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首页> 外文期刊>Analytica chimica acta >Development and evaluation of a novel nucleic acid sequence-based amplification method using one specific primer and one degenerate primer for simultaneous detection of Salmonella Enteritidis and Salmonella Typhimurium
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Development and evaluation of a novel nucleic acid sequence-based amplification method using one specific primer and one degenerate primer for simultaneous detection of Salmonella Enteritidis and Salmonella Typhimurium

机译：一种新的基于核酸序列的扩增方法的开发和评估，该方法使用一种特异性引物和一种简并引物同时检测肠炎沙门氏菌和鼠伤寒沙门氏菌

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Salmonella Enteritidis and Salmonella Typhimurium are the most widespread causes of salmonellosis and gastrointestinal diseases worldwide. Thus, their simple and sensitive detection is significantly important in biosafety and point-of-care diagnostics. In that regard, although present nucleic acid-based attempts are mainly focused on the detection methods encompassing all Salmonella enterica members in a single reaction, serotypes other than S. Enteritidis and S. Typhimurium are clinically and epidemiologically rare to humans. Therefore, regarding high ribosomal RNA (rRNA) copy numbers in a cell, isothermal nucleic acid sequence-based amplification (NASBA) technique was employed for simple, sensitive and simultaneous detection of the bacteria. However, due to high sequence homology among 16S rRNA genes and consequently, very few specific regions, we developed a novel NASBA method called "single specific primer-NASBA or SSP-NASBA" in which the specificity of the antisense primer is sufficient to perform a specific NASBA reaction. Accordingly, we designed highly specific NASBA antisense and degenerate sense primers for a segment of 16S rRNA variable region by universal sequence alignment to simultaneously detect S. Enteritidis and S. Typhimurium. Meanwhile, the approach was successfully evaluated for various Salmonella as well as closely related non-Salmonella serovars. Specific and simultaneous detection of both bacteria was achieved with the designed primer set in a single reaction environment with a detection limit of less than 10 CFUs mL~(-1). The developed NASBA assay should facilitate the overall process and provide a simple, fast, specific and sensitive approach for molecular diagnostics of pathogens under various circumstances, e.g. outbreaks.
机译：肠炎沙门氏菌和鼠伤寒沙门氏菌是全世界沙门氏菌病和胃肠道疾病的最广泛原因。因此，它们的简单而灵敏的检测在生物安全和即时诊断中非常重要。在这方面，尽管当前基于核酸的尝试主要集中于在单个反应中涵盖所有肠炎沙门氏菌成员的检测方法，但除肠炎沙门氏菌和鼠伤寒沙门氏菌以外的血清型在临床和流行病学上对于人类都是罕见的。因此，关于细胞中核糖体RNA（rRNA）的高拷贝数，采用了基于等温核酸序列的扩增（NASBA）技术对细菌进行简单，灵敏和同时的检测。但是，由于16S rRNA基因之间的序列高度同源，因此几乎没有特定区域，我们开发了一种新颖的NASBA方法，称为“单特异性引物-NASBA或SSP-NASBA”，其中反义引物的特异性足以完成特定的NASBA反应。因此，我们通过通用序列比对设计了高度特异性的NASBA反义和简并引物，用于16S rRNA可变区片段，以同时检测肠炎沙门氏菌和鼠伤寒沙门氏菌。同时，该方法已成功评估了各种沙门氏菌以及密切相关的非沙门氏菌血清型。使用设计的引物组在单一反应环境中实现了对两种细菌的​​特异性和同时检测，检测极限小于10 CFUs mL〜（-1）。开发的NASBA测定法应有助于整个过程，并提供一种简单，快速，特异和灵敏的方法在各种情况下对病原体进行分子诊断，例如：爆发。

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