MOE Key Laboratory of Laser Life Science & Institute of Laser Life Science, College of Biophotonics, South China Normal University, Guangzhou, China.
PLoS One. 2009 Nov 26;4(11):e8074. doi: 10.1371/journal.pone.0008074.
The safety of genetically modified organisms (GMOs) has attracted much attention recently. Polymerase chain reaction (PCR) amplification is a common method used in the identification of GMOs. However, a major disadvantage of PCR is the potential amplification of non-target DNA, causing false-positive identification. Thus, there remains a need for a simple, reliable and ultrasensitive method to identify and quantify GMO in crops. This report is to introduce a magnetic bead-based PCR-free method for rapid detection of GMOs using dual-color fluorescence cross-correlation spectroscopy (FCCS). The cauliflower mosaic virus 35S (CaMV35S) promoter commonly used in transgenic products was targeted. CaMV35S target was captured by a biotin-labeled nucleic acid probe and then purified using streptavidin-coated magnetic beads through biotin-streptavidin linkage. The purified target DNA fragment was hybridized with two nucleic acid probes labeled respectively by Rhodamine Green and Cy5 dyes. Finally, FCCS was used to detect and quantify the target DNA fragment through simultaneously detecting the fluorescence emissions from the two dyes. In our study, GMOs in genetically engineered soybeans and tomatoes were detected, using the magnetic bead-based PCR-free FCCS method. A detection limit of 50 pM GMOs target was achieved and PCR-free detection of GMOs from 5 microg genomic DNA with magnetic capture technology was accomplished. Also, the accuracy of GMO determination by the FCCS method is verified by spectrophotometry at 260 nm using PCR amplified target DNA fragment from GM tomato. The new method is rapid and effective as demonstrated in our experiments and can be easily extended to high-throughput and automatic screening format. We believe that the new magnetic bead-assisted FCCS detection technique will be a useful tool for PCR-free GMOs identification and other specific nucleic acids.
最近，人们对转基因生物（GMO）的安全性给予了极大的关注。聚合酶链反应（PCR）扩增是鉴定 GMO 常用的方法。然而，PCR 的一个主要缺点是可能会扩增非靶 DNA，导致假阳性鉴定。因此，仍然需要一种简单、可靠和超灵敏的方法来鉴定和定量作物中的 GMO。本报告介绍了一种基于磁珠的无 PCR 方法，用于使用双色荧光相关光谱（FCCS）快速检测 GMO。该方法以转基因产品中常用的花椰菜花叶病毒 35S（CaMV35S）启动子为靶标。通过生物素标记的核酸探针捕获 CaMV35S 靶标，然后通过生物素-链霉亲和素连接使用链霉亲和素包被的磁性珠进行纯化。纯化的靶 DNA 片段与两种分别用罗丹明绿和 Cy5 染料标记的核酸探针杂交。最后，通过同时检测两种染料的荧光发射，使用 FCCS 检测和定量目标 DNA 片段。在我们的研究中，使用基于磁珠的无 PCR FCCS 方法检测了转基因大豆和番茄中的 GMO。实现了 50 pM GMO 靶标的检测限，并且使用磁性捕获技术从 5μg 基因组 DNA 中实现了无 PCR 的 GMO 检测。此外，通过使用来自 GM 番茄的 PCR 扩增靶 DNA 片段在 260nm 处的分光光度法验证了 FCCS 方法确定 GMO 的准确性。新方法在我们的实验中证明是快速有效的，并且可以很容易地扩展到高通量和自动化筛选格式。我们相信，新的基于磁珠的 FCCS 检测技术将成为无 PCR GMO 鉴定和其他特定核酸的有用工具。
