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Optimisation of phage quantification method with quantitative PCR for environmental samples

Starting date Planned:
May to October 2018 (Flexible)

Master Thesis Proposal

Proposed Topic

Optimisation of phage quantification method with quantitative PCR for environmental samples.

Project summary

Bacteriophages, viruses that infect bacteria, are being increasingly used as biocontrol agents to restrain the growth of undesired microorganisms. The use of phages as control agents requires as well, the development of tools to quantify them fast and accurate since the traditional gold standard method, agar overlay, takes minimum 18 hours to be completed. An alternative approach is quantitative real-time polymerase chain reaction (qPCR) that requires approximately 4 hours (Anderson et al., 2011). This method allows for the quantification of intracellular and extracellular phages (Lindell et al., 2007). Furthermore, it is also possible to count phages to which their host cannot grow on solid agar plates (Refardt, 2012). The purpose of this project is to validate the qPCR method for the quantification of environmental phages. The project is divided into 2 phases: first, the technique will be optimised using Escherichia coli as host and the phage T4. Second, qPCR will be tested with environmental samples.

Methodology:

In the development of this project, the student will have the opportunity to learn classical microbiological methods, including agar overlay method, most probable number (MPN), dilution methods, cultivation and phage maintenance. The student will also learn molecular methods like DNA extraction, gel electrophoresis and qPCR.

 

References / Bibliography:

Anderson, B., Rashid, M. H., Carter, C., Pasternack, G., Rajanna, C., Revazishvili, T., … Sulakvelidze, A. (2011). Enumeration of bacteriophage particles: Comparative analysis of the traditional plaque assay and real-time QPCR- and nanosight-based assays. Bacteriophage, 1(2), 86–93. doi.org/10.4161/bact.1.2.15456 [1]

Lindell, D., Jaffe, J.D., Coleman, M.L., Futschik, M.E., Axmann, I.M., Rector, T., Kettler, G., Sullivan, M.B., Steen, R.,Hess, W.R., Church, G.M.,Chisholm, S.W. (2007) Genome-wide expression dynamics of a marine virus and host reveal features of co-evolution. Nature 449,83 Doi:10.1038/nature06130

Refardt, D. (2012). Real-time quantitative PCR to discriminate and quantify lambdoid bacteriophages of Escherichia coli K-12. Bacteriophage, 2(2), 98–104. doi.org/10.4161/bact.20092 [2]

Kontakt / Contact

Dr. rer. nat. Jimena Barrero Canosa
(+49) 30 314 26827
Raum BH-N 605
E-Mail-Anfrage [3]
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