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Title:	Polymerase chain reaction and high-resolution melting based gene screening system for adverse drug reactions detection
Authors:	Chong Kim Soon (P80413)
Supervisor:	Gan Kok Beng, Assoc. Prof. Dr.
Keywords:	Genes Polymerase chain reaction Drugs Universiti Kebangsaan Malaysia -- Dissertations Dissertations, Academic -- Malaysia
Issue Date:	17-Jan-2019
Description:	Allopurinol, a drug used in the treatment of chronic gout and hyperuricemia, is one of the leading causes of drug-induced severe cutaneous adverse drug reactions (SCARs) reported in Malaysia and Singapore. Steven-Johnson Syndrome, Toxic Epidermal Necrolysis and Hypersensitivity Syndrome are the most life-threatening SCARs. Recent pharmacogenetic studies have shown the human leukocyte antigen-B*58:01 (HLA-B*58:01) is the strong association signal for allopurinol-induced SCARs. The screening of the HLA-B*58:01 before the medication may reduce the risk of SCARs. However, the HLA-B*58:01 screening is a cost-prohibitive test and time-consuming for routine clinical practice. The available screening test requires a specific and expensive instrument which is mainly used in medical research rather than in clinical practice. In this project, a low-cost gene screening system using high-resolution melting methods had been designed and developed for HLA-B*58:01 allele detection. The system consisted of a thermal cycler chamber and a fluorescence detection chamber. The thermal cycler chamber was developed to amplify the amount of the deoxyribonucleic acid sample by using polymerase chain reaction. The fluorescence detection chamber was developed to acquire the fluorescence signal during the highresolution melting process. The system was powered by a 230 VAC power supply. A 6-well Aluminum heating block was designed to fit snugly with the 0.1 milliliter microcentrifuge tubes. The block was heated and cooled by using Peltier. The Peltier had controlled by a proportional-integral temperature feedback controller. The temperature of the block temperature had measured by a negative temperature coefficient thermistor as feedback to the temperature controller. The optical fluorescence detector was developed to detect the high-resolution melting signal from each microcentrifuge tube. An excitation light (470 nm) from a light emitting diode passed through an excitation filter to excite reaction mixture in the microcentrifuge tube. The fluorescent light signal (525 nm) emitted from the reaction mixture passed through the emission filter and detected by a silicon photomultiplier. A data acquisition device was used to record the fluorescence signal and block's temperature during the screening process. The acquired data were transferred to a personal computer using a universal serial bus and saved in a text file. A graphical user interface was developed using LabVIEW. An algorithm was developed to classify the present and absent of HLA-B*58:01 allele into a positive or negative result. The results had benchmarked against Eco Real-Time PCR system. A pilot study was conducted using six sets of high-resolution melting assays. The high-resolution melting assays consisted of three sets of assays with the HLA-B*58:01 allele and three sets of assays without the HLA-B*58:01 allele. The results showed that the developed system was able to classify the HLA-B*58:01 allele correctly compared to the Eco Real-Time Polymerase Chain Reaction system. The developed system required 1 hour 28 minutes 5 seconds to complete the HLA-B*58:01 screening test. The dimension of the development system was 22cm×20cm×36cm (Width×Depth×Height). The temperature ramp rates of the developed system were 5.5°C/s (heating) and 4.4°C/s (cooling), respectively. The temperature range of the system was 50°C to 100°C. The temperature uniformity of the system was ± 0.1°C.,Ph.D.
Pages:	117
Call Number:	QH447.C486 2019 3 tesis
Publisher:	UKM, Bangi
Appears in Collections:	Faculty of Engineering and Built Environment / Fakulti Kejuruteraan dan Alam Bina

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