Evaluation of DNA extraction methods for use in combination with SYBR Green I real-time PCR to detect Salmonella enterica serotype enteritidis in poultry
Evaluation of DNA extraction methods for use in combination with SYBR Green I real-time PCR to detect Salmonella enterica serotype enteritidis in poultry
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DOI：
10.1128/aem.69.6.3456-3461.2003
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发表时间：
2003-06-01
影响因子：
4.4
通讯作者：
Toti, L
中科院分区：
文献类型：
--
作者：
De Medici, D;Croci, L;Toti, L
本研究的目的是建立一种快速、重复性好、稳定的方法检测家禽样品中的肠道沙门氏菌血清型。首先，对于从预富集培养物中提取和纯化DNA，比较了四种方法（煮沸、碱裂解、Nucleospin和Dynabeads DNA Direct System 1）。然后将最有效的方法与基于与ABI Prism 7700系统一起使用的双链DNA结合染料SYBR绿色I的实时PCR方法组合。通过获得的扩增子的解链温度（T-m）确定反应的特异性。实验在实验性地被肠杆菌血清型污染的鸡样品和市售家禽样品上进行，其也用于与标准培养方法（即，ISO 6579/2001）。四种DNA提取方法的比较结果显示，除煮沸法和Nucleospin法（产生最低阈值循环的两种方法）的结果外，其他方法之间存在显著差异。选择煮沸作为首选提取方法，因为它最简单、最快速。然后将该方法与SYBR绿色I实时PCR结合，使用引物SEFA-1和SEFA-2。通过T-m证实了反应的特异性，T-m对所得扩增子始终具有特异性;用血清型肠炎沙门氏菌特异性曲线获得的平均峰T-m为82.56 +/-0.22 ℃。使用平均阈值循环和不同浓度的血清型肠杆菌（范围为10（3）至10（8）CFU/ml）构建的标准曲线显示良好的线性（R-2 = 0.9767）和小于10（3）CFU/ml的灵敏度限值。本研究的结果表明，SYBR绿色I实时荧光定量PCR构成了一种有效的和易于执行的方法，用于检测家禽样品中的血清型肠炎。
The objective of this study was to develop a rapid, reproducible, and robust method for detecting Salmonella enterica serotype Enteritidis in poultry samples. First, for the extraction and purification of DNA from the preenrichment culture, four methods (boiling, alkaline lysis, Nucleospin, and Dynabeads DNA Direct System 1) were compared. The most effective method was then combined with a real-time PCR method based on the double-stranded DNA binding dye SYBR Green I used with the ABI Prism 7700 system. The specificity of the reaction was determined by the melting temperature (T-m) of the amplicon obtained. The experiments were conducted both on samples of chicken experimentally contaminated with serotype Enteritidis and on commercially available poultry samples, which were also used for comparisons with the standard cultural method (i.e., ISO 6579/2001). The results of comparisons among the four DNA extraction methods showed significant differences except for the results from the boiling and Nucleospin methods (the two methods that produced the lowest threshold cycles). Boiling was selected as the preferred extraction method because it is the simplest and most rapid. This method was then combined with SYBR Green I real-time PCR, using primers SEFA-1 and SEFA-2. The specificity of the reaction was confirmed by the T-m, which was consistently specific for the amplicon obtained; the mean peak T-m obtained with curves specific for serotype Enteritidis was 82.56 +/- 0.22degreesC. The standard curve constructed using the mean threshold cycle and various concentrations of serotype Enteritidis (ranging from 10(3) to 10(8) CFU/ml) showed good linearity (R-2 = 0.9767) and a sensitivity limit of less than 10(3) CFU/ml. The results of this study demonstrate that the SYBR Green I real-time PCR constitutes an effective and easy-to-perform method for detecting serotype Enteritidis in poultry samples.