Efficiency and sensitivity of the digital droplet PCR for the quantification of antibiotic resistance genes in soils and organic residues
2016
Cave, Laura | Brothier, Elisabeth | Abrouk, Danis | Bouda, Panignimyandé Salomon | Hien, Edmond | Nazaret, Sylvie | Laboratoire d'Ecologie Microbienne - UMR 5557 (LEM) ; Institut National de la Recherche Agronomique (INRA)-Université Claude Bernard Lyon 1 (UCBL) ; Université de Lyon-Université de Lyon-Ecole Nationale Vétérinaire de Lyon (ENVL)-VetAgro Sup - Institut national d'enseignement supérieur et de recherche en alimentation, santé animale, sciences agronomiques et de l'environnement (VAS)-Centre National de la Recherche Scientifique (CNRS) | Research Group on Environmental Multi-Resistance and Efflux Pump ; Ecole Nationale Vétérinaire de Lyon (ENVL) | IBIO - Plateforme de bio-imagerie en sciences médicales ; Université de Lyon | Centre National de la Recherche Scientifique (CNRS) | UMR Ecologie et Sols ; Université Joseph Ki-Zerbo de Ouagadougou = University of Ouagadougou (UJZK) | CNRS; ADEME; Campus France; IRD
Droplet digital PCR (ddPCR) allows absolute quantification and tolerance to inhibitors and has been proposed as the method of choice to overcome limitations of qPCR. The aim of this study was to evaluate ddPCR and qPCR performances to detect low copy number and copy number variation of antibiotic resistance genes (sul1 and qnrB genes encoding for resistance to sulfonamides and quinolones, respectively) using bacterial genomic DNA (gDNA) and metagenomic DNA extracted from soil and organic residue samples. With gDNA, qPCR showed a better range of quantification but the lower limit of quantification was at 15 copies of qnrB target vs. 1.6 in ddPCR. In the presence of background DNA or inhibitors, we observed a high loss of sensitivity in qPCR and an overestimation of target sequences. When using high amount of environmental DNA templates (70 ng per reaction), ddPCR was still allowing accurate quantification without adding PCR facilitator (i.e., T4 Gene 32 protein). Sensitivity to detect copy number variation was tenfold higher in ddPCR than in qPCR. Finally, the advantages of using ddPCR in environmental studies were confirmed with the quantification of sul1 and qnrB in soils, manures, or urban wastes.
اظهر المزيد [+] اقل [-]الكلمات المفتاحية الخاصة بالمكنز الزراعي (أجروفوك)
المعلومات البيبليوغرافية
تم تزويد هذا السجل من قبل Institut national de la recherche agronomique