Quantitative Polymerase Chain Reaction Coupled With Sodium Dodecyl Sulfate and Propidium Monoazide for Detection of Viable Streptococcus agalactiae in Milk

Abstract

Streptococcus agalactiae is an important pathogen causing bovine mastitis. The aim of this study was to develop a simple and specific method for direct detection of S. agalactiae from milk products. Propidium monoazide (PMA) and sodium dodecyl sulfate (SDS) were utilized to eliminate the interference of dead and injured cells in qPCR. Lysozyme (LYZ) was adopted to increase the extraction efficiency of target bacteria DNA in milk matrix. The specific primers were designed based on cfb gene of S. agalactiae for qPCR. The inclusivity and exclusivity of the assay were evaluated using 30 strains. The method was further determined by the detection of S. agalactiae in spiked milk. Results showed significant differences between the SDS-PMA-qPCR, PMA-qPCR and qPCR when a final concentration of 10 mg/ml (R ² = 0.9996, E = 95%) of LYZ was added in DNA extraction. Viable S. agalactiae was effectively detected when SDS and PMA concentrations were 20 μg/ml and 10 μM, respectively, and it was specific and more sensitive than qPCR and PMA-qPCR. Moreover, the SDS-PMA-qPCR assay coupled with LYZ was used to detect viable S. agalactiae in spiked milk, with a limit of detection of 3 × 10³ cfu/ml. Therefore, the SDS-PMA-qPCR assay had excellent sensitivity and specificity for detection of viable S. agalactiae in milk.

Keywords: Streptococcus agalactiae; milk; propidium monoazide; qPCR; sodium dodecyl sulfate.

FIGURE 1

Optimization of SDS concentration. Samples of 8 × 10⁷ cfu/ml of S. agalactiae were treated with different SDS concentrations. Error bars indicate standard deviations. Values with the same letter indicate no significant differences (P > 0.05); different lower and upper case letters indicate significant differences at P < 0.05 and P < 0.01, respectively. Color version available online.

FIGURE 2

(A) The standard curve for S. agalactiae at 5 mg/ml of lysozyme. (B) The standard curve for S. agalactiae at 10 mg/ml of lysozyme. (C) The standard curve for S. agalactiae at 15 mg/ml of lysozyme. (D) The standard curve for S. agalactiae at 20 mg/ml of lysozyme.

FIGURE 3

Optimization of propidium monoazide (PMA) concentration that inhibits amplification of dead S. agalactiae cells. Samples were treated with 0, 10, 20, 30, 40, and 50 μM PMA. Error bars indicate standard deviations. Values with the same letter indicate no significant difference (P > 0.05); different lower and upper case letters indicate significant differences at P < 0.05 and P < 0.01, respectively. Color version available online.

FIGURE 4

Standard curve for S. agalactiae of SDS–PMA–qPCR assay, plotted values represented the mean value and standard deviations obtained from three triplicate tests. Cq = threshold cycle. Color version available online.

FIGURE 5

Quantification of viable and viable/dead mix of S. agalactiae cells by qPCR, PMA–qPCR, and SDS–PMA–qPCR methods. Milk samples inoculated with (I) 3 × 10³ cfu/ml of viable cells, (II) 3 × 10³ cfu/ml of viable cells and 3 × 10⁴ cfu/ml of dead cells, and (III) 3 × 10⁴ cfu/ml of viable cells and 3 × 10³ cfu/ml of dead cells. All tests were carried out in duplicate. Methods for the preparation of dead cells and mixed viable and dead cells are described in Materials and Methods. Values within the same group (I, II, and III) with different lower (a, b) and upper (A, B, C) case letters are significantly different by Duncan’s multiple range test at P < 0.05 and P < 0.01, respectively.