The Application Of Highly Sensitive Electrochemiluminescence Method In Plant Viruses And Genetic Modified Organism Detction

Plant viruses can have a considerable impact on the general public with respectto environment, food supply, and also safety, quality and diversity of food available.In economic terms, viruses are only of importance if it is likely that they will spreadto crops during their commercial lifetime, which varies greatly between very shortextremes in horticultural production and very long extremes in forestry. There are noup-to-date estimates of world losses attributable to plant viruses, the ever increasingworld population requires higher productivity on a constantly declining area ofagricultural land, a effective viruses control measures, will play a vital role inproviding the world with an adequate and varied supply of safe, high quality food[4].The variety of techniques that have been developed for the detection of plantviruses includes: electron microscopy coupled with serological techniques as inimmunoelectron microscopy; direct observation of viral double stranded RNAsfollowing gel electrophoresis; use of serological techniques (ELISA); detection of theviral genome using molecular hybridization technology [8]; detection of the viralgenome using polymerase chain reaction (PCR) technology. Naturally, every methodhas its own specificities and limitations. But, a common shortcoming of thesemethods is that they are usually tedious, multi-stage, low-sensitivity, high cost.Electrochemiluminescence PCR method is a high sensitive nucleic acidanalytical method. There is described in detail in our previous research. In this paper,we improved electrochemiluminescence PCR method and used it to detect plantviruses for the first time. Electrochemiluminescence assay was coupled with nucleic acid probes hybridization applied to detect PCR products. Whether the plants infectedviruses was discriminated by detecting the special nucleic acid sequences of viruses.The PCR products hybridize with a pair of probes designed specially can specificselect the target for detection, thus can avoid pseudo-positive result. Afterhybridization, the PCR products caught by the probes were collected and then theluminescence signal was detected using ECL system. The ECL signals of infectedwith viruses' samples would be much higher than those of healthy samples. So,according to the threshold value of ECL signal we can tell whether the sample wasinfected or not. This method has high sensitivity, stability, simple operation, accurateresult, and is a high effective method to detection plant viruses.Since the polymerase chain reaction (PCR) was introduced in1985, it hadsignificantly impacted nucleic acid analysis and gene detection. However, PCR isoften criticized for its complex, expensive, time-comsuming and labor-intensiveprocedure. Many detection assays have been developed for nucleic acid detection, but,few methods can approach PCR sensitivity. If one could develop a methodology thatcan amplify signals like PCR and eliminate the drawbacks of PCR, which would beextremely beneficial. Fortunately, Mirkin had reported a bio-barcode method fornucleic acid and protein detection, this method has been shown to be extraordinarysensitivity like PCR.Herein, we developed an Electrochemiluminescence (ECL)-based bio-barcodemethod for the detection of genetically modified organism. This method consisted ofrestriction endonuclease digestion, nucleic acid hybridization, ECL detection and thebiotin-label was used for selective immunomaget capture by streptavidin-coated paramagnetic beads in detection cell. Compared with ordinary bio-barcodes method,this assay was simple, fast, stability, and can directly detect GMO from raw materials.