The DNA Detection Methods Based On Magnetic Particle And Flourencense Beads

As new carriers or reporters, micro- or nano-sized particles/beads have been widely used in biomedical research areas. Some techniques for biomolecule detection based on particles/beads have been commercialized. Herein, the DNA fragment of 23S DNA of E. Coli was detected using the magnetic particle. On the other hand, using flourensence beads as carriers, high-risk Human papillomavirus (HPV) were genotyped by Luminex system.5'- amino group labeled specific oligonucleitide was immobilized onto the surface of the carboxyl terminated magnetic particles and hybridized with DNA fragment amplified from 23S gene of E. Coli by asymmtric PCR. Then the single base chain extension (SBCE) reaction was carried out on magnetic particle surface in four tubes and the biotin-labeled ddNTP was introduced, respectively. Finally, the conjugate of streptavidin-phycoerythrin (SA-PE) was added to each tube and the fluorescence signal on magnetic particle was analyzed by biochip scanner. The results show that 0.65 nmol of 5'- amino labeled probe could be immobilized onto 1 mg of carboxyl terminated magnetic particles and the efficiency of immobilization was 97.60ï¼…. Based on SBCE desighed for G, the fluorescence signal for single base (C) was 21 times more intensive than the other three bases (A, T, G).A pair of conservative primers Gp5/Gp6 (Gp6 5'-biotin) was used to amplify HPV DNA with 150 bp from L1 region of HPV standard plasmid, then the PCR products was hybridized with three oligonucleitide probes (HPV18, 33, 35) coupled on three kinds of fluorescent microspheres. The SA-PE was introduced in the system and the emmision signal at different wavelength was read by Luminex^TM100 according the addressable fluorescent microsphere and the fluorescent signal from hybridization reaction the HPV genotype can be read, the ratio of fluoresence for posotive and negative is in the range of 8-72, all three different types of HPV genotype can be read correctly with 100ï¼….Compared with traditional methods, the DNA detection based on particles/beads have advantages of rapid performance, easy operation, high sensitive, and less time consuming, and have the potetial application in clinics.