A major-capsid-protein-based multiplex PCR assay for rapid identification of selected virulent bacteriophage types

Abstract

Bacteriophages represent a promising alternative for controlling pathogenic bacteria. They are ubiquitous in the environment, and their isolation is usually simple and fast. However, not every phage is suitable for biocontrol applications. It must be virulent (i.e., strictly lytic), non-transducing, and safe. We have developed a method for identifying selected types of virulent phages at an early stage of the isolation process to simplify the search for suitable candidates. Using the major capsid protein (MCP) as a phylogenetic marker, we designed degenerate primers for the identification of Felix O1-, GJ1-, N4-, SP6-, T4-, T7-, and Vi1-like phages in multiplex PCR setups with single phage plaques as templates. Performance of the MCP PCR assay was evaluated with a set of 26 well-characterized phages. Neither false-positive nor false-negative results were obtained. In addition, 154 phages from enrichment cultures from various environmental samples were subjected to MCP PCR analysis. Eight of them, specific for Salmonella enterica, Escherichia coli, or Erwinia amylovora, belonged to one of the selected phage types. Their PCR-based identification was successfully confirmed by pulsed-field gel electrophoresis of the phage genomes, electron microscopy, and sequencing of the amplified mcp gene fragment. The MCP PCR assay was shown to be a simple method for preliminary assignment of new phages to a certain group and thus to identify candidates for biocontrol immediately after their isolation. Given that sufficient sequence data are available, this method can be extended to any phage group of interest.

Fig. 1

Dotplot analysis of MCP sequences used for the primer design. The order of the phages is according to Table 1

Fig. 2

Phylogenetic analysis of novel phages. New MCP sequences were aligned with those used for primer design. A neighbor-joining tree was constructed with 1,000 bootstrap replicates. Branches with values < 50% were collapsed. Nodes are colored according to phage groups. Purple, N4; grey, Vi1; blue, T4; yellow, GJ1; green, SP6; brown, FO1; red, T7. Open nodes indicate novel phages