Enhancement of the sensitivity of chytrid infection detection in large algae cultures

• Main Author: Östlund, Andreas
• Format: Others
• Language: English
• Published: 2018
• Subjects: Biochemistry and Molecular Biology; Biokemi och molekylärbiologi
• Online Access: http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-364459

Large-scale culturing of algae with the aim of production of economically valuable substances is a growing industrial sector. A common problem of such industrial algae cultures is that they are prone to suffer from fungal infections causing growth inhibition of the algae and reduced production of the target substances. Having sensitive and target specific methods for the early detection of these infections are of great economic importance since in later stages infections may be very hard to treat. There are several potential methods that can be used for infection detection such as visual detection through microscopy or DNA amplification based methods like PCR. This report presents a novel protocol for the early detection of the parasitic fungi Paraphysoderma sedebokerense infecting the green micro algae Haematococcus pluvialis. DNA from infected frozen algae cultures collected from the Gustavsberg production plant of Astareal AB was extracted using ZR Fungal /Bacterial DNA MiniPrep kit. Conventional PCR and SYBR Green quantitative PCR were further compared to each other and optimized to be more sensitive in detecting parasitic DNA. The annealing temperature and primer concentration were optimized. In addition, two different SYBR Green PCR kits and the use of fresh versus frozen algae cultures were compared. The quantitative PCR method was able to detect as low as 300 DNA copies accurately but occasionally was capable to give a signal corresponding to as little as 4 DNA copies in a reaction. Meanwhile the conventional PCR method could accurately detect as low as 600 DNA copies and occasionally 60 DNA copies. This report shows the possibility of improving the detection of P. sedebokerense infection in industrial H. pluvialis cultures by PCR technique, emphasizes the differences between conventional PCR and quantitative PCR and presents a working protocol for the quantitative PCR based monitoring of P. sedebokerense infections.