Development of a Sensitive Real-Time Quantitative RT-PCR Assay for the Detection of Pear Chlorotic Leaf Spot-Associated Virus

Abstract

Pear chlorotic leaf spot associated virus (PCLSaV) belongs to the genus Emaravirus and possesses a genome composed of five negative-sense single-stranded RNA (-ssRNA) segments. This study developed a SYBR green-based reverse transcription quantitative PCR (RT-qPCR) assay for the detection of PCLSaV infecting pear trees. A set of two primers q5-F2/q5-R2 designed based on the viral RNA5 sequences showed high specificity and feasibility for PCLSaV detection. The standard curve was established. RT-qPCR assays showed that PCLSaV content was greatly higher in diseased branch and symptomatic leaf samples than that in un-diseased branch and asymptomatic leaf samples. The RT-qPCR was reliability in the detection of the virus in field and in-vitro cultured pear samples. This technique would be useful for the supervision of the viral disease and the certification of pear planting materials.

Keywords: Pear, Pear chlorotic leaf spot-associated virus, Reverse transcription quantitative PCR, RT-PCR.