养殖沉积环境中气单胞菌属细菌的实时荧光定量PCR检测方法的建立

Establishment of the real-time fluorescent quantitative PCR method for detecting Aeromonas in sediment environment of aquaculture

中文关键词: GyrB基因  沉积环境  气单胞菌属  实时荧光定量PCR

英文关键词: GyrB gene  Sediment environment of aquaculture  Aeromonas  Real-time fluorescent quantitative PCR

中文摘要:

      利用GyrB基因的特异性引物建立沉积环境中气单胞菌属细菌的实时荧光定量PCR检测方法。将已知浓度的气单胞菌菌液加入灭菌的沉积物样品中，作为模拟沉积物样品。通过选择和优化沉积物DNA提取方法、特异性引物、标准曲线模板，建立沉积环境中气单胞菌属细菌准确检验的实时荧光定量PCR方法，同时验证该方法的特异性、灵敏性、重复性。结果表明，采用改进的溶菌酶-SDS温和裂解法提取沉积物DNA，以扩增GyrB基因片段的IAF和IAR为特异性引物，并直接以模拟沉积物样品DNA为标准品构建标准曲线，可以建立适用于定量检测沉积环境中气单胞菌属细菌的实时荧光定量PCR方法。该方法可灵敏、特异、准确地定量检测刺参养殖池塘底泥中气单胞菌属中不同种的细菌，检出效率可达103 CFU/g。统计分析显示，变异系数为0.21%-0.80%，均小于5%，表明重复性良好。

英文摘要:

      Aeromonas is one of the pathogens causing skin ulcer syndrome in cultured Apostichopus japonicas. It is important to monitor the population of these bacteria in the culture environment for disease control. Unfortunately, current real-time quantitative PCR detection method is mostly based on the pure culture and cannot truly reflect the bacteria population in the sediment environment. Therefore, we established a real-time fluorescent quantitative PCR method to improve the pathogen detection. The simulative sediment samples were made by adding known-concentration bacteria into the sterilyzed sediment; the DNA extraction method was selected by comparing three improved DNA extraction methods; genus-specific primers were selected based on the GyrB gene of Aeromonas. Meanwhile, PCR reaction conditions and systems were optimized and a standard curve based-on extracted sediment DNA was utilized to examine the bacteria in the sediment. The results indicated that the modified lysozyme-SDS gentle lyse method and genus-specific primers (IAF and IAR based on GyrB gene of Aeromonas) were efficient and unique to detect Aeromonas. In addition, the standard curve was set up by using the extracted DNA from the simulated sediment samples as template. The new real-time fluorescent quantitative PCR protocol was highly sensitive and specific to detect different species of Aeromonas from the sediment with the quantification limit of 103 CFU/g. The coefficient of variation fell between 0.21% and 0.80% by statistical analysis (less than 5%), which demonstrated the good repeatability of the method. Thus, we have established a highly sensitive, specific, and reproducible method to measure Aeromonas in sediment environment of aquaculture even with low quantification.