A multiplex RT-PCR assay for simultaneous detection of four viruses from sweet cherry

• A multiplex RT-PCR assay was developed for simultaneous detection of four stone fruit viruses, PNRSV, PDV, LChV-2 and CGRMV.
• Primers concentration and reaction conditions were optimized for the multiplex RT-PCR.
• The protocol could detect the viruses at a dilution of 10−6 of cDNA and 10−1 of RNA.
• The assay was evaluated using sweet cherry field samples naturally infected with target viruses.
• The multiplex RT-PCR assay is simple, sensitive and reliable for detection of four target viruses in sweet cherry.

A multiplex reverse transcription polymerase chain reaction (mRT-PCR) assay was developed for simultaneous detection of four viruses: Prunus necrotic ringspot virus (PNRSV), Prune dwarf virus (PDV), Little cherry virus-2 (LChV-2) and Cherry green ring mottle virus (CGRMV). Random hexamer primer was used for cDNA synthesis. Detection primers were designed against the genomic sequences of four viruses. The reaction conditions were firstly optimized by selecting primers combinations and standardizing the individual primer proportion and PCR protocols. Then, sensitivity of multiplex RT-PCR was evaluated. The assay could detect all four viruses in diluted cDNA (10−6, about 8.64 × 10−4 ng μL−1) and RNA (10−1, about 0.2 μg). The reliability of the assay was evaluated by cloning and sequence alignments. The developed multiplex RT-PCR method was then used to test virus infections from field samples of sweet cherry in this paper. It will be quite helpful for plant quarantine and certification programs. To our knowledge, it is the first report of the multiplex RT-PCR assay for simultaneous detection of PNRSV, PDV, LChV-2 and CGRMV.