Quantification of resistance genes in veal manure by qPCR “But first we have to have some conditions clear”

Abstract

The relation with the big therapeutic use of antibiotics in human and veterinary medicine and the growing problem of resistance is getting more and more attention world wide. In the Netherlands, a political agreement has been reached to decrease antibiotic use in veal farming immediately. The aim of this research is to determine the qPCR detection limit of antibiotic resistance genes (tet(S)) in veal feces, to choose between two (absorbing overshoe and plastic cup) sampling methods and to investigate PCR inhibition and its removal by dilution. Also a start will be made in the setup of a protocol for quantification of Integrase genes (intI1) in feces samples. Integrons are intermediates in the pickup and expression of resistance genes and are often found on plasmids. The sampling with a plastic cup will gave the best qPCR data. Because of inhibition factors, which are present in feces, a ten fold dilution of the DNA was necessary for an optimal qPCR reaction. The detection limit for tet(S) was determined as 10^5 spiked genes per gram feces, which is a workable detection limit for veal feces. Unstable standard dilutions were seen during this research, reasons and solutions for this are discussed.