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Development and application of loop-mediated isothermal amplification for detecting the highly benzimidazole-resistant isolates in Sclerotinia sclerotiorum.

Duan YB, Yang Y, Wang JX, Liu CC, He LL, Zhou MG - Sci Rep (2015)

Bottom Line: The point mutation at codon 198 (GAG → GCG, E198A) occurs in more than 90% of field resistant populations in China.Traditional detection methods of benzimidazole-resistant mutants of S. sclerotiorum are time-consuming, tedious and inefficient.This method had a good specificity, sensitivity, stability and repeatability.

View Article: PubMed Central - PubMed

Affiliation: College of Plant Protection, State &Local Joint Engineering Research Center of Green Pesticide Invention and Application, Nanjing Agricultural University, Nanjing, 210095, China.

ABSTRACT
Resistance of benzimidazole fungicides is related to the point mutation of the β-tubulin gene in Sclerotinia sclerotiorum. The point mutation at codon 198 (GAG → GCG, E198A) occurs in more than 90% of field resistant populations in China. Traditional detection methods of benzimidazole-resistant mutants of S. sclerotiorum are time-consuming, tedious and inefficient. To establish a suitable and rapid detection of benzimidazole-resistant mutants of S. sclerotiorum, an efficient and simple method with high specificity was developed based on loop-mediated isothermal amplification (LAMP). Eight sets of LAMP primers were designed and four sets were optimized to specially distinguish benzimidazole-resistant mutants of S. sclerotiorum. With the optimal LAMP primers, the concentration of LAMP components was optimized and the reaction conditions were set as 60-64 °C for 60 min. This method had a good specificity, sensitivity, stability and repeatability. In the 1491 sclerotia, 614 (41.18%) were positive by LAMP, and 629 (42.19%) positive by MIC. Therefore, the LAMP assay is more feasible to detect benzimidazole-resistant mutants of S. sclerotiorum than traditional detection methods.

No MeSH data available.


Related in: MedlinePlus

Specificity of LAMP detection of the E198A mutant in S. sclerotiorum. Assessment was based on (A) HNB visualization of color change or (B) gel electrophoresis analysis of the LAMP products. M, 2K plus; 1, wild type of S. sclerotiorum; 2, E198A genotype of S. sclerotiorum; 3, F200Y genotype of S. sclerotiorum; 4, E198K genotype of F. graminearum; 5, E198L genotype of F. graminearum; 6, E198V genotype of B. cinerea; 7, E198A genotype of B. cinerea; 8, E198K genotype of B. cinerea; 9, ddH2O.
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f5: Specificity of LAMP detection of the E198A mutant in S. sclerotiorum. Assessment was based on (A) HNB visualization of color change or (B) gel electrophoresis analysis of the LAMP products. M, 2K plus; 1, wild type of S. sclerotiorum; 2, E198A genotype of S. sclerotiorum; 3, F200Y genotype of S. sclerotiorum; 4, E198K genotype of F. graminearum; 5, E198L genotype of F. graminearum; 6, E198V genotype of B. cinerea; 7, E198A genotype of B. cinerea; 8, E198K genotype of B. cinerea; 9, ddH2O.

Mentions: The F200Y mutation genotype of S. sclerotiorum, the E198K, E198L mutation genotypes of F. graminearum, and the E198V, E198A, E198K mutation genotypes of B. cinerea were used as negative controls to determine the specificity of LAMP. From HNB-visualization (Fig. 5A) or gel electrophoresis (Fig. 5B), LAMP was positive only for the E198A mutant TZ25 of S. sclerotiorum, but not for other mutants or isolates. This indicated that the established LAMP method had a high level of specificity for detecting the E198A mutation genotype of S. sclerotiorum.


Development and application of loop-mediated isothermal amplification for detecting the highly benzimidazole-resistant isolates in Sclerotinia sclerotiorum.

Duan YB, Yang Y, Wang JX, Liu CC, He LL, Zhou MG - Sci Rep (2015)

Specificity of LAMP detection of the E198A mutant in S. sclerotiorum. Assessment was based on (A) HNB visualization of color change or (B) gel electrophoresis analysis of the LAMP products. M, 2K plus; 1, wild type of S. sclerotiorum; 2, E198A genotype of S. sclerotiorum; 3, F200Y genotype of S. sclerotiorum; 4, E198K genotype of F. graminearum; 5, E198L genotype of F. graminearum; 6, E198V genotype of B. cinerea; 7, E198A genotype of B. cinerea; 8, E198K genotype of B. cinerea; 9, ddH2O.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4660316&req=5

f5: Specificity of LAMP detection of the E198A mutant in S. sclerotiorum. Assessment was based on (A) HNB visualization of color change or (B) gel electrophoresis analysis of the LAMP products. M, 2K plus; 1, wild type of S. sclerotiorum; 2, E198A genotype of S. sclerotiorum; 3, F200Y genotype of S. sclerotiorum; 4, E198K genotype of F. graminearum; 5, E198L genotype of F. graminearum; 6, E198V genotype of B. cinerea; 7, E198A genotype of B. cinerea; 8, E198K genotype of B. cinerea; 9, ddH2O.
Mentions: The F200Y mutation genotype of S. sclerotiorum, the E198K, E198L mutation genotypes of F. graminearum, and the E198V, E198A, E198K mutation genotypes of B. cinerea were used as negative controls to determine the specificity of LAMP. From HNB-visualization (Fig. 5A) or gel electrophoresis (Fig. 5B), LAMP was positive only for the E198A mutant TZ25 of S. sclerotiorum, but not for other mutants or isolates. This indicated that the established LAMP method had a high level of specificity for detecting the E198A mutation genotype of S. sclerotiorum.

Bottom Line: The point mutation at codon 198 (GAG → GCG, E198A) occurs in more than 90% of field resistant populations in China.Traditional detection methods of benzimidazole-resistant mutants of S. sclerotiorum are time-consuming, tedious and inefficient.This method had a good specificity, sensitivity, stability and repeatability.

View Article: PubMed Central - PubMed

Affiliation: College of Plant Protection, State &Local Joint Engineering Research Center of Green Pesticide Invention and Application, Nanjing Agricultural University, Nanjing, 210095, China.

ABSTRACT
Resistance of benzimidazole fungicides is related to the point mutation of the β-tubulin gene in Sclerotinia sclerotiorum. The point mutation at codon 198 (GAG → GCG, E198A) occurs in more than 90% of field resistant populations in China. Traditional detection methods of benzimidazole-resistant mutants of S. sclerotiorum are time-consuming, tedious and inefficient. To establish a suitable and rapid detection of benzimidazole-resistant mutants of S. sclerotiorum, an efficient and simple method with high specificity was developed based on loop-mediated isothermal amplification (LAMP). Eight sets of LAMP primers were designed and four sets were optimized to specially distinguish benzimidazole-resistant mutants of S. sclerotiorum. With the optimal LAMP primers, the concentration of LAMP components was optimized and the reaction conditions were set as 60-64 °C for 60 min. This method had a good specificity, sensitivity, stability and repeatability. In the 1491 sclerotia, 614 (41.18%) were positive by LAMP, and 629 (42.19%) positive by MIC. Therefore, the LAMP assay is more feasible to detect benzimidazole-resistant mutants of S. sclerotiorum than traditional detection methods.

No MeSH data available.


Related in: MedlinePlus