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