Development and preliminary validation of a real-time RT-PCR based method targeting tmRNA for the rapid and specific detection of Salmonella

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• 作者：Sheila McGuinness^a ; ^{sheila.mcguinness@nuigalway.ie} ; Thomas Barry^a ; ^b ; ^{thomas.barry@nuigalway.ie} ; Justin O'Grady^a ; ¹ ; ^{j.ogrady@ucl.ac.uk}
• 关键词：Salmonella ; Real-time RT-PCR ; tmRNA ; ssrA gene ; Molecular diagnostics
• 刊名：Food Research International
• 出版年：2012
• 期刊代码：53_09639969
• 类别：abs
• 出版时间：March, 2012
• 卷：45
• 期：2
• 页码：989-992
• 文件大小：161 K

摘要

In this study, a real-time reverse transcriptase PCR (real-time RT-PCR) assay for the detection of Salmonella was designed and developed. The real-time RT-PCR assay targeted the multi-copy RNA, tmRNA, had an inclusivity and exclusivity of 100%and a sensitivity of 鈮?#xA0;1 cell equivalent. The assay was combined with culture enrichment and an initial validation was performed in accordance with ISO 16140: 2003. Culture enrichment required 18 h primary enrichment in buffered peptone water (BPW) and 6 h selective enrichment in Rappaport-Vassiliadis Soya Peptone Broth (RVS). The combined culture enrichment/real-time RT-PCR method had a relative specificity of 100%when compared to the traditional culture method. When compared to an assay targeting the ssrA gene, which encodes for tmRNA, an approximate 1000-fold increase in sensitivity of detection was observed. Targeting the multi-copy tmRNA transcript rather than its encoding gene improves assay sensitivity which should enable reduction in turn-around time of this alternative method for Salmonella testing.