Study Of Novel Approaches For PPRV Nucleic Acid Detection By Gole-nanotechnology

Gold nanoparticles (AuNP)is small gold particles ranging from lnm to100nm in diameter, which is widely used in molecular biology, showing its superiority as emerging material. This study conducts two approaches and discusses their superiority, aming to investigate the feasibility of peste des petits ruminants virus (PPRV) nucleic acid detection by gold nanotechnology.1、Preparation and characterization of gold nanoparticlesIn order to obtain uniform sized, good monodispersity, stable property gold nanoparticles for subsequent experiments, gold nanoparticles is prepared by sodium citrate reduction of chloroauric acid and characterized by full wavelength scanning and transmission electron microscope. The particle diameter is about13nm,which are suited for nucleic acid probe labeling.2、Research on nucleic acid detection of PPRV based on gold nanoparticles and magnetic microparticlesA method for rapid detection of PPRV nucleic acid is established by AuNP, MMP and HRP. The concentration of some components are optimized. The detection signal correlates well with the concentration of target from1μM to10fM. In the ssDNA detection of PPRV N gene there is good linear relationship between OD450nm and the target from100ng/μl to20ng/μl in the case of PPRV RNA detection. It takes about45min for the detection process with magnetic separation. It is time saving, highly sensitive, fast and convenient.3、Study on the detection of PPRV nucleic acid with oligonucleotide probe labeled gold nanoparticlesThe method is constituted by gold nanoparticles,biotin-avidin and silver nitrate.The hybridization temperature, time and the silver staining component and silver staining time are optimized respectively. PPRV N gene ssDNA detection shows that the lowest concentration is lOfM, and the time required is1.5h. The established approach is sensitive, easy to operate, which would provide data and technical support for the nucleic acid and clinical detection of PPRV.Gold nanoparticles-magnetic microparticles-HRP signal amplification measure and gold nanoparticles-silver staining signal amplification to detect PPRV N gene are established and evaluated, and implemented to of PPRV. It may enrich the application of gold nanotechnology in biomolecule, and would provide clues for methodological study.