The Allele-Specific Probe and Primer Amplification Assay, a New Real-Time PCR Method for Fine Quantification of Single-Nucleotide Polymorphisms in Pooled DNA

Abstract

The evolution of fungicide resistance within populations of plant pathogens must be monitored to develop management strategies. Such monitoring often is based on microbiological tests, such as microtiter plate assays. Molecular monitoring methods can be considered if the mutations responsible for resistance have been identified. 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. We describe here a new real-time PCR method, the allele-specific probe and primer amplification assay (ASPPAA PCR). This method makes use of mixtures of allelespecific 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. 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. 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. 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%. Fungicide resistance and its management are of great importance in crop protection. The monitoring of this resistance is a crucial area of research, one on which our knowledge of the distribution, evolution, and effect of fungicide resistance in the field depends. In most cases, the degree of sensitivity of fungal populations to one or more fungicides is assessed by biological methods (17). These bioassays, conducted in vitro or in vivo, have been miniaturized (i.e., microtiter plate methods), but nonetheless they consume considerable resources and time. When the molecular mechanisms of resistance are known (e.g., target mutation, target overexpression, and increased drug efflux), and particularly when the underlying DNA polymorphisms (single-nucleotide polymorphism [SNPs], deletions, or insertions) have been defined, various molecular methods can be used to monitor antimicrobial resistance (8, 14, 15). The principle methods for quantifying resistance are based on real-time PCR technology. Alleles are amplified in a specific manner, either independently or in multiplex systems, with allele-specific probes or primers. Polymorphic alleles then are quantified by the cycle of quantification values and compared to the wild-type values (Cq; i.e., at a given threshold, Cq is the number of PCR cycles at which reporter fluorescence becomes significant or is distinguishable from the backg...

Molecular characterisation and detection of resistance to succinate dehydrogenase inhibitor fungicides in Botryotinia fuckeliana (Botrytis cinerea)

“…and Venturia inaequalis (Cooke) G. Winter . Recently, quantitative PCR assays based on the use of AS probes have been proposed for accurate quantification of SNP frequencies responsible for a high level of resistance to fenhexamid in B. fuckeliana populations …”

Using next‐generation sequencing to detect mutations endowing resistance to pesticides: application to acetolactate‐synthase (ALS)‐based resistance in barnyard grass, a polyploid grass weed

“…19,20 Following technology evolution, allele-specific PCR has been at the basis of 'hi-tech', high-throughput assays implementing quantitative PCR. 21,22 Other genotyping techniques allowing high-throughput detection of mutations at known positions have also been used to develop pesticide resistance diagnosis, such as MALDI-TOF, 23 TaqMan, 24,25 SimpleProbe, 26 DNA microarrays 27 or SNaPshot. 28 A common feature of all the techniques described above is that they only provide information about the mutation(s) present at a given nucleotide position or codon, which generally requires identification of the mutation(s) in question beforehand.…”

Resistance profiles of Botrytis cinerea populations to several fungicide classes on greenhouse tomato and strawberry in Lebanon

“…Traditional methods for detection and quantification of fungicide resistance in fungal populations are biological assays based on colony growth and spore germination inhibition on media amended with fungicides. Additionally, when the molecular mechanisms of resistance are known, several molecular techniques based on the detection of single‐nucleotide polymorphisms (SNPs) are used (De Miccolis Angelini et al ., 2010, 2012; Billard et al ., 2012a; Samaras et al ., 2016).…”