Quantitative analysis of nucleic acid extraction methods for Vibrio cholerae using real-time PCR and conventional PCR
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The devastating diarrheal disease cholera is caused by the bacterium Vibrio cholerae. Nucleic acid extraction is the primary step for several molecular detection approaches. In order to identify the sources of cholera illness, an efficient, fast and easy DNA extraction method for toxigenic Vibrio cholerae is desired which can be applied on diverse type of samples. This methodology developmental setup study was performed in the Environmental Microbiology Lab, Department of Microbiology, University of Dhaka, Bangladesh from March 2015 to May 2015 attempted to compare three DNA extraction methods for efficient detection and quantification of Vibrio cholerae. Three nucleic acid extraction methods: (Boiled template, Phenol: Chloroform: Isoamyl alcohol, QiaAmp® mini kit), were assessed for four, routinely tested, templates: crude culture, suspension in water, phosphate buffer saline (PBS), and food by conventional and real time PCR targeting the toxin-coding ctxA gene. Finally, the results were compared in context of processing time and overall cost. Cycle threshold (Ct) values were within acceptable parameters by real time PCR (5 to 35 cycles). All the three extraction methods produced sufficient yield of DNA and copy number for detection by real time and conventional PCR. The boiled template method for water samples yielded low amount of DNA in comparison to the other methods, and is therefore sensitive to detect by non-quantitative, conventional PCR only. Despite an overall low detectability from water samples, our comparison reveals that the boiled template method is the most suitable method for high quality and quantity pathogenic DNA particularly in light of limited access to expensive kits and reagents, time constraints, and high sample load.
|Tidsskrift||Mymensingh Medical Journal|
|Status||Udgivet - 2018|