PCR-free detection of genetically modified organisms using magnetic capture technology and fluorescence cross-correlation spectroscopy

Abstract

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.

Figure 1. Schematic of the fluorescence correlation spectrometer.

An argon-ion laser (488 nm) and a helium-neon laser (633 nm) are used for the excitation of the RG dye and the Cy5 dye in the DNA sample through Dichroic Mirrors 1 and 2. The sample is contained in a chamber with a cover glass and is placed on the stage of an inverted microscope. Fluorescent light from the sample is collected by a high numerical aperture objective lens and transmitted by Dichroic Mirror 2. After passing through Dichroic Mirror 3, the residual laser excitation light and Raman scattered light are removed by additional band-pass filters. Green fluorescence between 505 nm and 550 nm is recorded by Detector 1 and red fluorescence above 650 nm is recorded by Detector 2. A computer was used to control the instrument and to obtain the auto- and cross-correlation functions of the fluorescent intensities, using the counted photoelectron pulses.

Figure 2. Proposed methodology for PCR-free identification of GMOs by magnetic capture-FCCS.

Genomic DNA is isolated from GMOs and then fragmented. Biotin-labeled DNA is hybridized with the 35S promoter region and streptavidin coated-magnetic beads are used to capture the targets from the sample and then washed. The single strand targets are released and hybridized with two fluorophore labeled probes for FCCS detection.

Figure 3. Auto-correlation functions and count rates (insets) of dye-labeled gene probes measured by FCS; (A) Rhodamine Green-labeled gene probes; (B) Cy5-labeled gene probes.

The measurement time was 60 seconds.

Figure 4. Cross-correlation functions of singly labeled gene probes.

(A) RG-labeled gene probe, excited by the 488 nm laser beam. (B) Non-hybridized RG-labeled and Cy5-labeled gene probes, excited simultaneously by 488 nm and 633 nm laser beams.

Figure 5. Electrophoresis detection of PCR amplified GMO in tomato samples.

Left column: the DNA marker; Column 1: negative control; Column 2: non-GM tomato sample; and Column 3: GM tomato sample.

Figure 6. Sensitivity and reliability assessment.

(A) Cross-correlation functions of PCR amplified target 195 bp fragments in GM tomatoes of different concentrations (the solid curve) and non-GM tomatoes control (the dash curve); (B) Correlation of PCR amplified GMO concentrations determined by FCCS and by spectrophotometry method at 260 nm.

Figure 7. FCCS detection without PCR.

Measured cross-correlation functions of the GMOs (the blue curve), non-GMOs (the black curve) with magnetic bead based samples treatments, and GMOs without magnetic bead based samples treatments (the red curve). (A) GM soybean and (B) GM tomato.