Two new real-time quantitative reverse transcription polymerase chain reaction assays with unique target sites for the specific and sensitive detection of lineages 1 and 2 West Nile virus strains.
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.
Eiden
Martin
M
Institute for Novel and Emerging Infectious Diseases, Friedrich-Loeffler-Institut, Federal Research Institute for Animal Health, Greifswald-Insel Riems, Germany.
Vina-Rodriguez
Ariel
A
Hoffmann
Bernd
B
Ziegler
Ute
U
Groschup
Martin H
MH
D020121:5' Untranslated Regions / Q000235:genetics; D000818:Animals; D001483:Base Sequence; D017931:DNA Primers; D015342:DNA Probes; D016679:Genome, Viral; D008969:Molecular Sequence Data; D009518:New York / Q000453:epidemiology; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods / Q000662:veterinary*; D012680:Sensitivity and Specificity; D014901:West Nile Fever / Q000453:epidemiology / Q000662:veterinary; D014902:West Nile virus / Q000235:genetics* / Q000302:isolation & purification
D020121:5' Untranslated Regions; D017931:DNA Primers; D015342:DNA Probes