Dataset Information

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.

SUBMITTER: Zhao Y

PROVIDER: S-EPMC6450196 | biostudies-other | 2019

REPOSITORIES: biostudies-other

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

Zhao Yankun Y Chen He H Liu Huimin H Cai Jianxing J Meng Lu L Dong Lei L Zheng Nan N Wang Jiaqi J Wang Cheng C

Frontiers in microbiology 20190329

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 < ...[more]

PMID: 30984156

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Project description:The number and type of synthetic chemicals that are being produced worldwide continues to increase significantly. While these industrial chemicals provide numerous benefits, there is no doubt that some have potential to damage the environment and health. Toxicity must be evaluated and use must be carefully controlled and monitored in order to minimize potential damage. DNA microarray technology has become an important new technique in toxicology. We are using the yeast Saccharomyces cerevisiae as a model organism for toxicological study because it is a simple, fast-growing eukaryote that has been thoroughly characterized. In order to evaluate toxicity by newly synthesized or mixture chemicals, toxicity-induced gene expression alteration profiles by known chemicals should be collected. In our study, cells need to be exposed with same experimental cellular condition, semi lethal (IC50), respectively. In the case of SDS (CAS; 151-21-3), the exposure dose was decided as 0.01% by growth curve with continuously diluted exposure. SDS is an anionic surfactant that is suspected to be gastrointestinal or liver toxicant. // Toxicity of anionic detergents determined by Saccharomyces cerevisiae microarray analysis: Sodium n-dodecyl benzene sulfonate (LAS) and sodium dodecyl sulfate (SDS) are popular anionic detergents (surfactants) that are used worldwide and the toxicities of these chemicals have been characterized. We applied these chemicals in a DNA microarray bioassay and determined that the microarray data reflects previous findings and also provides some new information about anionic detergent toxicity. The mRNA expression profiles suggest that LAS and SDS cause damage to membranes and alterations in carbon metabolism, and induce the oxidative stress response. We also found that LAS and SDS induce the pleiotropic drug-resistance network, and that LAS and SDS may be pumped out of yeast cells by this network. Hierarchical clustering of the expression profiles showed that LAS and SDS cause similar features of toxicity and that the toxicity is similar to that of capsaicin but different from that of cadmium and mercury. Keywords: stress response

Dataset Information

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

Publications

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

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