Development of a real-time PCR assay based on primer-probe energy transfer for the detection of swine vesicular disease virus.
A real-time PCR assay based on primer-probe energy transfer (PriProET) was developed to detect swine vesicular disease virus (SVDV). Specificity tests of SVDV and heterologous virus showed specific amplification of SVDV strains only. The amplification plot for the closely related Coxsackievirus B5 remained negative. The sensitivity of assay was five copies of viral genome equivalents. A key point of the assay is tolerance toward mutations in the probe region. Melting curve analysis directly after PCR, with determination of probe melting point, confirmed specific hybridisation of the SVDV strains. Eight of twenty SVDV strains tested, revealed shifted melting points that indicated mutations in the probe region. All predicted mutations were confirmed by nucleotide sequencing. With the PriProET system there is a chance to identify phylogenetically divergent strains of SVDV, which may appear negative in other probe-based real-time PCR assays. At the same time, any difference in melting points may provide an indication of divergence in the probe region. The high sensitivity, specificity, and tolerance toward mutations in the probe region of the SVDV PriProET assay may improve the early and rapid detection of a wide range of SVDV strains, allowing reduced turnaround time and the use of high-throughput, automated technology.
Hakhverdyan
M
M
Joint Research and Development Division, Department of Virology, The National Veterinary Institute and the Swedish University of Agricultural Sciences, Uppsala, Sweden. mikhayil.hakhverdyan@sva.se
Rasmussen
T B
TB
Thorén
P
P
Uttenthal
A
A
Belák
S
S
D000818:Animals; D020137:Base Pair Mismatch; D001483:Base Sequence; D017931:DNA Primers; D004735:Energy Transfer; D029822:Enterovirus B, Human / Q000145:classification / Q000235:genetics* / Q000302:isolation & purification / Q000472:pathogenicity; D005843:Geography; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction / Q000379:methods*; D012367:RNA, Viral / Q000235:genetics / Q000302:isolation & purification; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods; D016415:Sequence Alignment; D012689:Sequence Homology, Nucleic Acid; D013552:Swine; D013553:Swine Diseases / Q000175:diagnosis / Q000821:virology; D013555:Swine Vesicular Disease / Q000175:diagnosis / Q000821:virology*
D017931:DNA Primers; D012367:RNA, Viral