title: Suspension Array for Multiplex Detection of Eight Fungicide-Resistance Related Alleles inBotrytis cinerea.
abstract: A simple and high-throughput assay to detect fungicide resistance is required for large-scale monitoring of the emergence of resistant strains ofBotrytis cinerea. Using suspension array technology performed on a Bio-Plex 200 System, we developed a single-tube allele-specific primer extension assay that can simultaneously detect eight alleles in one reaction. These eight alleles include E198 and 198A of the β-Tubulingene(BenA), H272 and 272Y of the Succinate dehydrogenase iron-sulfur subunit gene (SdhB), I365 and 365S of the putative osmosensor histidine kinase gene (BcOS1), and F412 and 412S of the 3-ketoreductase gene (erg27). This assay was first established and optimized with eight plasmid templates containing the DNA sequence variantsBenA-E198,BenA-198A,SdhB-H272,SdhB-272Y,BcOS1-I365,BcOS1-365S,erg27-F412, anderg27-412S. Results indicated that none of the probes showed cross-reactivity with one another. The minimum limit of detection for these genotypes was one copy per test. Four mutant plasmids were mixed with 10 ng/μL wild-type genomic DNA in different ratios. Detection sensitivity of mutant loci was 0.45% forBenA-E198A,BcOS1-I365S, anderg27-F412S, and was 4.5% forSdhB-H272Y. A minimum quantity of 0.1 ng of genomic DNA was necessary to obtain reliable results. This is the first reported assay that can simultaneously detect mutations inBenA,SdhB,BcOS1, anderg27.
authors.affiliation: College of Life Science and Bioengineering, Beijing University of Technology Beijing, China.
authors.forename: Xin
authors.initials: X
authors.lastname: Zhang
authors.affiliation: College of Life Science and Bioengineering, Beijing University of Technology Beijing, China.
authors.forename: Fei
authors.initials: F
authors.lastname: Xie
authors.affiliation: College of Life Science and Bioengineering, Beijing University of Technology Beijing, China.
authors.forename: Baobei
authors.initials: B
authors.lastname: Lv
authors.affiliation: College of Life Science and Bioengineering, Beijing University of Technology Beijing, China.
authors.forename: Pengxiang
authors.initials: P
authors.lastname: Zhao
authors.affiliation: College of Life Science and Bioengineering, Beijing University of Technology Beijing, China.
authors.forename: Xuemei
authors.initials: X
authors.lastname: Ma
keywords: ASPE PCR; Botrytis cinerea; fungicide resistance; multiplex detection; suspension array