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首页> 美国卫生研究院文献>Applied and Environmental Microbiology >Agreement between amoA Gene-Specific Quantitative PCR and Fluorescence In Situ Hybridization in the Measurement of Ammonia-Oxidizing Bacteria in Activated Sludge
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Agreement between amoA Gene-Specific Quantitative PCR and Fluorescence In Situ Hybridization in the Measurement of Ammonia-Oxidizing Bacteria in Activated Sludge

机译：在测定活性污泥中氨氧化细菌时amoA基因特异性定量PCR和荧​​光原位杂交之间的协议

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Microbial abundance is central to most investigations in microbial ecology, and its accurate measurement is a challenging task that has been significantly facilitated by the advent of molecular techniques over the last 20 years. Fluorescence in situ hybridization (FISH) is considered the gold standard of quantification techniques; however, it is expensive and offers low sample throughput, both of which limit its wider application. Quantitative PCR (qPCR) is an alternative that offers significantly higher throughput, and it is used extensively in molecular biology. The accuracy of qPCR can be compromised by biases in the DNA extraction and amplification steps. In this study, we compared the accuracy of these two established quantification techniques to measure the abundance of a key functional group in biological wastewater treatment systems, the ammonia-oxidizing bacteria (AOB), in samples from a time-series experiment monitoring a set of laboratory-scale reactors and a full-scale plant. For the qPCR analysis, we tested two different sets of AOB-specific primers, one targeting the 16SrRNA gene and one targeting the ammonia monooxygenase (amoA) gene. We found that there was a positive linear logarithmic relationship between FISH and the amoA gene-specific qPCR, where the data obtained from both techniques was equivalent at the order of magnitude level. The 16S rRNA gene-specific qPCR assay consistently underestimated AOB numbers.
机译：微生物丰度是大多数微生物生态学研究的核心，其准确测量是一项具有挑战性的任务，在过去的20年中，分子技术的出现极大地促进了微生物的丰度。荧光原位杂交（FISH）被认为是定量技术的金标准。但是，它价格昂贵且样品通量低，这两者都限制了其更广泛的应用。定量PCR（qPCR）是一种替代方法，可提供更高的通量，并且已广泛用于分子生物学中。 DNA提取和扩增步骤中的偏差可能会损害qPCR的准确性。在这项研究中，我们比较了这两种已建立的定量技术的准确性，这些技术用于测量生物废水处理系统中关键功能基团（氨氧化细菌（AOB））的丰度，该样本来自一系列监测一系列时间的实验实验室规模的反应堆和大型工厂。对于qPCR分析，我们测试了两组不同的AOB特异性引物，一组针对16SrRNA基因，另一组针对氨单加氧酶（amoA）基因。我们发现FISH与amoA基因特异性qPCR之间存在正线性对数关系，其中从两种技术获得的数据在数量级上是等效的。 16S rRNA基因特异性qPCR测定法始终低估了AOB数。

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