PCR-free quantitative detection of genetically modified organism from raw materials. An electrochemiluminescence-based bio bar code method

Abstract

A bio bar code assay based on oligonucleotide-modified gold nanoparticles (Au-NPs) provides a PCR-free method for quantitative detection of nucleic acid targets. However, the current bio bar code assay requires lengthy experimental procedures including the preparation and release of bar code DNA probes from the target-nanoparticle complex and immobilization and hybridization of the probes for quantification. Herein, we report a novel PCR-free electrochemiluminescence (ECL)-based bio bar code assay for the quantitative detection of genetically modified organism (GMO) from raw materials. It consists of tris-(2,2'-bipyridyl) ruthenium (TBR)-labeled bar code DNA, nucleic acid hybridization using Au-NPs and biotin-labeled probes, and selective capture of the hybridization complex by streptavidin-coated paramagnetic beads. The detection of target DNA is realized by direct measurement of ECL emission of TBR. It can quantitatively detect target nucleic acids with high speed and sensitivity. This method can be used to quantitatively detect GMO fragments from real GMO products.

Figure 1

A schematic of the ECL-based bio-barcode method for quantitative detection of GMO.

Figure 2

(a): Effect of the concentration of streptavidin-coated beads on ECL intensity. Streptavidin-coated beads of 1μl was added to 25μl of three-strand complex. The concentration of beads is 1-7×10⁵ beads/μl. (b): Effect of the TBR-to-capture probe ratio on ECL intensity. The TBR-to-capture probe concentration ratio is in the range of 40 to 100 (40μmol/l, 1μl).

Figure 3

Calibration curve for the ECL-based bio-barcode assay. (a): The calibration curve was obtained by measuring a series of purified GM soybean samples containing CaMV35S molecules with a quantity ranging from 1 fmol to 100 pmol in a volume of 25μl. (b): The calibration curve was obtained by measuring a series of samples containing 0.1% to 100% genomic DNA from GM soybean.

Figure 4

(a): ECL signals of three purified GMO samples (solid bars), in comparison with three non-GMO samples (white bars). The DNA samples were digested by restriction endonucleases. Then, the digested products were subjected to electrophoresis in 2% agarose gel and the 169 bp DNA fragments of CaMV35S were collected and detected by the ECL-based bio-barcode assay. The dashed line represents the threshold value for purified GMO samples detection. (b): ECL signals of three GMO samples from raw materials (solid bars), in comparison with three non-GMO samples (white bars). The DNA samples were digested by restriction endonucleases and directly detected by the ECL-based bio-barcode assay. The dashed line represents the threshold value for non-purified GMO samples detection.