摘要
Edwardsiella tarda is one of the most important emerging pathogens in tile 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 （logl0n~ as x; n~ 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.
Edwardsiella tarda is one of the most important emerging pathogens in tile 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 （logl0n~ as x; n~ 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.
基金
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
