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

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

doi:10.3389/fmicb.2019.00661

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 (R2 = 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.

Highlights

Streptococcus agalactiae is a Gram-positive, pathogenic bacterium that commonly cause subclinical mastitis in dairy cattle (Santos et al, 2013)
The results showed that amplification of cfb was positive only in S. agalactiae strains, and no amplified signals were observed from DNA of Cronobacter sakazakii, Bacillus cereus, Escherichia coli, Shigella flexneri, Cronobacter muytjensii, Salmonella enterica, Lactobacillus plantarum, Enterococcus faecalis, Staphylococcus aureus, and Streptococcus salivarius
The results indicated that the primers were highly specific for S. agalactiae, with no cross-reactivity to non-target bacteria

Summary

Introduction

Streptococcus agalactiae is a Gram-positive, pathogenic bacterium that commonly cause subclinical mastitis in dairy cattle (Santos et al, 2013). The naked DNA can persist in the environment long after cell death, qPCR, and M-qPCR generate high false-positive rates due to DNA persistence after cell death and overestimate infection risks (Niu et al, 2018). To overcome this limitation, DNA-intercalating dyes such as propidium monoazide (PMA) are a promising way to selectively detect viable cells. The PMA forms covalent bonds with DNA under intense light, PCR amplification of such modified DNA is strongly inhibited, allowing unbound DNA from viable cells to be amplified and detected in subsequent PCR (Nocker et al, 2006, 2009)

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