All samples were tested at least
in triplicates. A sample was classified as nptII and/or nptIII positive,
if PCR amplification occurred before crossing point (¼Cp) 40. A
single positive replicate was sufficient to classify the sample as
positive. Samples producing only replicates with amplification
curves above Cp 40 were re-extracted. A sample was rated as
nptII and/or nptIII negative only, if the 16S rRNA TaqMan assay was
positive (see Section 2.6) and all nptII/nptIII replicates showed no
amplification in the respective PCR assay. All samples were checked
for inhibition by analyzing 1:5 and 1:25 serial dilutions. Taking into
account a DNA recovery rate from soil of approx. 60% as established
for the PowerSoil procedure (see 2.3) and according to the validation
results of the quantitative PCR assays (see 2.7) the 95%
detection limit for nptII and nptIII was approx. 500e700 copies per
gram soil.