Ultrasensitive detection of genetically modified plant by fluorescence cross-correlation spectroscopy

In this study, a novel method for the direct detection of GMP without amplified by the general method of PCR is firstly presented and proved by experiments. in our method, fluorescence correlation spectroscopy, cleaving nucleic acid by restriction endonuclease and two nucleic acid probe hybridization techniques are combined to distinguish the cauliflower mosaic virus (CaMV) 35S promoter and determine whether samples contain genetically modified components. The detection principle is as follows: firstly, two restriction endonucleases FOKI and BsrDIare used to cleave the genomic DNA and the 169bp fragments of CaMV 35S promoter are retrieved; secondly, two nucleic acid probes labeled by Rhodamine Green and Cy5 dyes respectively hybridize with cleaved 169bp fragments of CaMV 35S promoter; thirdly, the hybridization products simultaneously with two dye-labeled probes are detected by fluorescence cross-correlation spectroscopy and GMP is distinguished. As the detection and analysis by FCS can be performed at the level of single molecule, there is no need for any type of amplification. Genetically modified tobaccos are measured by this method. The results indicate this method can detect CaMV 35S promoter of GNU exactly and the sensitivity can be down to 3.47X10(-10)M. Because no any type of amplification is involved, this method can avoid the non-specific amplification and false-positive problems of PCR. Due to its high-sensitivity, simplicity, reliability and little need for sample amounts, this method promises to be a highly effective detection method for GW.