High-throughput detection of highly benzimidazole-resistant allele E198A with mismatch primers in allele-specific real-time polymerase chain reaction
2009
Chen, Changjun | Zhao, Wei | Lu, Yuejian | Wang, Jianxin | Chen, Yu | Li, Hongxia | Zhou, Mingguo
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 MBCHR mutation (E198A) in the β-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 MBCHR and benzimidazole-sensitive MBCS 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.
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