Evaluation of methods of DNA extraction from Staphylococcus aureus in milk for use in real-time PCR
Evaluation of methods of DNA extraction from Staphylococcus aureus in milk for use in real-time PCR
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DOI：
10.4238/2015.january.16.6
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发表时间：
2015-01-01
影响因子：
0.4
通讯作者：
Santos, M. V.
中科院分区：
文献类型：
--
作者：
Dibbern, A. G.;Botaro, B. G.;Santos, M. V.
本研究的目的是评估从患有亚临床乳腺炎的奶牛的牛奶样本中提取金黄色葡萄球菌 (SAU) 和编码牛线粒体细胞色素 B (BMCB) 的基因的 4 种方法的重复性和性能，用于通过实时聚合酶链反应进行扩增。从自然感染金黄色葡萄球菌的奶牛中获取两份牛奶样品，并进行以下提取方法：Qiagen DNA提取试剂盒； Axyprep DNA 提取试剂盒；二氧化硅柱沸腾法和二氧化硅柱法。重复提取后，对洗脱液进行纯化和沉淀，通过分光光度法测定纯度（A（260）/A（280）比）和浓度（μg/μL），并通过目标基因（SAU和BMCB）的实时聚合酶链反应进行扩增。 DNA 提取方法对 BMCB 和 SAU 的 DNA 浓度和阈值循环没有影响。与其他测试方法相比，使用 Qiagen DNA 提取时的纯度比 (A(260)/A(280)) 更高 (1.76 +/- 0.136)。我们的结果表明，与其他方法相比，Qiagen 的 DNA 提取试剂盒产生的样品纯度最高。
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 (A(260)/A(280) ratio) and concentration (mu g/ mu L) 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 (A(260)/A(280)) 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.