Quantitative multiplex assay for simultaneous detection and identification of Indiana and New Jersey serotypes of vesicular stomatitis virus.
In order to establish a rapid and reliable system for the detection of vesicular stomatitis virus (VSV), we developed a quantitative reverse transcription-PCR assay for the detection, quantification, and differentiation of the major serotypes, VSV Indiana and VSV New Jersey, using a closed-tube multiplex format. The detection system is based on the recently invented primer-probe energy transfer (PriProET) system. A region of the gene encoding the RNA-dependent RNA polymerase was amplified by using VSV-specific primers in the presence of two serotype-specific fluorescent probes. By incorporating nucleotide analogues in the primers, both serotypes were amplified with similar efficiencies. The generation of specific amplicons resulted in fluorescent signals for either of the two serotypes, and the specificities of the reactions were confirmed from the melting temperature profiles of the fluorescent probes. The limits of detection were found to be less than 10 50% tissue culture infective doses/ml for both serotypes. The diagnostic value of the new method was tested with clinical materials from experimentally infected pigs, and it is concluded that the method is a powerful tool for the rapid identification of VSV.
Rasmussen
Thomas B
TB
Department of Virology, Danish Institute for Food and Veterinary Research, Lindholm, DK-4771 Kalvehave, Denmark.
Uttenthal
Ase
A
Fernández
Jovita
J
Storgaard
Torben
T
D000818:Animals; D001483:Base Sequence; D017931:DNA Primers; D004735:Energy Transfer; D008969:Molecular Sequence Data; D012367:RNA, Viral / Q000032:analysis; D012324:RNA-Dependent RNA Polymerase / Q000737:chemistry / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D018353:Rhabdoviridae Infections / Q000175:diagnosis / Q000662:veterinary* / Q000821:virology; D013280:Stomatitis / Q000175:diagnosis / Q000662:veterinary* / Q000821:virology; D013552:Swine; D013553:Swine Diseases / Q000175:diagnosis* / Q000821:virology*; D014721:Vesicular stomatitis Indiana virus / Q000145:classification* / Q000235:genetics / Q000302:isolation & purification*; D018116:Vesiculovirus; D014764:Viral Proteins / Q000737:chemistry / Q000235:genetics
D017931:DNA Primers; D012367:RNA, Viral; D014764:Viral Proteins; C040530:L protein, vesicular stomatitis virus; D012324:RNA-Dependent RNA Polymerase