基于recA基因的SYBR Green I Real-time qPCR与RAA-LFD检测鳗败血假单胞菌方法的建立与应用

Establishment and application of SYBR Green I Real-time qPCR and RAA-LFD based on recA gene for detection of Pseudomonas anguilliseptica

  • 摘要: 为对鳗败血假单胞菌 (Pseudomonas anguilliseptica, PA) 的早期感染进行快速诊断,基于recA基因建立了SYBR Green I实时荧光定量PCR (SYBR Green I Real-time quantitative PCR, qPCR) 和重组酶介导等温扩增结合侧流层析试纸条 (Recombinase-mediated isothermal amplification combined with lateral flow dipstick, RAA-LFD) 2种检测方法。以PA的管家基因recA为靶标,设计筛选出1对qPCR特异性引物、1对RAA特异性引物和RAA探针,并通过同源重组构建标准品质粒pUC18-recA,以建立2种检测方法。将建立的方法应用于检测PA感染的大口黑鲈 (Micropterus salmoides) 组织样本,并测定PA载量。建立的qPCR方法最低DNA检测浓度为2.816×102 拷贝·μL−1,模板量与Ct值在构建的标准曲线中具有很好的线性关系 (r2=0.999 2),且具有较强的特异性和稳定性;建立的RAA-LFD方法最低DNA检测浓度为2.816×104 拷贝·μL−1,检测时效最快达15 min,且显色较为稳定、特异性强。应用结果显示,qPCR方法和RAA-LFD方法阳性样本检出率分别为87.50%和85.00%,相较普通PCR方法有明显提高;建立的qPCR方法可准确测定PA感染宿主组织中的菌体载量,其中肾中的载量最高,达3.533×107 拷贝·ng−1。建立的2种方法特异性较好,其中qPCR灵敏性更高,RAA-LFD则时效性更强,两者均可用于PA早期感染的检测,且建立的qPCR方法还可对感染宿主体内的菌体载量进行定量分析。

     

    Abstract: For rapid diagnosis of early infection with Pseudomonas anguilliseptica (PA), we established SYBR Green I Real-time quantitative PCR (qPCR) and recombinase-mediated isothermal amplification combined with lateral flow dipstick (RAA-LFD) based on recA gene for rapid and specific detection. A pair of qPCR specific primers, a pair of RAA specific primers and a RAA probe were designed and screened based on the recA gene of PA. The standard quality plasmid pUC18-recA was constructed by homologous recombination to establish the two detection methods. The established methods were applied to detect PA-infected largemouth bass (Micropterus salmoides) tissue samples and PA load was determined. The minimum DNA detection concentration of the qPCR method was 2.816×102 copies·μL−1. There was a good linear relationship between the template amount and Ct value in the standard curve (r2=0.999 2), and the method had strong specificity and stability. The minimum DNA detection concentration of the RAA-LFD method was 2.816×104 copies·μL−1. The detection time of the RAA-LFD method was 15 min, and the color development was relatively stable and the specificity was strong. The application results show that the positive detection rates of qPCR and RAA-LFD were 87.50% and 85.00%, respectively, which was significantly higher than that of the common PCR method. The established qPCR method could accurately measure the bacterial load in the tissues of PA infected hosts. The highest bacterial load was found in the kidney (3.533×107 copies·ng−1). Both methods can be used for rapid detection of early PA infection. The established qPCR can also be used to quantitatively analyze the bacterial load in infected hosts.

     

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