Nucleic acid based in-vitro diagnostic device

Tezin Türü: Doktora

Tezin Yürütüldüğü Kurum: Orta Doğu Teknik Üniversitesi, Fen Edebiyat Fakültesi, Biyolojik Bilimler Bölümü, Türkiye

Tezin Onay Tarihi: 2018




Commonly used nucleic acid based in-vitro diagnostic systems utilize either quantitative PCR (polymerase chain reaction) or conventional PCR. Quantitative PCR is a fast and reliable method; however, the prices of quantitative PCR devices are relatively expensive due to the cost of highly sensitive sensors. Equipment using conventional PCR is lower in price but have several disadvantages such as long analysis periods, contamination risk, false positivity risk, and usage of carcinogenic chemicals. The purpose of the study is to design, manufacture, and validate a nucleic acid based in-vitro diagnostic device operating with a novel microparticle based method. In this microparticle based method, PCR is performed with the presence of microparticles and oligonucleotide probes. Oligonucleotide probes that are unused in PCR adsorb onto the microparticles; however, fluorophore molecules released with the exo-nuclease activity of Taq. DNA polymerase does not adsorb onto microparticles. By this way, background fluorescence is reduced and the need for sensitive sensors for detecting the fluorescence difference disappears. For this purpose, a prototype device including a LED light source, two filter sliders, two excitation band-pass filters, a 24 sample carousel, two emission band-pass filters, and a CCD camera was designed and manufactured. Microparticle batch equilibrium analysis was performed and using DNA eluates from 105 HBV negative-diagnosed and 187 HBV positive-diagnosed samples, prototype device validation study including cutoff score, analytical sensitivity, analytical specificity, linear range, precision, robustness, diagnostic sensitivity, and diagnostic specificity was conducted. The findings suggest that low-cost prototype device combines high sensitivity, specificity, reproducibility and accuracy for HBV DNA quantitation in a high linear range.