Detection of lactococcal 936-species bacteriophages in whey by magnetic capture hybridization PCR targeting a variable region of receptor-binding protein genes

Abstract

Aims: To develop PCR assays able to distinguish between groups within lactococcal 936-species bacteriophages, as defined by their different receptor-binding protein (RBP) genes. Methods and Results: DNA sequences of RBP genes from 17 lactococcal bacteriophages of the 936-species were compared, and six phage groups were identified. For each phage group a specific primer pair targeting a variable region of the RBP genes was designed. In nine of 20 whey samples, from dairies with recorded phage problems, between one and six phage groups were identified by conventional PCR. The sensitivity and specificity of the method was improved by magnetic capture hybridization (MCH)-PCR using a capture probe targeting an 80-bp highly conserved region just upstream from the RBP gene in all the investigated phages. The MCH-PCR was performed on 100 ll whey samples and the detection limit of the assay was 10 2 -10 3 PFU ml )1 as opposed to the detection limit of 10 4 PFU ml )1 for conventional PCR performed on 1-ll whey samples. Conclusions:In this study, PCR assays have been developed to detect six different types of RBP genes in lactococcal 936-species bacteriophages.Significance and Impact of the Study: The PCR assays have practical applications at cheese plants for detection of 936-species phages with different RBP and thereby potentially with different host ranges. This knowledge will make it possible to improve starter culture rotation systems in the dairy industry.

Receptor-Binding Protein of Lactococcus lactis Phages: Identification and Characterization of the Saccharide Receptor-Binding Site

“…Recently, Dupont et al (13) reported six different types of RBP genes of lactococcal 936 species bacteriophages and developed a PCR assay to detect them. They suggested that the PCR assay could be used in cheese plants for phage detection.…”

Multiplex Fast Real-Time PCR for Quantitative Detection and Identification of cos - and pac -Type Streptococcus thermophilus Bacteriophages

“…(8), but these methods are time-consuming. To speed up the analysis, PCR techniques have been used to detect phages in different kinds of dairy samples (1,4,6,7,10,12). Increasing demand for quantitative, more sensitive, and quicker procedures is prompting the development of real-time quantitative PCR (qPCR) methods.…”

Bacteriophages in Dairy Industry: PCR Methods as Valuable Tools

“…Special care must be taken when dairy samples are used directly as templates, because the presence of inhibitors may interfere with the PCR amplification and leads false negative results (Wilson, 1997;Ercolini et al, 2004). Magnetic capture hybridization (Dupont et al, 2005) or a previous DNA isolation from the sample, as is described for detecting pathogens in dairy samples (Cremonesi et al, 2007), are some of the additional steps that could be included in the PCR methods to remove PCR inhibitors and/or reduce the effect of the components of the dairy matrix.…”

“…Moreover, several protocols have been published to detect and identify the three species of Lc. lactis bacteriophages most frequently found in milk plants (c2, 936 and 335), in cheese whey samples (Labrie & Moineau, 2000;Dupont et al, 2005;Deveau et al, 2006;Szczepańska et al, 2007;Suárez et al, 2008;Kleppen et al, 2011), milk (del Río et al, 2007 and even in the factory equipment (Verreault et al, 2011;Kleppen et al, 2011) or air samples (Verrault et al, 2011).…”

“…The technique has been adapted to detect phages in milk samples (Binetti et al, 2005;Dupont et al, 2005;del Río et al, 2007), in cheese whey (Brüssow et al, 1994;Labrie and Moineau, 2000), in the equipment within the factory (Verreault et al, 2011, Kleppen et al, 2011 or even from air samples (Verrault et al, 2011) with a detection limit of 10 3 -10 4 phage particles per milliliter. S. thermophilus bacteriophages have been identified in industry milk fermentation samples as yogurt and cheese whey, after phage DNA extraction and subsequent PCR (Brüssow et al, 1994) or by direct PCR of milk (Binetti et al, 2005;Dupont et al, 2005, del Río et al, 2007. A PCR protocol to detect Lb.…”