摘要

    环境中出现新出现的抗生素抗性基因（ARG），尤其是那些赋予对最后手段的抗生素碳青霉烯（blaKPC）和粘菌素（mcr-1）的抗性的基因，已经成为一个重要的环境问题。实时聚合酶链反应（qPCR）方法通常用于量化环境中新兴的ARGs，一些研究报告其丰度很高。由于DNA模板的高度多样性和环境样品中的复杂性，由于潜在的非特异性扩增，可能会高估目标基因，甚至检测到假阳性。这项研究比较了基于染料的qPCR和基于探针的qPCR分析在活性污泥（AS）样品中检测blaKPC和mcr-1的性能，这表明基于染料的qPCR对blaKPC和mcr-1的检测与基于探针的qPCR结果相比，该检测可能为假阳性。 qPCR反应的下一代测序（NGS）将引物二聚体和非特异性扩增子鉴定为假阳性检测的主要原因。 NGS还检测了阴性反应中的目标扩增子，表明基于探针的qPCR分析可能存在假阴性检测。加标样品的测试表明，基于染料的qPCR分析的假阳性检测和过高估计主要发生在目标DNA浓度较低的情况下，而基于探针的qPCR的假阴性检测主要是由于环境样品中扩增效率降低引起的。在一起，结果确定了qPCR方法在复杂环境样品中的局限性，并证明了NGS在通过qPCR定量新兴ARG中的修复效用。

    Occurrence of emerging antibiotic resistance genes (ARGs) in the environment, especially those conferring resistance to the last resort antibiotic carbapenems (blaKPC) and colistin (mcr-1), has become an important environmental issue. Real-time polymerase chain reaction (qPCR) methods were commonly used to quantify emerging ARGs in the environment, with some studies reporting high abundance. Due to the high diversity of DNA templates and complexity in environmental samples, overestimation or even false-positive detection of target genes may occur due to potential non-specific amplification. This study compared the performance of dye-based qPCR and probe-based qPCR assays for the detection of blaKPC and mcr-1 in activated sludge (AS) samples, which showed that the detection of blaKPC and mcr-1 by the dye-based qPCR assays was likely false-positive when compared with probe-based qPCR results. Next generation sequencing (NGS) of the qPCR reactions identified primer dimers and non-specific amplicons as the primary causes for the false-positive detection. NGS also detected target amplicons in the negative reactions, indicating potential false-negative detection by the probe-based qPCR assays. Testing of spiked samples showed false-positive detection and overestimation by the dye-based qPCR assays primarily occurred at low concentrations of target DNA, while false-negative detection by probe-based qPCR was caused primarily by reduced amplification efficiencies in the environmental samples. Together, the results identified the limitations of the qPCR methods in complex environmental samples and demonstrated the remedial utility of NGS in quantifying emerging ARGs by qPCR.

    https://link.springer.com/article/10.1007/s00253-021-11202-4