Molecular detection tools for blueberry mosaic associated virus and blueberry shoestring virus

Blueberry mosaic-associated virus (BlMaV) and blueberry shoestring virus (BSSV) are significant pathogens affecting blueberry production in Michigan. This study aimed to develop and validate TaqMan-based reverse transcription quantitative PCR (real time RT-PCR) assays for the detection and quantification of BlMaV and BSSV. Leaf samples from symptomatic and asymptomatic plants were collected from commercial blueberry farms in Michigan, and total RNA was extracted. Specific primers and probes targeting the RNA-dependent RNA polymerase (RdRp) and a 23kDa protein of BlMaV, and RdRp and coat protein (CP) gene of BSSV were optimized and validated for sensitivity, specificity, and efficiency. All of the developed real time RT-PCR assays were sensitive, specific, and could detect as few 0.01 fg of BlMaV and BSSV RNA or as low as 4.6 copies of BlMaV and BSSV DNA with an average cycle threshold (Ct) value of 35. The optimized BlMaV assays were found to be sensitive and comparable to end-point RT-PCR. The BSSV assays were more consistent and sensitive compared to DAS-ELISA. The assays were able to detect BlMaV and BSSV in both flower and leaf tissue. The real time RT-PCR assays provide a rapid, sensitive, and specific method for the detection and quantification of these viruses to assist laboratories and nurseries in the production of virus-free planting material.