תפריט נגישות
ניגודיות עדינהניגודיות גבוהה
מונוכרום
הדגשת קישורים
חסימת אנימציה
פונט קריא
סגור
איפוס הגדרות נגישות
להורדת מודול נגישות חינם תפריט נגישותניגודיות עדינהניגודיות גבוההמונוכרוםהדגשת קישוריםחסימת אנימציהפונט קריאסגוראיפוס הגדרות נגישותלהורדת מודול נגישות חינםRocha, J.; Cacace, D.; Kampouris, I.; Guilloteau, H.; Jäger, T.; Karaolia, P.; Manaia, C.M.; Merlin, C.; Fatta-Kassinos, D.; Schwartz, T.; Berendonk, T.U.
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, bla TEM, qnrS, sul1, bla CTXM-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 bla TEM, while bla CTXM-32 and vanA were below the limit of quantification in most or all the samples. For the genes 16S rRNA, sul1, intI1, bla TEM 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. © 2018 Elsevier Ltd.
Universidade Católica Portuguesa, CBQF - Centro de Biotecnologia e Química Fina - Laboratório Associado, Escola Superior de Biotecnologia, Rua Arquiteto Lobão Vital, 172, 4200-374 Porto, Portugal; Environmental Sciences Technische Universität Dresden, Institute for Hydrobiology, Dresden, Germany; CNRS-Université de Lorraine, Laboratory of Physical Chemistry and Microbiology for the Environment, UMR 7564, Institut Jean Barriol, Vandoeuvre-lès-Nancy, France; Karlsruhe Institute of Technology, Institute of Functional Interfaces, Karlsruhe, Germany; Institute of Soil Water and Environmental Sciences, Volcani Center, Agricultural Research Organization, Rishon Lezion, Israel; Department of Civil Engineering and Environmental Engineering and Nireas-International Water Research Centre, University of Cyprus, P.O. Box 20537, 1678, Nicosia, Cyprus
Rocha, J.; Cacace, D.; Kampouris, I.; Guilloteau, H.; Jäger, T.; Karaolia, P.; Manaia, C.M.; Merlin, C.; Fatta-Kassinos, D.; Schwartz, T.; Berendonk, T.U.
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, bla TEM, qnrS, sul1, bla CTXM-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 bla TEM, while bla CTXM-32 and vanA were below the limit of quantification in most or all the samples. For the genes 16S rRNA, sul1, intI1, bla TEM 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. © 2018 Elsevier Ltd.
