Direct real-time taqman PCR and PCR methods for bacterial pathogens in human blood samples

• Main Author: Koh, Yong Chuan
• Other Authors: Ayi Teck Choon @ Yeo Teck Choon
• Format: Final Year Project
• Language: English
• Published: 2014
• Subjects: DRNTU::Science::Biological sciences::Molecular biology
• Online Access: http://hdl.handle.net/10356/60729
• Institution: Nanyang Technological University

Traditional clinical diagnosis of bacterial pathogens involves cell culturing which could take days. Direct Polymerase Chain Reaction (Direct PCR) is a new method in detecting pathogens by directly putting samples into PCR without DNA extraction. However, not much work has been done for Direct real-time Taqman PCR for blood samples due to PCR inhibitors and quenchers present in the blood. Here, we showed that two polymerases, A and B, when used together on spiked blood samples, could give consistent Cycle Threshold (CT) values. This was an improvement compared to using Polymerase A alone. CT values were also comparable with traditional diagnostic method of PCR using PlatinumTaq DNA Polymerase on extracted DNA samples. Furthermore, we explored a Fast Direct PCR method on Lightcycler and used dipsticks as an alternative to electrophoresis. Results showed Fast Direct PCR was completed within 16 minutes, and sensitivity was not compromised as compared to traditional Direct PCR. Dipsticks showed equal sensitivity with electrophoresis results. These two findings opened up great potential for Direct PCR developments for clinical diagnostics with the advantage of cost and time savings, thus increasing capabilities to handle emergency outbreaks.