scholarly journals Fluorescence resonance energy transfer-based real-time polymerase chain reaction method without DNA extraction for the genotyping of F5, F2, F12, MTHFR, and HFE

2014 ◽  
pp. 99 ◽  
Author(s):  
Jordi Martinez-Serra ◽  
Juan Robles ◽  
Antoni Nicolas ◽  
Antonio Gutierrez ◽  
Teresa Ros ◽  
...  
Keyword(s):  
Polymerase Chain Reaction ◽  
Energy Transfer ◽  
Real Time ◽  
Dna Extraction ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Polymerase Chain Reaction Method ◽  
Chain Reaction ◽  
Reaction Method ◽  
Polymerase Chain

scholarly journals Genotyping of Benzimidazole-Resistant and Dicarboximide-Resistant Mutations in Botrytis cinerea Using Real-Time Polymerase Chain Reaction Assays

2008 ◽  
Vol 98 (4) ◽  
pp. 397-404 ◽  
Author(s):  
Shinpei Banno ◽  
Fumiyasu Fukumori ◽  
Akihiko Ichiishi ◽  
Kiyotsugu Okada ◽  
Hidetoshi Uekusa ◽  
...  
Keyword(s):  
Polymerase Chain Reaction ◽  
Real Time ◽  
Botrytis Cinerea ◽  
Melting Curve ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Cross Resistance ◽  
Melting Curve Analysis ◽  
Chain Reaction ◽  
Polymerase Chain

Botrytis cinerea, an economically important gray mold pathogen, frequently exhibits multiple fungicide resistance. A fluorescence resonance energy transfer-based real-time polymerase chain reaction assay has been developed to detect benzimidazole- and dicarboximide-resistant mutations. Three benzimidazole-resistant mutations—198Glu to Ala (E198A), F200Y, and E198K—in β-tubulin BenA were detected using a single set of fluorescence-labeled sensor and anchor probes by melting curve analysis. Similarly, three dicarboximide-resistant mutations—I365S, V368F plus Q369H, and Q369P—in the histidine kinase BcOS1 were successfully distinguished. Unassigned melting profiles in BenA genotyping assay resulted in the identification of a new benzimidazole-resistant BenA E198V mutation. This mutation conferred resistance to carbendazim as do E198A and E198K mutations. The isolates with BenA E198V mutation showed a negative cross-resistance to diethofencarb, but to a lesser extent than the E198A mutants. A survey of 210 B. cinerea field isolates revealed that most of benzimidazole-resistant isolates possessed the E198V or E198A mutation in the BenA gene, and the I365S mutation in the BcOS1 gene was also frequently observed in Japanese isolates. However, benzimidazole-resistant isolates with BenA F200Y or E198K mutations, which confer the diethofencarb-insensitive phenotype, were rare. Our BenA and BcOS1 genotyping is a rapid and reliable method that is suitable for monitoring the fungicide-resistant field population.

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