Multiplex PCR for detection and identification of lactococcal bacteriophages

Abstract

Three genetically distinct groups of Lactococcus lactis phages are encountered in dairy plants worldwide, namely, the 936, c2, and P335 species. The multiplex PCR method was adapted to detect, in a single reaction, the presence of these species in whey samples or in phage lysates. Three sets of primers, one for each species, were designed based on conserved regions of their genomes. The c2-specific primers were constructed using the major capsid protein gene (mcp) as the target. The mcp sequences for three phages (eb1, Q38, and Q44) were determined and compared with the two available in the databases, those for phages c2 and bIL67. An 86.4% identity was found over the five mcp genes. The gene of the only major structural protein (msp) was selected as a target for the detection of 936-related phages. The msp sequences for three phages (p2, Q7, and Q11) were also established and matched with the available data on phages sk1, bIL170, and F4-1. The comparison of the six msp genes revealed an 82. 2% identity. A high genomic diversity was observed among structural proteins of the P335-like phages suggesting that the classification of lactococcal phages within this species should be revised. Nevertheless, we have identified a common genomic region in 10 P335-like phages isolated from six countries. This region corresponded to orfF17-orf18 of phage r1t and orf20-orf21 of Tuc2009 and was sequenced for three additional P335 phages (Q30, P270, and ul40). An identity of 93.4% within a 739-bp region of the five phages was found. The detection limit of the multiplex PCR method in whey was 10(4) to 10(7) PFU/ml and was 10(3) to 10(5) PFU/ml with an additional phage concentration step. The method can also be used to detect phage DNA in whey powders and may also detect prophage or defective phage in the bacterial genome.

FIG. 1

Protein profiles of 936-related phages as determined by SDS–15% PAGE. The gel was stained with Coomassie blue. Lane 1, phage p2; lane 2, Q7; lane 3, Q11; lane 4, Q42; lane 5, Q46; lane M, prestained molecular mass markers (New England Biolabs). Arrow, major structural protein used for N-terminal sequencing and encoded by the msp gene.

FIG. 2

Nucleotide sequence alignment of the gene coding for the major structural proteins of six members of the 936 species. The sequence differences are indicated by boldface. The start and nonsense codons are underlined. The regions selected for the PCR primers are indicated by a double underline (936A and 936B).

FIG. 3

SDS–15% PAGE of CsCl-purified P335-related phages. Lane 1, Q30; lane 2, φ31; lane 3, P270; lane 4, ul36; lane M, prestained molecular mass markers (New England Biolabs).

FIG. 4

Multiplex PCR competition assay for different combinations of phage species in the same sample. Phage Q7 represents the 936 species, Q30 represents the P335 species, and Q38 represents the c2 species. Reactions were carried out with 1.25 (A and C) and 2.50 (B and D) U of Taq DNA polymerase. Lanes (boldface, phage concentration of 10⁸ PFU/ml; lightface, phage concentration of 10⁷ PFU/ml): 1, 14, 15, and 28, 100-bp DNA ladder (Gibco/BRL, Burlington, Ontario, Canada); 2, 936 plus c2; 3, 936 plus P335; 4, c2 plus P335; 5, 936 plus c2 plus P335; 6, 936 plus c2; 7, 936 plus P335; 8, 936 plus P335; 9, c2 plus P335; 10, 936 plus P335; 11, c2 plus P335; 12, 936 plus c2 plus P335; 13, 936 plus c2 plus P335; 16, 936 plus c2 plus P335; 17, 936 plus c2 plus P335; 18, 936 plus c2 plus P335; 19, 936 plus c2 plus P335; 20, 936 plus c2 plus P335; 21, 936; 22, c2; 23, P335; 24, 10 pg of 936 DNA; 25, 10 pg of c2 DNA; 26, 10 pg of P335 DNA; 27, negative control.