Saudi Universities Theses & Dissertations
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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 Identifying Pathogenic Variations on Early Stage of Pregnancy Using Invasive and Non-Invasive Prenatal Samples(2022) Sonbol, Bayan Sameer Mohammed; Borgio, Francis; AbdulAzeez, SayedVarious factors can increase the risk of fetal genetic abnormalities during pregnancy, such as maternal age and family history. Prenatal diagnosis is a critical clinical practice for couples at risk of genetic disorders in the fetus. The study aims to identify pathogenic gene variations in the early stage of fetal development using invasive and non-invasive prenatal samples by developing fetal cell isolation, gender determination, and gene variation screening. Different samples were collected from various sources, maternal blood (n=10), serum (n=8), paternal blood (n=7), maternal buccal swab (n=8), maternal urine (n=9), and amniotic fluid (n=6). Coelomic fluid (n=1) was also collected for the first time in the history of Saudi Arabia. Different methods were used for fetal cell isolation: morphology based using a designed manual micromanipulator from invasive samples; Cluster of differentiation 71 (CD71) microbeads mediated magnetic cell sorting and percoll based subfractionation for fetal nucleated red blood cells (fNRBCs) from invasive and non-invasive samples. In-house multiplex gender PCR was used for the fetus gender determination. Nested and Amplification refractory mutation system (ARMS) PCR with newly designed primers carried out for detecting sickle cell disease (SCD) mutation (HBB:c.20A>T) in the isolated fetal cells. Our study has successfully identified a heterozygous sickle mutation in HBB gene from amniotic fluid cells and fetal DNA sample (n=3) using ARMS PCR. The study has also determined the fetal gender in single cell and fetal DNA using the gender PCR multiplex. All fetal DNA of fetal cells had a high accuracy than cell free fetal DNA (cffDNA). This study highlights that single-cell isolation from amniotic fluid and coelomic fluid could be used for accurate genetic screening for early diagnosis upon largescale validation.24 0Item Restricted Study of antibiotic resistance genes of Candida auris and nanoparticles as a potential therapeutic(2022) Al-Dossary, Hanan Abdulrahman Nasser; بيرجيو، فرانسيسIn recent years, Candida auris (C. auris), a nosocomial infection, has emerged as a challenge, due to the limited therapeutic options and multidrug-resistance leading to a higher mortality rate. C. auris is reported to have developed resistance to various antifungal drugs such as fluconazole, amphotericin B, 5-fluorocytosine, caspofungin, etc., Hence, it becomes imperative to study and detect different resistant genes occurring among different strains isolated from the nosocomial infections. On the other hand, nanomaterials are known to have an exclusive characteristic in antimicrobial therapy through their controlled size and targeted drug delivery. Therefore, this study was conducted with the aim of identifying nineteen C. auris strains collected from King Fahd Hospital of the University and King Fahad Specialist Hospital in Dammam then identified by 18S rRNA gene and ITS region sequencing technique and detecting the drug resistance-associated mutations in ERG11, TAC1B and FUR1 genes to get an insight into the pattern of drug resistance. Molecular identification was successfully achieved using 18S rRNA gene and ITS region and 5 drug resistance associated missense variants identified in ERG11 (F132Y and K143R) and TAC1B (H608Y, P611S and A640V) genes of C. auris strains. Additionally, the current study offers the prophylactic and therapeutic application of hydrothermally synthesized Ag-silicalite-1 (Si/Ag ratio 25) nanomaterial against the nineteen clinical strains of C. auris, TiZSM-5, 4wt%Ag/TiZSM-5 were prepared using impregnation technique, Ag-silicalite-1 (25) and Ag-silicalite-1 (100) nanoformulations were characterized using different phases determination (XRD), surface area analysis (BET), diffuse reflectance UV-Visible spectroscopy (DRS-UV-Vis), thermogravimetric analysis and differential thermal analysis (TGA-DTA), morphological studies [(scanning electron microscopy/energy dispersive X-Rays spectroscopy (SEM/EDX) and transmission electron microscope (TEM)]. All the collected C. auris strains were subjected to antifungal efficacy by colony forming unit (CFU) technique and antibiofilm activity by employing cell kill assay, scanning electron microscopy (SEM) and light microscopy. Across all the nineteen C. auris strains, 4wt% Ag/TiZSM-5 and Ag-silicalite-1 significantly inhibited the growth of the majority of C. auris strains and a significant inhibitory effect on biofilm’s survival rate, the lowest inhibition value was (10 %) with Ag-silicalite-1 at 24 and 48h incubation. A profound change in morphogenesis and in addition to the reduction in the number of C. auris cells was shown by SEM and light microscopy. The presence of high surface area and uniform dispersion of nanosized Ag species displays an enhanced anti-Candida activity and therefore it has a great potential against the emerging multidrug-resistant C. auris.13 0Item Restricted The Impact of Gold Nanoparticles on Colon Cancer: An In Vitro Cellular and Molecular Analysis(2021) Aldahhan, Razan Abdulhameed; المهيزعي، دانة عبد الرحمن1 0Item Restricted The Impact of Biologically Synthesized Nanoparticles on Colon Cancer Cells: In Vitro Cellular and Morphological Investigation(2022) Rasdan, Alia Saeed Mohammed; خان، فردوس علمAbstract: Colorectal cancer (CRC) attacks the colon or rectal region of the large intestine. CRC is the third globally prevalent cause of cancer mortality. Although current treatments are effective, they have several drawbacks such as systemic toxicity. Therefore, nanoparticles (NPs) are being investigated as delivery systems. Green synthesized NPs are eco-friendly, non-toxic, and safe to manufacture, making them a better option than chemically synthesized NPs. In this project, we tested green vs. chemically synthesized NPs with and without date extract to study their cytotoxic effects. The sol-gel approach was used to create CoFe2O4/(Ni0.4Cu0.2Zn0.4Fe1.8O4( NPs. Characterization was performed using XRD, SEM, TEM, and zeta potential to investigate the NP structure and morphology. These assays confirmed the chemical composition with size ranging from 24 to 41 nm. Furthermore, we performed MTT, DAPI, and cell migration on HCT116 (colorectal cancer) and HEK293 (human embryonic kidney) cell lines. Treatment groups included: (BS1, BS2, BS3, BS4, BS5, BS6, BS7, BS8, and S9) at varying concentrations for 48h. For HCT116, MTT results revealed that the lowest viability was 20.1% after treatment with BS8 and the highest was 23.76% in BS1. Whereas for HEK293, the lowest viability was 21.72% in S9 and the highest was 25.84% in BS6. The results obtained from DAPI assay suggest that all NPs inhibit the growth of both cell lines with significantly shrunken and fragmented nuclei. In cell migration, treatment with BS1, BS2, BS3, BS7, BS8, and S9 showed very few cells migrated across the empty areas in HCT116. Cell migration was observed in HEK293 following treatment with BS1, BS2, BS3, BS4, BS6, BS7, BS8, and S9 NPs. Thus, these results suggest that BS1, BS2, BS3, BS7, BS8, and S9 can be good candidates for CRC therapy. This research will help find a new therapy for CRC treatment.1 0