基于苹果DNA的NFC苹果汁鉴伪方法的建立

Establishment of Authentication Detection Methods for NFC Apple Juice Based on DNA of Apples

[目的]通过分析比较不同品种苹果的NFC与FC果汁在DNA方面的差异,为NFC果汁的真伪鉴别和NFC果汁标准的建立提供科学方法和试验依据。[方法]在传统CTAB提取DNA方法的基础上加入纳米硅基磁珠(Si-OH@Fe_(3)O_(4)NPs)以提高苹果汁DNA的提取浓度,开发实时定量PCR,以鉴别NFC苹果汁和掺假(AAJ)果汁。[结果]试验确定的磁珠提取DNA最佳条件是样品用量1 mL和颠倒混匀3 min,提取出的DNA浓度均在50~60 ng/mL;在NFC苹果汁中连续稀释DNA的RT-PCR扩增标准曲线是y=-2.095 9x+30.714 0,R^(2)=0.998 5;不同程度掺假NFC苹果汁中DNA的RT-PCR扩增曲线为y=0.968 3x+28.591 0,相关性为R^(2)=0.981 9。[结论]这种基于新的DNA提取方法的RT-PCR技术在NFC苹果鉴伪中有着良好的稳定性、可重复性、可靠性;试验建立的方法可为NFC果汁的鉴伪检验提供有用工具。

[Objective]The differences of DNA between NFC juice and FC juice from different apple varieties were analyzed and compared to provide scientific method and experimental basis for the authentication detection of NFC juice and the establishment of NFC juice standard. [Method]The extraction concentration of juice DNA can be greatly improved after adding silicon based magnetic beads(Si-OH@Fe_(3)O_(4)NPs) based on CTAB for DNA traditional extraction method, then the real-time quantitative PCR was developed to identify NFC apple juice and adulterated apple juice. [Result]The experiment results showed the optimal conditions for extracting DNA in NFC apple juice by magnetic beads were 1 mL of sample dosage and 3 min of reverse mixing, and the extracted DNA concentration was 50-60 ng/mL under above operating conditions. The standard curve for RT-PCR amplification of continuously diluted DNA from NFC apple juice was y =-2.095 9x +30.714 0, R^(2)=0.998 5. The RT-PCR amplification curve of DNA in adulterated fruit juice(AAJ) with different degrees adulterated FC juice was y = 0.968 3x+28.591 0, R^(2)=0.981 9. [Conclusion]RT-PCR based on the new DNA extraction method has good stability, repeatability and reliability in NFC apple forgery identification. The method established in this experiment can provide useful tools for NFC apple fruit juice authentication detection.