DEVELOPMENT OF A MINOR GROOVE BINDER ASSAY FOR SENSITIVE REAL-TIME ONE-STEP RT-PCR DECTECTION OF SWINE VESICULAR DISEASE VIRUS

摘要

Minor groove binder (MGB)1 probes are short oligonucleotide probes that can function as a reporter in real-time PCR. At one end of the oligonucleotide there is a fluorophore and at the other a quencher. The probe also has a minor groove binding molecule attached. The probes are designed to bind to a sequence situated between a set of PCR primers. When unbound the MGB probe is quenched due to the proximity between fluorophore and quencher. During PCR the MGB probe hybridizes to a specific target, increasing the distance between the fluorophore and quencher and resulting in a detectable increase in fluorescence. Incorporation of the MGB probes minor groove binding molecule into the DNA a-helix, increasing the melting temperature of the complex. This enables the design of shorter probes. Here we describe the application of this MGB technology to the one-step real-time RT-PCR detection of Swine Vesicular Disease Virus (SVDV).