey milk was 0.1% due to few interference factors, while the limit of detection of double PCR was 0.5% in raw milk, 2.0% in pasteurized milk, high-temperature sterilized milk and freeze-dried donkey milk. Compared with single PCR, double PCR greatly reduced the detection time and cost. Conclusion This study provides an accurate, sensitive and economical method for the detection of cow milk adulteration in donkey milk, and has practical reference value.