利用聚合 ? 連鎖反應偵測臨床葡萄球菌菌株之評估

1. 利用聚合?連鎖反應偵測臨床葡萄球菌菌株之評估利用聚合?連鎖反應偵測臨床葡萄球菌菌株之評估 • Whether the widely-effective antibiotics can be used to cure the infection of Staphylococcus has been a long-concerned question. Traditionally, investigators in microbiology laboratories use oxacillin to inspect the fitness of using cephalosporin and general laboratories adopt traditional phenotyping to identify MRS (methicillin resistant Staphylococcus). But some strains are not correctly identified causing a little confusion. • When MRS is wrongly identified as MSS (methicillin susceptible Staphylococcus), it will lead to the failure of treatment. Furthermore, if there is no efficient infection control, it will cause the spread of MRS strains. When MSS is wrongly identified as MRS, it will cause money waste resulting from unnecessary isolation of healthy people. Therefore, it’s necessary to correctly identify MRS. • In this study, we adopt NCCLS guideline of 2004, with which cefoxitin and oxacillin are both used to identify MRSA (methicillin resistant Staphylococcus aureus) and MRCNS (methicillin resistant coagulase negative Staphylococcus) strains. Besides, Then compare with using PCR technique to identify mecA gene as gold standard by two different primer pairs. At the same time, detect clfA and nucA gene (also by PCR) to monitor the accuracy of S. aureus identification, and using the house-keeping gene in the conserved regions of Eubacterial 16S rRNA and Staphylococcal 16S rRNA to be a positive control. We can evaluate whether cephalosporin is a efficient treatment of S. aureus directly or indirectly by assaying these six related gene. In this report, bother Staph Latex kit and DNase test were evaluated for their sensitivity and specificity on the identification of S. aureus by using it separately or combined together. The results showed that the sensitivity and accuracy are 99.2% and 99.4% respectively and could be improved to 100% by combining two tests. For identifying of MRSA or MRCNS, both cefoxitin and oxacillin were tested. The results indicated that in cefoxitin alone, the sensitivity and specificity are 96.1% and 100% respectively and in oxacillin alone the sensitivity and specificity are 94.7% and 97.8% respectively. When two tests were combined, the sensitivity and specificity could be improved to 98.7% and 97.8% respectively and the accuracy could be increased to 98.2%.

2. Evaluation on the detection of clinical Staphylococcus isolates using polymerase chain reaction • Ovarian cancer has a high death rate because most patients do not have obvious symptom and lack the proper tumor marker for detection. Previous experiment showed that OVTA-2 protein can be recognized by antibodies of ovary cancer patients so we believe this protein is an ovarian tumor associated antigen. In this research, our aim is to use a recombinant OVTA-2 protein fragment to immunize chicken and constructed an antibody library by phage display technology. By screening of a scFv (single-chain variable fragment) phage library, the OVTA-2 protein specific scFv antibodies can be isolated. The full length OVTA-2 gene is 4150 bp but in this study, we cloned 600 bp DNA fragment into pGEX vector and the recombinant GST-OVTA-2 protein fragment is 35 kDa. After expression in a large amount in E.coli, we used GST Sepharose 4B to purify GST-OVTA-2 protein than use coomassie blue stain and western blot to detect the purified GST-OVTA-2 fusion protein. These purified GST-OVTA-2 fusion protein was mixed with adjuvant and the mixture was injected intramuscularly into Leghorn chicken. Polyclonal IgY antibodies were purified from the immunized chicken and examined by enzyme-linked immunosorbent assay (ELISA) and western blot analysis. The result indicated that IgY polyclonal antibodies can recognize GST-OVTA-2 protein fragment. Chicken''s spleen mRNA was isolated and chicken heavy chain and light chain antibody fragments were generated by use PCR. Antibody fragments were cloned into pComb3X vector and a chicken antibody library was constructed (3.2×103 library size). These antibody fragments were displayed at the phage tail and they were used for phage panning. Fourteen recombinant phages were randomly selected after 4 times panning steps. These phages were analyzed for their binding ability to OVTA-2 protein by ELISA test. Our results showed that several isolated recombinant phages have particular binding ability to GST-OVTA-2 protein. Further scfv was identified binding ability by western blot. We found scFv can recognize OVTA-2 protein fragment specifically. We hope to use these scFv antibody molecule apply to clinic and experiment and study in the future