Three genetic sub-populations (clade I, II and III) of Pseudo-nitzschia pungens, the potential toxic marine diatom, are known to have distinguishable growth characteristics under different culture conditions and distinct distributed patterns in the world. However, to date their exact eco-physiological traits are unrevealed in fields due to lack of the method to detect and/or measure abundances of each sub-populations, hence, the qPCR (quantitative real-time polymerase chain reaction) assay was developed to detect and quantify the P. pungens cells of each clade. Designed two specific primer sets, Pcla12F/R (for clade I and II) and Pcla3F/R (for clade III) only could amplify each target genomic DNA. The, significant linear relationships (R2>0.998) was established between Ct (threshold cycle) value and the log of cell abundance for each clade. Through the melting curve analysis, comparisons for gene copy numbers among the three clades and spike test for field study, our qPCR assay was reliable to quantify the cell numbers of each clade. There was strong linear correlation (R2>0.990) between cell abundances as estimated by qPCR assay and direct counting via light microscope in spike test, and 0.24 (clade I), 0.25 (clade II) and 0.33 (clade III) P. pungens cells per mL were detected markedly upon the use of specific two-primer set. Finally, developed qPCR assay was applied on field samples successfully. Our study implicate that our qPCR assay is an accurate and sensitive technique to estimate the cell abundances of each clade of P. pungens in field works.

译文

:假性尼氏假单胞菌(潜在的有毒海洋硅藻)的三个遗传亚群(I,II和III类)在世界上不同的培养条件和不同的分布方式下具有明显的生长特征。然而,由于缺乏检测和/或测量每个亚群的丰度的方法,迄今为止,尚未在田间揭示它们的确切生态生理特性,因此,开发了qPCR(实时定量聚合酶链反应)测定法检测并量化每个进化枝的P. pungens细胞。设计的两个特异性引物组,Pcla12F / R(对于进化枝I和II)和Pcla3F / R(对于进化枝III)只能扩增每个靶基因组DNA。 Ct(阈值周期)值与每个进化枝的细胞丰度对数之间建立了显着的线性关系(R2> 0.998)。通过解链曲线分析,比较三个进化枝之间的基因拷贝数以及进行实地研究的加标测试,我们的qPCR分析法能够可靠地量化每个进化枝的细胞数。经qPCR分析估计并在加标测试中通过光学显微镜直接计数,细胞丰度之间存在强线性相关性(R2> 0.990),而P.pungens细胞为0.24(I类),0.25(II类)和0.33(III类)细胞。使用特定的双引物组可显着检测出每mL的含量。最终,将开发的qPCR测定法成功地应用于野外样品。我们的研究表明,我们的qPCR测定法是一种准确而灵敏的技术,可以在野外工作中估算每个P. pungens枝的细胞丰度。

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