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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

Optimization of LAMP reaction temperature.(A) Assessment was based on HNB visualization of color change of the LAMP products. (B) Assessment was based on gel electrophoresis analysis of the LAMP products.
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f3: Optimization of LAMP reaction temperature.(A) Assessment was based on HNB visualization of color change of the LAMP products. (B) Assessment was based on gel electrophoresis analysis of the LAMP products.

Mentions: With genomic DNA of the mutant TZ25 as the templates, optimization of reaction temperature and time was performed on basis of the above optimized LAMP reaction components. Color change and the ladder-like pattern of the LAMP products were observed at 60–64 °C (Fig. 3A) and the intensity of DNA was similar (Fig. 3B). With the appropriate temperature (62 °C), the reaction time was optimized and the positive LAMP products were observed from 60 to 120 min by HNB-visualized color change (Fig. 4A) and from 45-120 min by gel electrophoresis (Fig. 4B). When the reaction time reached to 45 min, the color of LAMP products was slightly changed and the intensity of ladder-like pattern on gel electrophoresis was low, indicating the amplification efficiency of LAMP was low at 45 min. Therefore, the appropriate reaction condition of the established LAMP method was set as 60–64 °C for 60 min.


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)

Optimization of LAMP reaction temperature.(A) Assessment was based on HNB visualization of color change of the LAMP products. (B) Assessment was based on gel electrophoresis analysis of the LAMP products.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f3: Optimization of LAMP reaction temperature.(A) Assessment was based on HNB visualization of color change of the LAMP products. (B) Assessment was based on gel electrophoresis analysis of the LAMP products.
Mentions: With genomic DNA of the mutant TZ25 as the templates, optimization of reaction temperature and time was performed on basis of the above optimized LAMP reaction components. Color change and the ladder-like pattern of the LAMP products were observed at 60–64 °C (Fig. 3A) and the intensity of DNA was similar (Fig. 3B). With the appropriate temperature (62 °C), the reaction time was optimized and the positive LAMP products were observed from 60 to 120 min by HNB-visualized color change (Fig. 4A) and from 45-120 min by gel electrophoresis (Fig. 4B). When the reaction time reached to 45 min, the color of LAMP products was slightly changed and the intensity of ladder-like pattern on gel electrophoresis was low, indicating the amplification efficiency of LAMP was low at 45 min. Therefore, the appropriate reaction condition of the established LAMP method was set as 60–64 °C for 60 min.

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