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.


CONCLUSIONS
ASQPCR-MP appears suitable for large epidemiological studies involving resistant genotypes and requiring high-throughout formats.
Chen
Changjun
C
College of Plant Protection, Nanjing Agricultural University, Nanjing 210095, China. ccj100cn@yahoo.com.cn
Zhao
Wei
W
Lu
Yuejian
Y
Wang
Jianxin
J
Chen
Yu
Y
Li
Hongxia
H
Zhou
Mingguo
M
D000483:Alleles; D000818:Animals; D001203:Ascomycota / Q000187:drug effects* / Q000235:genetics; D001562:Benzimidazoles / Q000494:pharmacology*; D025141:Drug Resistance, Fungal; D005659:Fungicides, Industrial / Q000494:pharmacology*; D017354:Point Mutation; D016133:Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity
D001562:Benzimidazoles; D005659:Fungicides, Industrial; C031000:benzimidazole