香鱼假单胞菌SYBR GreenⅠ荧光定量PCR检测方法的建立

Development of a SYBR GreenⅠreal-time PCR assay for specific detection of Pseudomonas plecoglossicida

为建立香鱼假单胞菌的快速定量检测方法,本研究根据Gen Bank中登录的rpo D基因序列设计了一对特异性引物,以该菌基因组DNA为模板通过PCR扩增其180 bp的rop D基因片段,并克隆于p MD18-T载体中,以纯化的重组质粒为标准品建立了香鱼假单胞菌SYBR GreenⅠ荧光定量PCR检测方法。结果显示在1.9×103拷贝/μL^1.9×108拷贝/μL范围内呈现良好的线性关系,相关系数为0.999,灵敏度可达1.9×103拷贝/μL;该方法对恶臭假单胞菌、荧光假单胞菌、创伤弧菌等病原菌DNA扩增结果均为阴性,特异性良好;组内和组间变异系数均小于2%,具有较高的重复性和稳定性。本研究建立的方法不仅能够快速检测香鱼假单胞菌,还能够对该病原菌感染的动态变化进行定量研究,为大黄鱼香鱼假单胞菌感染导致的内脏白点病的早期诊断及防控提供一个新的检测技术。

To develop a specific assay for detection of Pseudomonas plecoglossicida, the SYBR green I real-time PCR assay were established with a pair of primers targeting rpoD gene of P.plecoglossicida. Then, a standard curve was constructed with series dilution of the recombinant plasmids containing the rpoD gene. The correlation coefficient of standard curve constructed using the threshold cycle （CT） versus log10 copy numbers of standard plasmid samples showed a good linearity （R2=0.999）, and the minimum level of detection was 1.9×10^3 copies/μL. The specificity of the assay was confirmed by the lack of cross-amplification with DNA templates from P.putica, Vibrio alginolyticus, V.parahaemolyticus, Streptococcus iniae, Aeromonas hyclrophilia, V. v-uln- ificus, Shcwanella alga, P.fluorescens and Nocardia seriolae. The coefficients of variance （CV） were 1.29% and 1.70% for the intra- and inter-assay, respectively, which indicated a good reliability and reproducibility. This detection assay provided a rapid means to quantitatively accessing the presence of P.plecoglossicida, which can be a useful tool for the quantification and monitoring of this bacterial pathogen in farmed fish, as well as the diagnosis of the P.plecoglossicida-related fish disease.