Development of Innovative Techniques for RNA Virus Diagnosis

• Main Authors: Huang, Rong-Yuan, 黃榮淵
• Other Authors: Chang, Margaret Dah-Tsyr
• Format: Others
• Language: en_US
• Published: 2009
• Online Access: http://ndltd.ncl.edu.tw/handle/87888364338501974344

博士 === 國立清華大學 === 分子與細胞生物研究所 === 97 === In vitro diagnostic device (IVD device) includes reagents, calibrators, sample collection device, control materials, and related instruments or apparatus according to the definition of U.S. Food and Drug Administration (USFDA). IVD device is now widely used for diagnostic, monitoring or preventive purposes. In this thesis investigation of in vitro diagnostic strategy for infectious RNA viruses leads to development of proto-type diagnosis including nucleic acid testing (NAT) system for Hepatitis C virus (HCV) and enzyme immunoassay (EIA) for Human immunodeficiency virus (HIV). At first Remus and MISA bioinformatics software were employed to analyze the 5’ untranslated region sequences in several HCV strains. Then the oligonucleotide primers and probes specifically reacted to six different HCV genotypes were designed and tested in a new HCV genotyping Polymerase Chain Reaction Enzyme-Linked Oligonucleotide-Sorbent Assay (PCR-ELOSA). Screening of 100 clinical serum samples using our novel PCR-ELOSA system indicated higher sensitivity and specificity than commercial NAT diagnostic kits. . It demonstrated the quality and performance of this new technique. In addition, in silico molecular modeling was carried out to investigate protein-glycan interaction. It was found that the structure of starch-binding domain (SBD) of Rhizopus oryzae glucoamylase (RoGA) was quite similar to the VH domain of human 2G12 monoclonal antibody, which recognized the HIV envelope glycoprotein. Here direct sandwich ELISA and in vitro glycan array analysis proved that RoSBD could bind to HIV1-gp120 glycoprotein through protein-polysaccharide interaction. Likewise, the SBD of Aspergillus niger glucoamylase (AnSBD) showed the same binding specificity. Hence we have firstly discovered and reported novel binding ability between RoSBD / AnSBD and a special glycan structure of HIV1 gp120 envelope glycoprotein, which leads to development of novel molecular diagnostic theory and reagents for detection of RNA virus, and may further contribute to the fields of biomedicine and biotechnology.