| Summary: | 碩士 === 國防醫學院 === 生理學研究所 === 106 === Periodontal disease (PD), a chronic inflammatory gum illness in the natural tooth or dental implants, cause gingivitis and periodontal tissue destruction by plaque microbiota. It will lead to tooth loss if left untreated appropriately. Porphyromonas gingivalis (P. gingivalis), a gram-negative anaerobe, is generally recognized as one of the major pathogen in the progression of PD. The progression of periodontal disease is closely related with the abnormal growth of P. gingivalis and they also penetrate into circulatory system via invading oral epithelial cells to initiate many of systemic diseases. At present, the diagnosis of PD is determined with the periodontal indexes (e.g. BOP and CAL) by the clinical dentist. But the diagnosis may be missed, when their signs are in the gray scale. Traditional methods that applied to help the clinical diagnosis include anaerobic culture, ELISA and PCR. However, these methods are tedious, time-consuming, and expensive. It is urgent to develop an ease of use, specific, sensitive and reliable method for clinical diagnosis of PD. In this study, 16S rRNA sequences of Porphyromonas gingivalis, Treponema denticola and Tannerella forsythia were collected from NCBI databank firstly, and aligned to screen the P. gingivalis-specific probes. The probes were modified with biotin and thiol group individually at one of the ends. One of the selected probe was modified with biotin as captured probe. And the thiol-modified probe was conjugated to gold nanoparticle as gold nanoparticle probe (GNP) which was blocked with bovine serum albumin (BSA). Both of them were applied to develop a nucleic acid-based lateral flow immunoassay (NABLFIA). After the hybridization with the target nucleic acids in an eppendorf, the streptavidin was also supplied to capture the biotin-GNP complexes. Then the nitrocellulose membrane that immobilized with both anti-streptavidin antibody (test line) and anti-BSA antibody (control line) was dipped into the eppendorf to complete the assay. And the red-band signals were visible with the naked eye. In our results of the synthesized single stranded DNA test, 0.3M of NaCl and the both of 5’ modified probes were the optimized conditions in this device. There is well linear correlation between 0~2,500 fmol (R2=0.9014) and the visual limit of detection was 10 fmol. Then the probes showed the high specificity in the simulated test by synthesized ssDNA of Treponema denticola, Tannerella forsythia and Porphyromonas gingivalis. All procedures could be completed in 40 minutes after the nucleic acid extraction without anaerobic culture. Thus the prototype of P. gingivalis-specific lateral flow immunoassay was successfully developed. It will be utilized to detect the clinical plaques to verify its practicability in following experiments.
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