Development of a loop-mediated isothermal amplification assay for detection of Cylindrocladium scoparium on Eucalyptus

Abstract:

[Objective] To establish a rapid detection technology LAMP for Eucalyptus dieback. [Methods] The pathogen against on Eucalyptus was studied. Based on the species-specific conserved region of beta-tubulin gene on Cylindrocladium scoparium, a set of primers (inner primer: FIP/BIP, outer primer: F3/B3, Loop primers: LooPF/LooPB) were designed, a LAMP assay for rapid detection of C. scoparium was developed, evaluated and optimized. [Results] The LAMP reaction system (50 μL) consisted of Bst DNA polymerase (8 U) 1 μL, Betaine solution (5 mol/L) 3.0 μL, dNTPs (2.5 mmol/L) 3.5 μL, 10×Thermopol Ⅱ 2.5 μL, MgCl2 (100 mmol/L) 2 μL, FIP/BIP (40 μmol/L) 1 μL, F3/B3 (10 μmol/L) 0.5 μL, LooPF/LooPB (10 μmol/L) 1 μL, DNA template 2 μL, with ddH2O up to the volume of 50 μL. Response procedures acted as the mixture was in 65 °C water bath pot for about 45 min first, and then inactivated 5 min under the 90 °C to end of reaction. Specificity assays showed that the amplification results of 12 test isolates can be distinguished though visual inspection, ultraviolet light, added fluorescent dye SYBR Green I, and electrophoresis test strip, and the 4 pathogens were positive, and that of the other 8 pathogens were negative. The detection limit of LAMP was 40 pg pure genomic DNA per 50 μL reaction, which fits entirely in the field test. The wild field effectiveness showed that the different physiological small kinds different regions of C. scoparium can be detected with the method of LAMP, meanwhile the ultraviolet light and electrophoresis test strip were significantly. [Conclusion] Summing up the above, the LAMP rapid detection can be applied in filed effectively.