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Rapid TaqMan-Based Quantification of Chlorophyll d-Containing Cyanobacteria in the Genus Acaryochloris

机译:基于Taqman的快速定量测定acaryochloris属中含叶绿素d的蓝细菌

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摘要

Reports of the chlorophyll (Chl) d-containing cyanobacterium Acaryochloris have accumulated since its initial discovery in 1996. The majority of this evidence is based on amplification of the gene coding for the 16S rRNA, and due to the wide geographical distribution of these sequences, a global distribution of Acaryochloris species was suggested. Here, we present a rapid, reliable, and cost-effective TaqMan-based quantitative PCR (qPCR) assay that was developed for the specific detection of Acaryochloris species in complex environmental samples. The TaqMan probe showed detection limits of10 16S rRNA gene copy numbers based on standard curves consisting of plasmid inserts. DNA from five Acaryochloris strains, i.e., MBIC11017, CCMEE5410, HICR111A, CRS, and Awaji-1, exhibited amplification efficiencies of>94% when tested in the TaqMan assay. When used on complex natural communities, the TaqMan assay detected the presence of Acaryochloris species in four out of eight samples of crustose coralline algae (CCA), collected from temperate and tropical regions. In three out of these TaqMan-positive samples, the presence of Chl d was confirmed via high-performance liquid chromatography (HPLC), and corresponding cell estimates of Acaryochloris species amounted to 7.6101 to 3.0103 per mg of CCA. These numbers indicate a substantial contribution of Chl d-containing cyanobacteria to primary productivity in endolithic niches. The new TaqMan assay allows quick and easy screening of environmental samples for the presence of Acaryochloris species and is an important tool to further resolve the global distribution and significance of this unique oxyphototroph.
机译:自1996年首次发现以来,含有叶绿素(Chl)d的蓝藻蓝藻的报道就积累了起来。大部分证据基于16S rRNA编码基因的扩增,以及这些序列的广泛地理分布,建议在全球范围内分布一草胺。在这里,我们介绍了一种基于TaqMan的快速,可靠且具有成本效益的定量PCR(qPCR)分析方法,该方法用于对复杂环境样品中的毒牙菌种进行特异性检测。 TaqMan探针根据包含质粒插入片段的标准曲线显示了10个16S rRNA基因拷贝数的检测极限。在TaqMan分析中进行测试时,来自五个醋利毒株(即MBIC11017,CCMEE5410,HICR111A,CRS和Awaji-1)的DNA表现出大于94%的扩增效率。当在复杂的自然社区中使用时,TaqMan分析法检测到从温带和热带地区收集的八份c壳珊瑚藻(CCA)样本中的四份中存在四氯化碳物种。在这些TaqMan阳性样本中,有三个样本通过高效液相色谱(HPLC)确认了Chl d的存在,相应的醋曲霉菌种细胞估计数为每毫克CCA 7.6101至3.0103。这些数字表明含Chl d的蓝细菌对内生石ni中初级生产力的重要贡献。新的TaqMan测定法可以快速,轻松地筛查环境样品中是否存在乙草胺物种,并且是进一步解决这种独特的光合养分的全球分布和重要性的重要工具。

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