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- Title
Development of a qPCR platform for quantification of the five bacteriophages within bacteriophage cocktail 2 (BFC2).
- Authors
Duyvejonck, Hans; Merabishvili, Maya; Pirnay, Jean-Paul; De Vos, Daniel; Verbeken, Gilbert; Van Belleghem, Jonas; Gryp, Tessa; De Leenheer, Julie; Van der Borght, Kelly; Van Simaey, Leen; Vermeulen, Stefan; Van Mechelen, Els; Vaneechoutte, Mario
- Abstract
To determine phage titers accurately, reproducibly and in a non-laborious and cost-effective manner, we describe the development of a qPCR platform for molecular quantification of five phages present in bacteriophage cocktail 2 (BFC2). We compared the performance of this molecular approach, with regard to quantification and reproducibility, with the standard culture-based double agar overlay method (DAO). We demonstrated that quantification of each of the five phages in BFC2 was possible by means of qPCR, without prior DNA extraction, but yields were significantly higher in comparison to DAO. Although DAO is assumed to provide an indication of the number of infective phage particles, whereas qPCR only provides information on the number of phage genomes, the difference in yield (qPCR/DAO ratio) was observed to be phage-dependent and appeared rather constant for all phages when analyzing different (freshly prepared) stocks of these phages. While DAO is necessary to determine sensitivity of clinical strains against phages in clinical applications, qPCR might be a valid alternative for rapid and reproducible quantification of freshly prepared stocks, after initial establishment of a correction factor towards DAO.
- Subjects
POLYMERASE chain reaction; BACTERIOPHAGES; NUCLEIC acid isolation methods; BACTERIAL cultures; GENETIC techniques
- Publication
Scientific Reports, 2019, Vol 9, Issue 1, pN.PAG
- ISSN
2045-2322
- Publication type
Article
- DOI
10.1038/s41598-019-50461-0