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High‐throughput detection of highly benzimidazole‐resistant allele E198A with mismatch primers in allele‐specific real‐time polymerase chain reaction
Author(s) -
Chen Changjun,
Zhao Wei,
Lu Yuejian,
Wang Jianxin,
Chen Yu,
Li Hongxia,
Zhou Mingguo
Publication year - 2009
Publication title -
pest management science
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.296
H-Index - 125
eISSN - 1526-4998
pISSN - 1526-498X
DOI - 10.1002/ps.1691
Subject(s) - genotyping , biology , variants of pcr , polymerase chain reaction , genotype , genetics , allele , point mutation , microbiology and biotechnology , primer (cosmetics) , genomic dna , gene , mutation , chemistry , organic chemistry
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 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 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. Copyright © 2009 Society of Chemical Industry