Open AccessTyping of bacteriophages by randomly amplified polymorphic DNA (RAPD)‐PCR to assess genetic diversity
Author(s) -
Gutiérrez Diana,
MartínPlatero Antonio M.,
Rodríguez Ana,
MartínezBueno Manuel,
García Pilar,
Martínez Beatriz
Publication year - 2011
Publication title -
fems microbiology letters
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.899
H-Index - 151
eISSN - 1574-6968
pISSN - 0378-1097
DOI - 10.1111/j.1574-6968.2011.02342.x
Subject(s) - biology , rapd , phage typing , genetics , typing , microbiology and biotechnology , dna , lactococcus lactis , lysogenic cycle , escherichia coli , bacteriophage , genetic diversity , bacteria , gene , lactic acid , population , demography , sociology
The recent boom in phage therapy and phage biocontrol requires the design of suitable cocktails of genetically different bacteriophages. The current methods for typing phages need significant quantities of purified DNA, may require a priori genetic information and are cost and time consuming. We have evaluated the randomly amplified polymorphic DNA (RAPD)‐PCR technique to produce unique and reproducible band patterns from 26 different bacteriophages infecting Staphylococcus epidermidis, Staphylococcus aureus, Lactococcus lactis, Escherichia coli, Streptococcus thermophilus, Bacillus subtilis and Lactobacillus casei bacterial strains. Initially, purified DNA and phage suspensions of seven selected phages were used as a template. The conditions that were found to be optimal 8 μM of 10‐mer primers, 3 μM magnesium oxalacetate and 5% dimethyl sulfoxide. The RAPD genomic fingerprints using a phage titer suspension higher than 10 9 PFU mL −1 were highly reproducible. Clustering by the Pearson correlation coefficient and the unweighted pair group method with arithmetic averages clustering algorithm correlated largely with genetically different phages infecting the same bacterial species, although closely related phages with a similar DNA restriction pattern were indistinguishable. The results support the use of RAPD‐PCR for quick typing of phage isolates and preliminary assessment of their genetic diversity bypassing tedious DNA purification protocols and previous knowledge of their sequence.