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2023 (1)

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Dissertation
Optimization of on-chip PCR protocols and amplification detection methods

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Abstract

As the first case of novel coronavirus disease (Covid-19) was reported in 2020 [1], it soon spread worldwide and turned into a public health emergency [2]. One key point to slow the spread of the disease is the rapid and accurate detection of the virus RNA. In this project, three three detection methods were compared to detect the DNA product resulting from LAMP-PCR using Covid-RNA target. The electrochemical test was designed to use Methylene blue as a redox reporter to bind on the gold surface upon DNA hybridization. However, the electrochemical results still show the amplification process with the peak height, which would be a result of the change in the pH. The advantage of the electrochemical biosensor is the high sensitivity and detailed information about the surface of the working electrode. The disadvantages of the electrochemical biosensor include a low tolerance for background noises, the requirement for pretreatment of the electrodes, and a potentiostat. In the colorimetric test, the color change is detectable after 15 minutes and was more obvious after 30 minutes. Yet the sensitivity is low as the naked eye cannot distinguish subtle color changes. Chambers should have a diameter larger than 2.5mm with a thickness of 2mm for results observable to the naked eye. The advantage of using colorimetric detection is that the detection is cheap and easy, and no extra machines are needed for detection. The fluorescent detection also shows signals after about 8 minutes. A higher increasing rate of the signals as well as an earlier reveal of signals were detected for the increasing starting concentration of RNAs. The advantage of using fluorescent detection would be that the changes in relative fluorescence units during the whole process could be monitored and the result could be precisely quantized. It also has a better sensitivity than the colorimetric while the disadvantage is the requirement for external devices.

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