The development of a rapid diagnostic system for difficult to culture human pathogens
The World Health Organization (WHO) has outlined a TB-free World strategy that aims to reduce tuberculosis prevalence by 2015. That could be achieved through the development of a specific and rapid detection system. In countries with a high incidence of tuberculosis, detection is solely based on spu...
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| Format: | Thesis |
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2013
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| Online Access: | http://eprints.uthm.edu.my/4615/ http://eprints.uthm.edu.my/4615/1/Balkis_A_Talip.pdf |
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| Summary: | The World Health Organization (WHO) has outlined a TB-free World strategy that aims to reduce tuberculosis prevalence by 2015. That could be achieved through the development of a specific and rapid detection system. In countries with a high incidence of tuberculosis, detection is solely based on sputum smear microscopy using Ziehl-Neelsen stain and chest radiography. This is followed by cultivation, which requires up to 12 weeks for confirmation. The diagnostic test available is insensitive, laborious, lacks specificity and does not even give unequivocal proof of infection. Consequently, this study was conducted to obtain confirmatory diagnostic information from Ziehl-Neelsen stained smears on glass slides. The fast-grower, non-pathogenic Mycobacterium smegmatis was used as the model organism due to similarity to the pathogenic strain, Mycobacterium tuberculosis. Laser capture microscopy (LCM) is primarily used as a microdissection tool in studies involving tissues and membranes. This technology was able to isolate intact individual cells fixed on archived glass slides regardless of how the smears and staining have been performed, confirmed by transmission electron microscopy (TEM) observation. Typically between 100 – 1,000 catapulted cells are enumerated using flow cytometry (FCM). A series of DNA extraction techniques which are heat-shock, heat-shock followed by ethanol precipitation and QIAamp® DNA Micro kit were compared and optimized for the lowest number of post-catapult cells. To maintain the detection of lowest number of catapulted cells, the rpoB-gene specific primers were designed for amplification using nested real-time PCR. Routinely, this system was able to detect as few as 30 catapulted cells per assay. This result demonstrated that it is possible to isolate the bacteria from glass slides and subsequently perform downstream molecular applications regardless of any inhibitory factors. In conclusion, we strongly recommend that this system may offer improved specificity and speed of tuberculosis detection with lower risk of exposure to infection through the use of stained slides. |
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