AUTHOR=Zhang Xin , Xie Fei , Lv Baobei , Zhao Pengxiang , Ma Xuemei 

TITLE=Suspension Array for Multiplex Detection of Eight Fungicide-Resistance Related Alleles in Botrytis cinerea

JOURNAL=Frontiers in Microbiology

VOLUME=7

YEAR=2016

URL=https://www.frontiersin.org/journals/microbiology/articles/10.3389/fmicb.2016.01482

DOI=10.3389/fmicb.2016.01482

ISSN=1664-302X

ABSTRACT=<p>A simple and high-throughput assay to detect fungicide resistance is required for large-scale monitoring of the emergence of resistant strains of <italic>Botrytis cinerea</italic>. 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-Tubulin <italic>gene</italic> (<italic>BenA</italic>), H272 and 272Y of the Succinate dehydrogenase iron–sulfur subunit gene (<italic>SdhB)</italic>, I365 and 365S of the putative osmosensor histidine kinase gene (<italic>BcOS1</italic>), and F412 and 412S of the 3-ketoreductase gene (<italic>erg27</italic>). This assay was first established and optimized with eight plasmid templates containing the DNA sequence variants <italic>BenA-</italic>E198, <italic>BenA-</italic>198A, <italic>SdhB-</italic>H272, <italic>SdhB-</italic>272Y, <italic>BcOS1-</italic>I365, <italic>BcOS1-</italic>365S, <italic>erg27</italic>-F412, and <italic>erg27</italic>-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% for <italic>BenA-</italic>E198A, <italic>BcOS1-</italic>I365S, and <italic>erg27</italic>-F412S, and was 4.5% for <italic>SdhB-</italic>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 in <italic>BenA</italic>, <italic>SdhB</italic>, <italic>BcOS1</italic>, and <italic>erg27</italic>.</p>