实时荧光定量PCR法快速检测赤潮环状异帽藻

Development of a real-time PCR assay for rapid detection and quantification of Heterocapsa circularisquama

以环状异帽藻(Heterocapsa circularisquama)ITS1区为靶序列设计了一对特异性引物,运用Real-time PCR方法,对环状异帽藻进行了定性定量检测。以其他9种赤潮微藻为对照验证其特异性。结果表明,仅含环状异帽藻DNA模板的样品有扩增,其他藻没有检测到扩增信号。以超声波破碎得到的藻液为标准品,所得标准曲线具有高度线性相关,相关系数R2为0.989,并且可以在30个循环内检测到0.5个细胞。对在不同培养条件下获得的环状异帽藻DNA样品进行检测,都能获得扩增信号。利用该方法建立的标准曲线对初步模拟现场样品进行定量检测,与显微镜计数结果基本一致。

A real-time polymerase chain reaction （PCR） assay was designed and evaluated for rapid detection and quantification of the toxic algae Heterocapsa circularisquama. A pair of specific primers was designed from the sequence of ITS 1 region, of which the PCR specificity was examined compared with the other nine dinoflagellates. PCR amplifications were detected only from samples which contained H. circularisquama cells, and specific signals were not detected from any other microalgae. Based on crushed algae by ultrasound for the standard, the standard curve we got has high linearity, and correlation coefficient R2 was 0.989. Moreover, this assay can detect 0.5 cell within 30 cycles, showing its high sensitivity. DNAs from H. circularisquama that had been cultured in different conditions, were assayed in the run. Using the developed standard curves, H. circularisquama could be quantified in agreement with the quantification by optical microscopy. [Journal of Fishery Sciences of China, 2010, 17 （ 2 ） ： 267-273]