| Author: | Chan, Kin-hung |
| Title: | Detection of rifampin resistant mycobacterium tuberculosis by nonradioactive polymerase chain reaction : single strand conformation polymorphism analysis |
| Degree: | M.Sc. |
| Year: | 2000 |
| Subject: | Mycobacterium tuberculosis Hong Kong Polytechnic University -- Dissertations |
| Department: | Multi-disciplinary Studies Department of Applied Biology and Chemical Technology |
| Pages: | xii, 65 leaves : ill. (some col.) ; 30 cm |
| Language: | English |
| Abstract: | Rifampin is an active anti-tubercular agent that is used in the short-course chemotherapy of Tuberculosis. The emergence of Multidrug Resistant Tuberculosis can be predicted by rifampin resistant in Mycobacterium tuberculosis. It is a burden to the public health as MDR-TB is an incurable disease with a high infectious rate. The detection of rifampin resistant M. tuberculosis is important in the treatment and control of the disease. The determination of drug susceptibility testing by conventional method is slow as time is needed for the recovery and growth of M. tuberculosis from the clinical specimens. It will be advantageous if a rapid detection of rifampin resistant M. tuberculosis can be performed directly in clinical specimens. Polymerase Chain Reaction - Single Strand Conformation Polymorphism analysis is one of the genotypic methods that are used in the rapid detection of mutations conferring to rifampin resistance in M. tuberculosis. Here, 23 rifampin sensitive and 16 rifampin resistant historical M. tuberculosis strains were tested for the presence of mutations by PCR-SSCP analysis. In the molecular mechanism of rifampin resistance, it is caused by mutations in the hot spot region of rpoB gene in M. tuberculosis. PCR-SSCP is used to detect mutations of single-base substitutions as well as a small deletions and insertions. In the study, four types of SSCP patterns were found which were corresponded to four different mutations in the rpoB gene. On the other hand, the SSCP patterns of 46 out of 51 clinical specimens that were positive for M. tuberculosis by culture were investigated. All the specimens were found to have the same wild type pattern as the control M. tuberculosis HRv37 strain. In the PCR-SSCP analysis, silver staining was used as the nonradioactive method in the detection of nucleic acids as it would be applicable in routine laboratory. Apart from PCR-SSCP analysis, Line Probe Assay is a commercial kit that is used for the detection of mutation in the rpoB gene of M. tuberculosis. The method is different from that of the PCR-SSCP as the type of mutations in four prevalent regions can be detected by the presence of specific probes in the test kit. In the study, three types of mutations Asp516→Val, His526→Asp and Ser531→Leu were detected by the probes in the assay. On the other hand, one type of mutation Leu533→Pro could only be detected by automated DNA sequencing as no specific probe was designed for the mutation. |
| Rights: | All rights reserved |
| Access: | restricted access |
Files in This Item:
| File | Description | Size | Format | |
|---|---|---|---|---|
| b1527696x.pdf | For All Users (off-campus access for PolyU Staff & Students only) | 2.8 MB | Adobe PDF | View/Open |
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