Abstract
Smut is a fungal disease with widespread prevalence in sugarcane planting areas. Early detection and proper identification of Sporisorium scitamineum are essential in smut management practices. In the present study, four specific primers targeting the core effector Pep1 gene of S. scitamineum were designed. Optimal concentrations of Mg2+, primer and Bst DNA polymerase, the three important components of the loop-mediated isothermal amplification (LAMP) reaction system, were screened using a single factor experiment method and the L16(45) orthogonal experimental design. Hence, a LAMP system suitable for detection of S. scitamineum was established. High specificity of the LAMP method was confirmed by the assay of S. scitamineum, Fusarium moniliforme, Pestalotia ginkgo, Helminthospcrium sacchari, Fusarium oxysporum and endophytes of Yacheng05-179 and ROC22. The sensitivity of the LAMP method was equal to that of the conventional PCR targeting Pep1 gene and was 100 times higher than that of the conventional PCR assay targeting bE gene in S. scitamineum. The results suggest that this novel LAMP system has strong specificity and high sensitivity. This method not only provides technological support for the epidemic monitoring of sugarcane smut, but also provides a good case for development of similar detection technology for other plant pathogens.
Highlights
Our previous work designed the specific primer bEQ-F/bEQ-R and the TaqMan probe targeting the bE gene, and we have developed a method to detect S. scitamineum based on real-time quantitative fluorescence PCR12
Loop-mediated isothermal amplification (LAMP) is an isothermal amplification technology invented by Notomi et al in 2000 which can complete automatic looping, strand displacement and DNA synthesis using Bst DNA polymerase and four specific primers[13]
In order to obtain a LAMP system suitable to detect S. scitamineum, a single factor optimization experiment was conducted on a Mg2+ concentration (5.75 mmol/L, 6.00 mmol/L, 6.25 mmol/L and 6.50 mmol/L), an inner vs. outer primer ratio (2:1, 4:1, 6:1 and 8:1) and the Bst DNA polymerase content (2.0 U, 4.0 U, 6.0 U and 8.0 U) in the LAMP system (Figs 1–3)
Summary
Our previous work designed the specific primer bEQ-F/bEQ-R and the TaqMan probe targeting the bE gene, and we have developed a method to detect S. scitamineum based on real-time quantitative fluorescence PCR12. By using this assay, the content of S. scitamineum in resistant/susceptible sugarcane genotypes could be detected, but it required expensive quantitative PCR instrument and suffered from the high cost of reagents and other consumables[12]. A released single-stranded DNA serves as a template for DNA synthesis facilitated by the second inner and outer primers that hybridize to the other end of the target This results in a DNA sequence which can form a stem-loop structure at both ends. This study aims to establish a LAMP system for rapid detection of S. scitamineum in sugarcane before symptoms of smut disease appear, laying a foundation for epidemiological studies on sugarcane, disease control and exit–entry quarantine in the exchange of sugarcane germplasms
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