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

Yankun Zhao; He Chen; Huimin Liu; Jianxing Cai; Lu Meng; Lei Dong; Nan Zheng; Jiaqi Wang; Cheng Wang

doi:10.3389/fmicb.2019.00661

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

Front Microbiol. 2019 Mar 29:10:661. doi: 10.3389/fmicb.2019.00661. eCollection 2019.

Authors

Yankun Zhao^{1

2

3}, He Chen^{1

2

3}, Huimin Liu^{1

2

3

4}, Jianxing Cai^{1

2

3}, Lu Meng⁴, Lei Dong⁴, Nan Zheng⁴, Jiaqi Wang⁴, Cheng Wang^{2

3}

Affiliations

¹ Institute of Quality Standard and Testing Technology for Agro-Products, Xinjiang Academy of Agricultural Sciences, Urumqi, China.
² Ministry of Agriculture and Rural Affairs-Laboratory of Quality and Safety Risk Assessment for Agro-Products, Urumqi, China.
³ Key Laboratory of Agro-Products Quality and Safety of Xinjiang, Urumqi, China.
⁴ Ministry of Agriculture Laboratory of Quality and Safety Risk Assessment for Dairy Products (Beijing), Institute of Animal Science, Chinese Academy of Agricultural Sciences, Beijing, China.

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.