摘要

       抗生素抗性细菌 (ARB) 和抗生素抗性基因 (ARG) 广泛分布在环境中，它们代表着潜在的公共健康威胁。定量 PCR (qPCR) 是检测和量化环境样品中 ARG 的合适方法。然而，不同实验室之间的基因量化数据的比较具有挑战性，因为数据主要是在非协调条件下使用不同的 qPCR 协议获得的。本研究旨在进行实验室间校准，以评估 qPCR 程序对 ARG 进行量化的固有可变性。为此，基于共同的 DNA 提取池和相同的协议以及不同的设备、试剂批次和操作员，对在三个不同国家收集的处理过的废水样本进行了分析。分析的基因是 16S rRNA、vanA、blaTEM、qnrS、sul1、blaCTXM-32 和 intI1，含有来自七个目标基因的片段的人工 pNORM1 质粒用作参考。在所有分析的样本中，16S rRNA 基因是最丰富的，其次是 intI1、sul1、qnrS 和 blaTEM，而 blaCTXM-32 和 vanA 在大多数或所有样本中都低于定量限。对于基因 16S rRNA、sul1、intI1、blaTEM 和 qnrS，实验室间变异低于 28%（分别为 3-8%、6-18%、8-21%、10-24%、15-28%） .虽然由于设备、试剂和操作员的差异，可能难以完全协调 qPCR 协议，但实验室间校准是提高不同环境区室和/或地理区域中 ARG 丰度比较数据可靠性的充分且必要的步骤。

       Antibiotic resistant bacteria (ARB) and antibiotic resistance genes (ARGs) are widely distributed in the environment where they represent potential public health threats. Quantitative PCR (qPCR) is a suitable approach to detect and quantify ARGs in environmental samples. However, the comparison of gene quantification data between different laboratories is challenging since the data are predominantly obtained under non-harmonized conditions, using different qPCR protocols. This study aimed at carrying out an inter-laboratory calibration in order to assess the variability inherent to the qPCR procedures for quantification of ARGs. With this aim, samples of treated wastewater collected in three different countries were analysed based on common DNA extract pools and identical protocols as well as distinct equipment, reagents batches, and operators. The genes analysed were the 16S rRNA, vanA, blaTEM, qnrS, sul1, blaCTXM-32 and intI1 and the artificial pNORM1 plasmid containing fragments from the seven targeted genes was used as a reference. The 16S rRNA gene was the most abundant, in all the analysed samples, followed by intI1, sul1, qnrS, and blaTEM, while blaCTXM-32 and vanA were below the limit of quantification in most or all the samples. For the genes 16S rRNA, sul1, intI1, blaTEM and qnrS the inter-laboratory variation was below 28% (3–8%, 6–18%, 8–21%, 10–24%, 15–28%, respectively). While it may be difficult to fully harmonize qPCR protocols due to equipment, reagents and operator variations, the inter-laboratory calibration is an adequate and necessary step to increase the reliability of comparative data on ARGs abundance in different environmental compartments and/or geographic regions.

https://www.sciencedirect.com/science/article/abs/pii/S2213343718300940