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Detection of two Prorocentrum species using sandwich hybridization integrated with nuclease protection assay

机译：夹心杂交结合核酸酶保护检测法检测两种原中心种

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摘要

A novel assay method using nuclease protection assay integrated with sandwich hybridization (NPA-SH) for qualitative and quantitative detection of microalgae has been developed. Two species-specific nuclease-protection-assay (NPA) probes targeted 28S ribosomal RNA of Prorocentrum minimum and Prorocentrum micans, respectively, were designed in this study. The assay consists of S1 nuclease protection, sandwich hybridization and signal detection. The specificity of the probes was verified with cultured algae in the laboratory and field sample from Jiaozhou Bay, and the quantity by NPA-SH analysis showed good agreement with that of cell-counting with a light microscope. The optical absorbance of probe binding on the target showed good linear fit with cell amount. A standard curve for P. minimum was established to correlate the optical absorbance to cell density on a basis in the linear range between 15 and 475 cells ml~(-1) seawater, and the equation deducted was 'y = 0.0053 x x + 0.0658' (R~2 = 0.992, n = 4). The assay was sensitive to detect 15 cells ml~(-1) seawater. And for P. micans, with linear range between 0.6 and 20 cells ml~(-1) seawater, the equation deducted was 'y = 0.1174 x x + 0.1106' (R~2 = 0.996, n = 4); the assay was sensitive to detect less than 1 cell ml~(-1) seawater. The inter-assay coefficients of variation (CVs) were 12.4 and 10.9%, respectively. The good specificity, sensitivity and reproducibility of the NPA-SH implied that this new technique could be extremely useful for qualitative and quantitative assay of P. minimum and P. micans at low abundance.
机译：开发了一种使用核酸酶保护分析法与三明治杂交法（NPA-SH）相结合的新方法，用于定性和定量检测微藻。在这项研究中，分别设计了两种分别针对最小原中心和原原微生物的28S核糖体RNA的物种特异性核酸酶保护测定（NPA）探针。该检测方法包括S1核酸酶保护，夹心杂交和信号检测。在胶州湾的实验室和田间样品中用培养的藻类验证了探针的特异性，并且通过NPA-SH分析的数量与用光学显微镜计数的细胞显示出良好的一致性。探针与靶标结合的吸光度与细胞数量呈良好的线性拟合。建立了最小体育标准曲线，以在15和475个细胞ml〜（-1）海水之间的线性范围为基础，将光吸收与细胞密度相关联，推导的方程式为'y = 0.0053 xx + 0.0658' （R〜2 = 0.992，n = 4）。该测定法灵敏地检测出15个细胞ml〜（-1）的海水。对于线性变化范围在0.6到20个细胞ml〜（-1）海水中的P. micans，推导的方程为'y = 0.1174 x x + 0.1106'（R〜2 = 0.996，n = 4）；该检测方法可检测不到1细胞ml〜（-1）的海水。批间变异系数（CVs）分别为12.4％和10.9％。 NPA-SH的良好特异性，灵敏性和可重复性表明，这项新技术对于低丰度小球藻和米克假单胞菌的定性和定量测定非常有用。

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