Development and validation of a TaqMan (TM) fluorescent quantitative real-time PCR assay for the rapid detection of Edwardsiella tarda

文献类型： 外文期刊

第一作者： Xie Guosi

作者： Xie Guosi;Huang Jie;Zhang Qingli;Han Nana;Shi Chengyin;Wang Xiuhua;Xie Guosi;Huang Jie

作者机构： Chinese Acad Fishery Sci, Key Lab Marine Fishery Resources Sustainable Util, Minist Agr, Yellow Sea Fisheries Res Inst, Qingdao 266071, Peoples R China;Shanghai Ocean Univ, Coll Fisheries & Life Sci, Shanghai 201306, Peoples R China;Shanghai Ocean Univ, Col

关键词： Edwardsiella tarda;TaqMan;real-time PCR;detection;16S rDNA

期刊名称：ACTA OCEANOLOGICA SINICA （ 2020影响因子：1.431； 五年影响因子：1.445 ）

ISSN： 0253-505X

年卷期： 2012 年 31 卷 4 期

页码：

收录情况： SCI

摘要： Edwardsiella tarda is one of the most important emerging pathogens in the global aquaculture industries. As such, an accurate diagnosis and quantitative analytical methods are urgently needed for this bacterium. In this study, primers and a TaqMan probe specific to the conservative sequences of the 16S rRNA gene of E. tarda were designed. The concentration of primers and TaqMan probe were optimized to 200 nmol/L and 120 nmol/L, respectively. The detection sensitivity of the FQ-PCR assay was determined to be as low as five copies of the target. sequence per reaction using the pGEM-16S rDNA recombinant plasmid as a template, which was 100 times more sensitive than conventional PCR. A standard curve by plotting the threshold cycle values (y) against the common logarithmic copies (log(10)n(c) as x; n(c) is copy number) of pGEM-16S rDNA was generated. The results of intra- and inter-assay variability tests demonstrate that the established FQ-PCR method was highly reproducible. The assay was specific for E. tarda as it showed that there was no cross-reactivity to eight additional bacterial pathogen strains in aquaculture. Thus, the FQ-PCR, assay has the potential for diagnostic purposes and for other applications, especially for the rapid detection and quantification of low-grade E. tarda infections.

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