Direct Quantitative Detection and Identification of Lactococcal Bacteriophages from Milk and Whey by Real-Time PCR: Application for the Detection of Lactococcal Bacteriophages in Goat's Raw Milk Whey in France

Abstract

The presence of Lactococcus bacteriophages in milk can partly or completely inhibit milk fermentation. To prevent the problems associated with the bacteriophages, the real-time PCR was developed in this study for direct detection from whey and milk of three main groups of Lactococcus bacteriophages, c2, 936, and P335. The optimization of DNA extraction protocol from complex matrices such as whey and milk was optimized allowed the amplification of PCR without any matrix and nontarget contaminant interference. The real-time PCR program was specific and with the detection limit of 10(2) PFU/mL. The curve slopes were -3.49, -3.69, and -3.45 with the amplification efficiency estimated at 94%, 94%, and 98% and the correlation coefficient (R(2)) of 0.999, 0.999, and 0.998 for c2, 936 and P335 group, respectively. This method was then used to detect the bacteriophages in whey and goat's raw milk coming from three farms located in the Rhône-Alpes region (France).

Figure 1

Melting curve analyses of the amplified PCR product of c2 (a) and the curve represents fluorescent changes over cycles of a 10-fold serial dilution of c2 (b).

Figure 2

Standard curves for bacteriophage c2 using SYBR in the real-time PCR.

Figure 3

The real-time PCR of c2 at different concentrations on agarose gel. Lane 1: DNA ladder (pb). Lane 2: negative control. Lane 3: positive control (10⁹ UFP/mL of c2 amplified by conventional PCR). Lane 4–13: concentration of c2 from 10⁹ to 10⁰ UFP/mL.

Figure 4

Amplification of DNA bacteriophages in the sample containing c2 alone at 10⁵ UFP/mL (1) and in the sample mixed of c2 at 10⁵ UFP/mL (2) with 10⁷ UFP/mL of 936 (3) and with 10⁷ UFP/mL of P335 (4).

Figure 5

The PCR products of the sample containing the phage alone at 10⁵ UFP/mL and in the sample mixed of three phages. Lane 1: DNA ladder (pb). Lane 2: c2 alone. Lane 3: c2 in the sample mixing three phages. Lane 4: 936 alone. Lane 5: 936 in the sample mixing three phages. Lane 6: P335 alone Lane 7: P335 in the sample mixing three.

Figure 6

Comparison of standard curves of pure culture and whey artificially contaminated with bacteriophages c2 (a), 936 (b), and P335 (c). Analysis of medium Ct value obtained in relation to bacteriophages concentrations log (PFU/mL).

Figure 7

Detection and quantification of three main groups of bacteriophages of Lactococcus in whey of goat's milk collected on different days (LS) of three farms in Rhône-Alpes: B2, B6 and B7.

Figure 8

Detection and quantification of three main groups of bacteriophages of Lactococcus in goat's raw milk collected on different days (L) on three farms in Rhône-Alpes: B2, B6 and B7.