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Published on: March 23, 2018
Evaluation of methods of DNA extraction from Staphylococcus aureus in milk for use in real-time PCR
A G Dibbern1, B G Botaro1, M P Viziack1
1Departamento de Nutrição e Produção Animal, Faculdade de Medicina Veterinária e Zootecnia, Universidade de São Paulo, Pirassununga, SP, Brasil.
Abstract:
The aim of this study was to evaluate the repeatability and performance of 4 methods of extracting DNA from Staphylococcus aureus (SAU) and the gene encoding bovine mitochondrial cytochrome B (BMCB) in milk samples from cows with subclinical mastitis for use in amplification by real-time polymerase chain reaction. Two milk samples were obtained from cows naturally infected with S. aureus and subjected to the following extraction methods: Qiagen DNA extraction kit; Axyprep DNA extraction kit; in silica column boil and in silica column method. After extraction in duplicate, eluates were subjected to purification and precipitation to determine purity (A₂₆₀/A₂₈₀ ratio) and concentration (μg/μL) by spectrophotometry and amplification by real-time polymerase chain reaction of target genes (SAU and BMCB). There was no effect of the DNA extraction method on DNA concentration and threshold cycle for BMCB and SAU. The purity ratio (A₂₆₀/A₂₈₀ ) was higher when using Qiagen DNA extraction (1.76 ± 0.136) compared to the other methods tested. Our results indicate that the DNA extraction kit from Qiagen produces samples of the highest purity ratio compared to other methods.
Insights
The Qiagen DNA extraction kit yielded the highest purity for Staphylococcus aureus and bovine mitochondrial cytochrome B DNA from mastitis milk samples. This method is recommended for real-time PCR applications.
Area of Science:
- Veterinary Microbiology
- Molecular Biology
- Biochemistry
Background:
- Subclinical mastitis in cows is often caused by Staphylococcus aureus (SAU).
- Accurate DNA extraction from milk is crucial for detecting SAU and host genes like bovine mitochondrial cytochrome B (BMCB) using real-time PCR.
- Evaluating different DNA extraction methods is essential for optimizing diagnostic sensitivity and specificity.
Purpose of the Study:
- To compare the performance and repeatability of four DNA extraction methods.
- To assess DNA purity and concentration for SAU and BMCB genes.
- To determine the optimal method for real-time PCR amplification in bovine mastitis milk samples.
Main Methods:
- DNA was extracted from S. aureus-infected milk using Qiagen, Axyprep kits, and two in-house silica column methods.
- Extracted DNA purity (A260/A280 ratio) and concentration were measured via spectrophotometry.
- Real-time PCR was used to amplify SAU and BMCB target genes.
Main Results:
- No significant difference in DNA concentration or real-time PCR threshold cycles was observed among the methods for SAU and BMCB.
- The Qiagen DNA extraction kit resulted in a significantly higher purity ratio (1.76 ± 0.136) compared to other methods.
- DNA purity is a critical factor for reliable downstream molecular analysis.
Conclusions:
- The Qiagen DNA extraction kit provides superior DNA purity for detecting Staphylococcus aureus and bovine mitochondrial cytochrome B in milk samples.
- While all tested methods yielded quantifiable DNA, the Qiagen kit is recommended for its higher purity, potentially improving real-time PCR assay robustness.
- Optimized DNA extraction is key for accurate diagnosis and monitoring of bovine mastitis.
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