Molecular biological methods using real-time polymerase chain reaction (RT-PCR) for detection of bacterial and viral genes in different environments have been developed into assays from different commercial sources. Applied Biosystems include and support two applications with their TaqMan instrument: the “Plus/Minus” and the “Allelic Discrimination” assays. These approaches are RT-PCR based, use short primers and fluorescent-labeled TaqMan probes and include three processes: a pre-read run, a PCR-amplification run, and a post-read run. In the “Plus/Minus” assay, samples and controls (distilled water) are loaded into the instrument, which calculates a positive or a negative outcome based on differences in signals between samples and the controls. When testing the “Plus/Minus” assay for detection of usp genes encoding a uropathogenic specific protein in Escherichia coli, an inordinately high proportion of false-positive signals was observed. This was shown to be due to a serious methodological deficiency. Our observations indicate that an adequate no-template control closely matching the target samples in all aspects, including amount of DNA, is required to establish a correct threshold in the pre-read run that forms the basis for further calculations in the post-read run of the “Plus/Minus” assay.