Imam Abdulrahman Bin Faisal University
Permanent URI for this collectionhttps://drepo.sdl.edu.sa/handle/20.500.14154/16
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Item Restricted Development of a Colorimetric SARS-CoV-2 Detection Technology Using Reverse-Transcription Loop-mediated Isothermal Amplification (RT-LAMP) Assay(2022) Alhamid, Galyah Mohammed Abdullah; اوغلو، حسين كومبولThe ongoing COVID-19 remains to spread worldwide, causing new cases and deaths daily since its declaration as a pandemic over two years ago. Diagnostic tests are the first line of defense against the transmission of this disease, with the reverse-transcription quantitative polymerase chain reaction (RT-qPCR) being the approved gold standard. However, due to the global demand for facilities, reagents, and healthcare workers needed for rapid population-based testing, alternative tests need to be invested. The most common limitation in using RT-qPCR is giving false diagnoses, which puts others at risk of infection. Efficient fighting against this pandemic correlates with easy and large-scale accessibility of detection tools. Therefore, we developed a reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay to diagnose COVID-19 by targeting and amplifying the SARS-CoV-2 E gene. The reaction takes place isothermally in a water bath set at 65 °C, and the positive results are read out with the naked eye via a color change from red to yellow, without requiring expensive and bulky instruments. In addition, a fluorometric RT-LAMP assay was developed to validate the results. Both colorimetric and fluorometric assays performed superbly when testing 150 clinical specimens, yielding 89.5% and 100% sensitivities, respectively. Optimizing the originally designed primer set (E-ID1) successfully detected SARS-CoV-2 in 20 minutes with a limit of detection of 500 copies/reaction, corresponding to 20 copies/µL, with no cross-reactivity observed among other respiratory viruses. No misamplification was evident after prolonged reaction times, which eliminates the most common limitation of potential false-positive diagnoses. Furthermore, we used a color sensor to demonstrate that the RGB codes gradually increase with the color change in positive RT-LAMP reactions, providing a simple alternative monitoring technique. Lyophilization of the colorimetric RT-LAMP reagents showed promising results to develop in-home and easy-to-store testing kit. These findings are essential to support the use of this technique in healthcare systems in fighting the COVID-19 pandemic, especially in resource-limited areas.11 0Item Restricted Comparing the Effect of Two Pyrimidine Analogues on Colorectal Cancer Cells(2021) Alshammari, Thamer Marhoun Bin Jarbou; ثابت، حسين حسن; المفتي، سارة أمينNowadays, people are living in an era with high standards of healthcare accessibility, which can remarkably improve the diagnostic tools and the treatment plans of different diseases. These standards contribute to the determination of the general life expectancy in many parts of the world. With cancer, however, the mortality rate has globally increased by almost 40% over the past four decades, and it is expected that the mortality will further increase to reach 60% in the current decade with an estimated death of 13 million cancer patients by the year 2030. Colorectal cancer (CRC) is considered the third most frequent type of cancer and the second leading cause of cancer-related deaths, for both sexes, in the world. The explanation of the increased mortality of colorectal cancer might be due to some environmental factors, such as; smoking, bad diet, obesity, and very low physical activity, in addition to genetic background. The major pathways of either environmental (epigenetic) or genetic causes of CRC include CpG hypermethylation and microsatellite instability (MSI) due to the mismatch repair (MMR) gene deficiency. Considering that CRC accounts for 10% of the most commonly diagnosed types of cancer, there is an urgent need for developing effective treatment plans for CRC patients. 5-Azacytidine (5-AZA), a cytidine pyrimidine nucleoside analogue, is an epigenetic drug that incorporates into DNA and blocks the activity of the DNMT enzyme and thereby inhibits DNA methylation that might cause CRC. In addition to 5-AZA, another chemotherapeutic drug named 5-fluorouracil (5-FU) was applied either synergistically with 5-AZA, or alone, in this study. Both drugs were applied to the human colorectal carcinoma cell line, HCT116, by performing several methods, such as MTT assay, wound healing assay, and colony formation assay to assess the cells viability and migration property before and after treatment to analyse their effect on CRC. In addition, whole exome sequencing (WES) was performed to assess the genetic changes after treatment with 5-FU. HEK293, which is the human embryonic kidney cell line, was used in the study as the control cell line receiving the same treatment regime as the HCT116 cells. The main result of this study shows that both drugs have a noticeable effect on the viability of HCT116 cells. 5-AZA alone possesses the highest efficacy on HCT116 cells, followed by the drugs combination and 5-FU alone, respectively. In addition, WES results show that 5-FU diminished the number of mutations in HCT116 cells. In a conclusion, 5-AZA alone as a treatment plan for CRC has more efficacy than either 5-FU alone or the drugs combination which leads to the recommendation of using it alone as an anticancer drug. Also, WES results suggest that the 5-FU mechanism of action as a DNA damage drug is proven.8 0