Usıng bacterıophages ın the extractıon of Brucella DNA from mılk and culture samples and ıts evaluatıon by multıplex PCR

Abstract

Molecular methods have been progressing at a noticeably rapid rate recently. Obtaining the DNA required for most of these methods is also trying to catch up with this progress. Although many different kits and processes are utilized for DNA extraction, bacterial isolation is still accepted as a gold standard in the diagnosis of brucellosis. This study aims to create a novel way that can be used as an extraction method including bacteriophages. Bacteriophages that are specific to Brucella (B.) genus and species are used commonly in conventional identification and biotyping. However, using bacteriophages in the extraction of the DNA of the target bacteria has not been tried so far. In this study, DNA was extracted from Brucella culture and milk samples through the use of Izat Nagar (Iz) phage with 102–104 routine test dilution. These milk samples were artificially infected by Brucella suis bacteria with the amounts changing between 3.68×109 and 0.44×107 cfu/ml. Positive results were obtained from multiplex PCR by observing the B. suis profile with 7 bands. The methodology of the study consisted of 4 main phases; namely, first centrifugation, phage addition, incubation and the second centrifugation. When the effects of phages on viable bacteria are considered, it might be said that a method, which could be named ‘Phage-Exsen’, and which supports the gold standard, has been suggested. This method combines the advantages of the conventional method and molecular ones and forms a basis for prospective studies. Moreover, it illustrates the use of bacteriophages for DNA extraction in an unusual way, which might form a new perspective.