![]() Some of the common detection quantitative PCR (qPCR) methods are restricted in the limit of detection (LoD) because of the high polymerase misincorporation rate in Taq DNA polymerases. It is essential to detect rare DNA-sequence variants for early cancer diagnosis or for drug-resistance mutations identification. tritici.Įffective polymerase chain reactions (PCR) are important in bio-laboratories. The method developed in this study can be readily adopted by other laboratories and used for multiple applications including resistance monitoring in field populations of Z. The frequency of S524T was highly contrasted in the airborne inoculum of all four countries, however, the H152R allele was never detected in the airborne inoculum. The S524T allele was present in all field samples and its proportion was significantly higher in Ireland than in Belgium, whereas the proportion of H152R was only sporadically present in both countries. The assay was then used on complex DNA samples originating from a spore trap network set up in Belgium, Denmark, Sweden and Ireland in 20 as well as on symptomatic leaf samples. tritici populations, a multiplex allele‐specific qPCR assay allowing for estimation of both allele frequencies in bulk DNA matrices was developed. To facilitate further studies on the monitoring and selection of these subsitutions in Z. Among the different amino acid substitutions in Zymoseptoria tritici associated with resistance to these fungicides, S524T in CYP51 (DMI target) and H152R in SdhC (SDHI target) are regarded as conferring the highest resistance factors to DMI and SDHI, respectively. However, multiple mutations have occurred over time in the genes encoding the targeted proteins which have led to a practical loss of fungicide efficacies. This new method can be used to quantify a complex quadriallelic SNP in a DNA pool with a false discovery rate of less than 1%.ĭemethylation inhibitor (DMI) and succinate dehydrogenase inhibitor (SDHI) fungicides are currently relied upon for the control septoria tritici blotch (STB) in European wheat fields. The levels of nonspecific amplification with the ASPPAA PCR were reduced at least four times below the level of currently available allele-specific real-time PCR approaches due to strong allele specificity in amplification cycles, including two allele selectors. It was developed for a single-nucleotide polymorphism (SNP) that is responsible for resistance to the sterol biosynthesis inhibitor fungicide fenhexamid, resulting in the replacement of the phenylalanine residue (encoded by the TTC codon) in position 412 of the enzymatic target (3-ketoreductase) by a serine (TCC), valine (GTC), or isoleucine (ATC) residue. This method makes use of mixtures of allele-specific minor groove binder (MGB) TaqMan probes and allele-specific primers for the fine quantification of SNPs from a pool of DNA extracted from a mixture of conidia. We describe here a new real-time PCR method, the allele-specific probe and primer amplification assay (ASPPAA PCR). Allele-specific real-time PCR approaches, such as amplification refractory mutation system (ARMS) PCR and mismatch amplification mutation assay (MAMA) PCR, are, despite their moderate efficacy, among the most precise methods for refining SNP quantification. Molecular monitoring methods can be considered if the mutations responsible for resistance have been identified. Such monitoring often is based on microbiological tests, such as microtiter plate assays. The evolution of fungicide resistance within populations of plant pathogens must be monitored to develop management strategies.
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