| Development and validation of a TaqManTM fluorescent quantitative real-time PCR assay for the rapid detection of Edwardsiella tarda |
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| 作者姓名： | XIE Guosi HUANG Jie ZHANG Qingli HAN Nan SHI Chengyin WANG Xiuhua |
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| 作者单位： | 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,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,Key Laboratory of Marine Fishery Resources Sustainable Utilization, Ministry of Agriculture, Yellow Sea Fisheries Research Institute, Chinese Academy of Fishery Sciences, Qingdao 266071, China,Key Laboratory of Marine Fishery Resources Sustainable Utilization, Ministry of Agriculture, Yellow Sea Fisheries Research Institute, Chinese Academy of Fishery Sciences, Qingdao 266071, China,Key Laboratory of Marine Fishery Resources Sustainable Utilization, Ministry of Agriculture, Yellow Sea Fisheries Research Institute, Chinese Academy of Fishery Sciences, Qingdao 266071, China,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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| 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. |
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| 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 （log10 nc 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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| 关 键 词： | 迟钝爱德华氏菌 PCR检测 快速检测 实时定量 TaqMan探针 rDNA序列 水产养殖业 检测灵敏度 |
| 收稿时间： | 2011/12/16 0:00:00 |
| 修稿时间： | 2012/4/22 0:00:00 |
Development and validation of a TaqManTM fluorescent quantitative real-time PCR assay for the rapid detection of Edwardsiella tarda |
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| XIE Guosi,HUANG Jie,ZHANG Qingli,HAN Nan,SHI Chengyin,WANG Xiuhua.Development and validation of a TaqManTM fluorescent quantitative real-time PCR assay for the rapid detection of Edwardsiella tarda[J].Acta Oceanologica Sinica,2012,31(4):140-148. |
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| Authors: | XIE Guosi HUANG Jie ZHANG Qingli HAN Nan SHI Chengyin and WANG Xiuhua |
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| Institution: | 1.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, China2.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. |
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| Keywords: | Edwardsiella tarda TaqMan real-time PCR detection 16S rDNA |
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