Research Of Detection Of Perv In Guangxi Bama Minipig Based On Magnetic Nanoparticles And Chemiluminescence

Xenotransplantation using zoic organs has the potential to resolve the severe shortage of human organ donors. However, porcine endogenous retrovims (PERV) are widely found in various swine breeds and could infect human cells, which is highly paid attention. Guangxi Bama minipigs could offer sufficient donors for xenotransplantation owing to the feature of small body, stable heredity and clear heredity background. To make full use of the Bama minipig resource and provide safe porcine donors, we established a PERV detecting approach based on Magnetic nanoparticles (MNPs) and Chemiluminescence (CL) for screening out Bama minipigs with low copy number of PERV provirus. The main study contents are as follows:1. Preparation of MNPs and its application in DNA extraction from bloodFe3O4nanoparticles were synthesized by the solvothermal method, and Fe3O4@SiO2MNPs were prepared after SiO2was packed on the surfaces. CMNPs were obtained after aminated and carboxylated modification. The results of the analysis of transmission electron microscope (TEM), scanning electron microscope (SEM), particle size, fourier transform infrared spectrometer (FT-IR) and vibrating sample magnetometer (VSM) showed that the MNPs had good shape, uniform diameter, good magnetization and were superparamagnetic. In additional, the prepared Fe3O4@SiO2MNPs could be well applied in genomic DNA extraction from Bama minipig blood.2. Establishment and Optimization of PERV detection method based on MNPs and CLCMNPs were used as solid-phase supports for the detection. Pol, env-A, env-B and env-C fragments were biotiylated by PCR amplification, solid-phase hybridization reaction, incubated with streptavidin-labeled alkaline phosphatase (SA-ALP), and the CL signals were immediately generated after3-(2’-spiroadamantane)-4-methoxy-4-(3"-phosphoryloxy) phenyl-1,2-dioxetane (AMPPD) were added into the magnetic complexes. PERV and PERV subtypes could be detected by this approach. The results indicated that the detection method was specific and highly sensitive. The optimal assay conditions were as follows:1mM for SA modification,10μM for probe modification,55℃(PERV),54℃(PERV-A),50℃(PERV-B),56℃(PERV-C) for hybridization temperature, and30min for hybridization time.3. CL detection of relative copy number of PERV in Guangxi Bama minipigGAPDH CL detection method was established and combined with the above PERV CL detection method for the analysis of relative copy number of PERV and3PERV subtypes provirus. The results showed that the relative copy numbers of PERVs were PERV (0.94±0.10-6.79±0.59), PERV-A (1.01±0.13-7.26±0.64), PERV-B (0.74±0.08-6.13±0.65), PERV-C (0.05±0.01-0.44±0.12), respectively. This indicated that the copy numbers of PERV provirus were greatly different in the genomic DNA of these20Bama minipigs, and the copy number of PERV-C provirus was extremely low.This established method could be applied to screen Bama minipigs and obain the ones with low copy number of PERV provirus, which provides suitable donors for xenotransplantation, and lays a foundation of breeding for Bama minipigs with low copy number of PERV provirus.