Yun, Foong Sui
(2005)
PCR-RDB assay for detection of vibrio cholerae.
Masters thesis, Universiti Sains Malaysia.
Abstract
A colorimetric detection method has been designed and optimized by
immobilizing a specific DNA probe onto Biodyne C membrane and hybridize it
with heat denatured biotin labeled PCR amplicon to detect the presence of the
hemM gene of Vibrio cholerae. Therefore it can be used to replaced agarose
gel electrophoresis and conventional culture method which are very low in
sensitivity for diagnosis of cholera. This method was refered to as PCR-RDB
assay. Optimization of the PCR-Rerverse Dot Blot assay include hybridization
temperature, amount of PCR amplicon, timing of the assay, optimizing of biotin
incorporation in PCR amplicon, dilution of streptavidin-alkaline phosphatase
conjugate used and development of a control positive for the assay. PCR-RDB
assay can be used in detection of biological or environmental samples to help in
control of cholera cases in high incidence area.
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