Development of DPO-PCR Detection Method for Tomato Spotted Wilt Virus

LIU Zhongmei; LUO Jia; PAN Zhongle; LIU Hongyi; LI Dandan; HAN Zhengkun; WEI Dongxu; MA Wei

doi:10.15933/j.cnki.1004-3268.2017.11.016

Abstract：In order to accurately and efficiently detect tomato spotted wilt virus(TSWV),in this study,a pair of dual-priming oligonucleotide (DPO) primers was designed based on TSWV coat protein (CP) gene by DPO technology and through optimization of reaction system factors including concentrations of primer,dNTPs and Taq DNA polymerase,a DPO-PCR detection method for TSWV was established.Its sensitivity,specificity and annealing temperature sensitivity were also evaluated.The results showed that the expected fragment of 208 bp was successfully amplified by the DPO-PCR system.The optimal DPO-PCR system (25 μL) was as follows:DNA 1 μL,10 × Buffer (Mg2+ Plus) 2.5 μL,dNTPs(2.5 mmol/L for each) 2.0 μL,Taq DNA polymerase(5 U/μL)0.2 μL,each primer(20 μ mol/L) 1.0 μL,with deionized water to supplement the system.The target gene could be efficiently amplified by the DPO primers in the annealing temperature range from 49.8 ℃ to 70.0 ℃,showing that the method was insensitive to annealing temperature.The detection sensitivity of the method was 7.5 ng when viral RNA was used as template.TSWV,tomato ringsopt virus,tobacco ring spot virus and tomato black ring virus were simultaneously detected by the method,and positive reaction appeared only for TSWV,indicating that the method had strong specificity.The established method can detect TSWV accurately and efficiently,and thus can be used for quarantine screening and disease diagnosis of entry and exit seedlings.