Abstract: 【Objective】The objective of this study is to develop a multiplex RT-PCR protocol to detect 3 sweet cherry virus species Prunus necrotic ringspot virus (PNRSV), Prune dwarf virus (PDV) and Little cherry virus-2 (LChV-2) simultaneously. 【Method】 The leaves of the sweet cherry (Prunus avium Lindl.) infected by 3 virus species were selected as the experimental materials. Total RNA was extracted using CTAB extraction buffer. For cDNA synthesis, reverse transcription was carried out using random hexamer primer. Six pairs of primers were designed according to the genome sequences of PNRSV, PDV and LChV-2 which were published in GenBank. Single RT-PCR and multiplex RT-PCR were carried out, respectively, to select the primer groups which could be used in the multiplex virus detection. Annealing temperature and the number of the PCR cycles were evaluated to optimize the multiplex RT-PCR conditions. The specificity of the primers was analyzed by using the single-virus infected samples, complex-virus infected samples and virus-free leaf samples. To analyze the sensitivity of the multiples RT-PCR, the transcript which was prepared from the complex-virus infected samples was diluted to 2-fold series. All of the reactions were carried out in the same reaction buffer and under the same conditions. To confirm the accuracy of the multiplex RT-PCR, each amplified fragment was purified by DNA gel extraction kit, cloned into pMD18-T vector and sequenced. In this paper, the multiplex RT-PCR was also used to detect virus infection in Chinese cherry (P. pseudocerasus Lindl.) trees which are cultivated in the orchards in Taian, Shandong Province. 【Result】 Two primer groups were selected and could be used in the multiplex RT-PCR detection. Group 1 including primers ‘PNRSV-S1/A1, PDV-S2/A2 and LChV2-S1/A1’ amplified the fragments of 733, 467 and 337 bp, respectively. Group 2 including ‘PNRSV-S1/A1, PDV-S3/A3, LChV2-S1/A1’ amplified the fragments of 733, 265 and 337 bp, respectively. The PCR products were consistent with the expected size. It was showed that both of the two primer groups could be used to detect 3 sweet cherry virus species with high specificity. Sensitivity analysis showed that virus fragments could be detected in the 23 dilution of the plant cDNA using both of the two primer groups. However, the intensity of each virus fragments was slightly different. The lowest concentration of the plant total RNA transcripts was 107.9 ng•μL-1. Sequence analysis confirmed that it was reliable and accurate to detect the viruses using these two primer groups. This method was then applied to detect the incidence of virus disease in Chinese cherry trees. The results indicated that all of the samples were infected by 2 virus species at least. Of the 9 detected samples, 5 of them were infected by 3 virus species simultaneously. Two of them were infected by PDV and LChV-2 and another two samples were infected by PNRSV and LChV-2. 【Conclusion】 The multiplex RT-PCR protocol can simultaneously detect PNRSV, PDV and LChV-2 with high stability, accuracy and sensitivity.

Key words: Prunus necrotic ringspot virus (PNRSV) , Prune dwarf virus (PDV) , Little cherry virus-2 (LChV-2) , multiplex RT-PCR , pathogen detection