Abstract:This study was conducted to develop a TaqMan Locked Nucleic Acid (LNA) probe real-time PCR assay that had a wide range of application, high sensitivity, and stability for the detection of Wheat dwarf vius (WDV) in the field. The specific primers and LNA probe (19 bp) were designed from conserved WDV-Rep sequences obtained from GenBank. The method was established to detect samples infected with WDV in the fields by TaqMan LNA probe real-time PCR. Matrix method was used to optimize the reaction system including the best concentration of the primers (0.5 μmol/L) and the probe (0.3 μmol/L). The slope and correlation coefficient of the standard curve were -3.424 3 and 0.997 3 , respectively, and the amplification efficiency was up to 96%. The coefficient of variation ranging from 0.11%-1.34% intra group and 0.44%-1.21% inter group indicated the excellent stability and reproducibility of the method. The sensitivity compared with the conventional PCR was analyzed by ten-fold dilution method. Results showed that the sensitivity of the former (55 copies/μL) was at least 100 times higher than the latter. In addition, the designment of probe become simpler and more convenient than TaqMan probe real-time PCR due to that the greatly shortened LNA probe is not prone to forming intermolecular and intramolecular secondary structures. Our results indicated that the detection method based on LNA probe is feasible for WDV early monitoring in the field, thus providing an efficient and rapid tool for WDV epidemiological investigation.