Two novel 1-step real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR) assays for the simultaneous detection of West Nile virus (WNV) lineage 1 and 2 strains were developed. Primers and the probe of assay 1 target the 5'-untranslated region (UTR), whereas the amplicon of assay 2 is located in the nonstructural region NS2A, which enables an unambiguous and independent WNV diagnosis based on 2 different amplicons. Both assays allow the detection of as few as 2-4 genome copies of WNV strains NY99, Uganda B956, Kunjin, and Sarafend (all cultured on Vero cells). A new synthetic RNA mutant of the 5'-UTR amplicon, which contains 6 twist inverted base-pair changes at the probe attachment site, was used as external calibrator control.