| XIE Guosi,HUANG Jie,ZHANG Qingli,HAN Nana,SHI Chengyin,WANG Xiuhua. 2012. Development and validation of a TaqManTM fluorescent quantitative real-time PCR assay for the rapid detection of Edwardsiella tarda. Acta Oceanologica Sinica, (4):140－148 |
| Development and validation of a TaqManTM fluorescent quantitative real-time PCR assay for the rapid detection of Edwardsiella tarda |
| Development and validation of a TaqManTM fluorescent quantitative real-time PCR assay for the rapid detection of Edwardsiella tarda |
| Received:December 16, 2011 Revised:April 22, 2012 |
| DOI：10.1007/s13131-012-0227-7 |
| Key words:Edwardsiella tarda TaqMan real-time PCR detection 16S rDNA |
| 中文关键词: Edwardsiella tarda TaqMan real-time PCR detection 16S rDNA |
| 基金项目:The Special Fund for Agro-scientific Research in the Public Interest under contract No. 201103034; Construction Special Fund of Modern Agriculture and Industrial Technology Research System under contract No.CARS-47. |
| Author Name | Affiliation | E-mail | | XIE Guosi | Key Laboratory of Marine Fishery Resources Sustainable Utilization, Ministry of Agriculture, Yellow Sea Fisheries Research Institute, Chinese Academy of Fishery Sciences, Qingdao 266071, China
College of Fisheries and Life Science, Shanghai Ocean University, Shanghai 201306, China | | | HUANG Jie | Key Laboratory of Marine Fishery Resources Sustainable Utilization, Ministry of Agriculture, Yellow Sea Fisheries Research Institute, Chinese Academy of Fishery Sciences, Qingdao 266071, China
College of Fisheries and Life Science, Shanghai Ocean University, Shanghai 201306, China | HuangJie@ysfri.ac.cn | | ZHANG Qingli | Key Laboratory of Marine Fishery Resources Sustainable Utilization, Ministry of Agriculture, Yellow Sea Fisheries Research Institute, Chinese Academy of Fishery Sciences, Qingdao 266071, China | | | HAN Nana | Key Laboratory of Marine Fishery Resources Sustainable Utilization, Ministry of Agriculture, Yellow Sea Fisheries Research Institute, Chinese Academy of Fishery Sciences, Qingdao 266071, China | | | SHI Chengyin | Key Laboratory of Marine Fishery Resources Sustainable Utilization, Ministry of Agriculture, Yellow Sea Fisheries Research Institute, Chinese Academy of Fishery Sciences, Qingdao 266071, China | | | WANG Xiuhua | Key Laboratory of Marine Fishery Resources Sustainable Utilization, Ministry of Agriculture, Yellow Sea Fisheries Research Institute, Chinese Academy of Fishery Sciences, Qingdao 266071, China | |
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| Abstract: |
| 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 FQPCR 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 (log10nc as x; nc 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. |
| 中文摘要: |
| 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 FQPCR 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 (log10nc as x; nc 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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