Naked-eye Detection Of Nucleic Acids Based-on Rolling Circle Amplification

In recent years, detection of nucleic acid plays more and more important role in disease diagnosis and therapy. Existing molecular biological detection technologies, such as quantitative PCR, usually require bulk, expensive and precise instruments, as well as professional trained operators. However in developing countries, as well as regions with limited settings and lack of funds and equipment, it is always a compelling requirement to develop portable, low-cost and equipment-free diagnostics products. Herein we are presenting a new method for sequence-specific and naked-eye detection of nucleic acids through isothermal amplification and magnetic particle mediated aggregation. A padlock probe was designed to hybridize to specific sequence on target genes followed by ligation and rolling circle amplification (RCA). Magnetic particles were then added into RCA products solution. Magnetic particles would physically combine with long linear DNA coils (such as RCA products) and form visible intertwined aggregates, while no aggregate was observed in the absence of high molecular weight NAs (>1kb). As few as0.62amol (124fM) of target DNA molecules were detected by naked eyes. It was further applied to detect Human papillomavirus typel8(HPV-18) gene in genomic DNA isolated from HeLa cells. This assay is sensitive and low-cost with minimal instrumentation, revealing great potential for molecular diagnostics in developing countries and regions with limited settings.In the first chapter, we summerized the principle, method and application of nucleic acid amplification techniques, such as PCR, Loop-mediated isothermal amplification (LAMP), RCA and so on. Furthermore, we discussed on the development, kinds, features and application of RCA in detail. At last, several kinds of techniques applied in POCT, including Lab on a chip, strip biosensor and colorimetry based on AuNPs, were demonstrated.In the second chapter, we established a homogeneous system for proof-in principle investigation of RCA method. At first, a DNA target was recognized and amplified through L-RCA reaction. Then, the ligation reaction and RCA reaction efficiency were analyzed by the gel electrophoresis. Exonuclease was employed to digest the linear DNA in the ligated reaction mixture, in order to prove that ligated products contained circular DNA. We also improved ligation conditions by changing padlock concentration to increase the ligation efficiency. In addition, we compared amplification efficiency and background interference between the single-primer RCA and double-primer RCA. RCA has proved strong ability to distinguish signal and background and could be used for nucleic acid detection.In the third chapter, we developed a nucleic acid detection method based on L-RCA reaction and magnetic particle mediated aggregation. Taq DNA ligase was used in ligation reaction instead of T4DNA ligase, which could significantly reduce nonspecific ligation and improve specificity. Then, we employed visual method based on magnetic particle mediated aggregation to detect HPV-18E7gene, instead of the conventional fluorescent labeling method. As few as0.62amol (124fM) of target DNA molecules were detected by naked eyes, which was at least1000times lower than previously reported methods. Finally, this method was successfully applied to detect HPV-18gene in the in-vitro culture cell lines (HeLa and Huh-7) and the target in the blood samples.To sum up, we developed a method for naked-eye detection of nucleic acids through isothermal amplification and magnetic particle mediated aggregation. Different from the systems based on miniaturization of traditional instrument, such as microchip qPCR technique. RCA-MPA method do not need the thermal cycler and fluorescence detection equipment, which greatly simplified the system. During the whole detecting process, minimal instrumentation was employed, including pipette, thermostatic equipment (water bath) and camera (mobile phone). Our method could not only greatly reduce the cost, but also keep the technology from environment and infrastructure limitation. We hope it could be employed as a useful tool for pathogen diagnostics and gene related disease screening by basic medical and health epidemic prevention departments in remote and developing areas.