Development of end-point and quantitative polymerase chain reaction assays to detect and discriminate between...

Blueberry scorch virus (BlScV) and blueberry virus S (BVS) are closely related carlaviruses with the latter being only recently characterized. Most available PCR assays for BlScV fail to detect some isolates of the virus, as well as BVS. Here, we describe PCR assays designed based on all available virus sequences in public databases that can discriminate BlScV from BVS. Initially, a triplex endpoint PCR assay was developed that yields different amplicon sizes for each virus and incorporates an internal control which assesses the quality of the nucleic acids and the efficiency of the enzymatic reactions. Subsequently, a duplex quantitative PCR was designed employing primers targeting both viruses and discriminatory probes specific to each. Comparison between end-point and qPCR assays revealed near parallel sensitivity. The availability of the two PCR formats reliably improves the detection assays of the viruses in diagnostic facilities, quarantine, and clean plant programs.