Real-time RT-PCR assay for rapid and specific detection of classical swine fever virus: comparison of SYBR Green and TaqMan MGB detection methods using novel MGB probes.
Real-time PCR is an accurate, rapid and reliable method that can be used for the detection and also for the quantitation of specific DNA molecules. The basic principle is the recurring measurement of a fluorescent signal, which is proportional to the amount of amplification product. In our trial two detection systems were tested for classical swine fever virus (CSFV) detection and for its discrimination from other pestiviruses; non-specific dsDNA-binding dye SYBR Green and specific fluorogenic TaqMan MGB probes. Real-time RT-PCR assays were evaluated for diagnostic sensitivity and specificity by different pestiviral reference and field strains. With both approaches, SYBR Green and TaqMan probes, respectively, all of the CSFV strains isolated on cell culture were detected and also clearly distinguished from other pestiviruses. However, the established one-step real-time TaqMan RT-PCR assay was shown to be more appropriate for pestivirus quantitation, it reduces the risk of contamination and is less time consuming.
Jamnikar Ciglenecki
Urska
U
Veterinary Faculty, University of Ljubljana, Gerbiceva 60, Ljubljana, Slovenia. urska.jamnikar@vf.uni-lj.si
Grom
Joze
J
Toplak
Ivan
I
Jemersić
Lorena
L
Barlic-Maganja
Darja
D
D000818:Animals; D006691:Classical Swine Fever / Q000821:virology*; D006692:Classical Swine Fever Virus / Q000302:isolation & purification*; D005456:Fluorescent Dyes; D015345:Oligonucleotide Probes; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D013552:Swine; D019914:Taq Polymerase
D005456:Fluorescent Dyes; D015345:Oligonucleotide Probes; D019914:Taq Polymerase