High‐throughput detection of highly benzimidazole‐resistant allele E198A with mismatch primers in allele‐specific real‐time polymerase chain reaction

BACKGROUND: A point mutation often confers resistance of organisms against medical drugs and agricultural pesticides. Allele-specific nucleotide polymerase chain reaction (ASPCR) and allele-specific quantitative real-time PCR using SYBR Green (ASQPCR) are widely and effectively applied to detect and monitor this type of resistance. However, the former is unsuitable for high-throughput detection, and the latter often reduces the accuracy of detection. RESULTS: In order to decrease background amplification, a rapid and high-throughput genotyping method with mismatch primers was developed (ASQPCR-MP) and applied specifically to survey the frequency of the highly benzimidazole-resistant MBC(HR) mutation (E198A) in the beta-tubulin gene of Sclerotinia sclerotiorum (Lib.) de Bary populations. Genomic DNA from 223 sclerotia was analysed. Similar genotype results were also obtained using ASPCR with mismatch primers and a mycelial growth inhibition assay. It was found that ASQPCR-MP clearly differentiated MBC(HR) and benzimidazole-sensitive MBC(S) phenotypes. Moreover, ASQPCR-MP took less than 6 h to complete. CONCLUSION: ASQPCR-MP appears suitable for large epidemiological studies involving resistant genotypes and requiring high-throughout formats.