Establishment of a Multiplex RT-PCR System for Rapid Detection of Four Major Rose Viruses in China

The rose industry in China faces significant challenges due to viral pathogens such as Prunus necrotic ringspot virus (PNRSV), apple stem grooving virus (ASGV), rose virus C (RVC), and Citrus tatter leaf virus (CTLV). This study aims to establish a rapid, efficient, and accurate detection method for these viral diseases to aid in their prevention and control. Based on morphological and microscopic observations, coat protein and polyprotein gene sequences of these viruses were retrieved from the NCBI database, and specific primers were designed based on highly conserved regions. Primer concentration, cDNA concentration, and annealing temperature were optimized to develop a multiplex RT-PCR detection system. The optimal reaction system was established with a total volume of 50 μl, with upstream and downstream primer concentrations of 0.3 μmol/liter for ASGV and CTLV, and 0.2 μmol/liter for PNRSV and RVC, with template cDNA at 2.0 ng/μl. Using touchdown PCR, we performed 10 cycles with an annealing temperature gradient of 65 to 55°C, decreasing by 1°C per cycle, followed by 20 cycles with a constant annealing temperature of 55°C. This method was successfully applied to 23 cut rose samples, yielding clear and distinct bands. These results demonstrate that the method can efficiently and accurately detect single or combined infections of the four rose viruses, offering a valuable tool for the rose industry in China.