title: Quantification of the pirimicarb resistance allele frequency in pooled cotton aphid (Aphis gossypii Glover) samples by TaqMan SNP genotyping assay.
abstract: BACKGROUND
  Pesticide resistance monitoring is a crucial part to achieving sustainable integrated pest management (IPM) in agricultural production systems. Monitoring of resistance in arthropod populations is initially performed by bioassay, a method that detects a phenotypic response to pesticides. Molecular diagnostic assays, offering speed and cost improvements, can be developed when the causative mutation for resistance has been identified. However, improvements to throughput are limited as genotyping methods cannot be accurately applied to pooled DNA. Quantifying an allele frequency from pooled DNA would allow faster and cheaper monitoring of pesticide resistance.
  
  
  RESULTS
  We demonstrate a new method to quantify a resistance allele frequency (RAF) from pooled insects via TaqMan assay by using raw fluorescence data to calculate the transformed fluorescence ratio k' at the inflexion point based on a four parameter sigmoid curve. Our results show that k' is reproducible and highly correlated with RAF (r >0.99). We also demonstrate that k' has a non-linear relationship with RAF and that five standard points are sufficient to build a prediction model. Additionally, we identified a non-linear relationship between runs for k', allowing the combination of samples across multiple runs in a single analysis.
  
  
  CONCLUSIONS
  The transformed fluorescence ratio (k') method can be used to monitor pesticide resistance in IPM and to accurately quantify allele frequency from pooled samples. We have determined that five standards (0.0, 0.2, 0.5, 0.8, and 1.0) are sufficient for accurate prediction and are statistically-equivalent to the 13 standard points used experimentally.
authors.affiliation: Elizabeth Macarthur Agricultural Institute, NSW Department of Primary Industries, Menangle, New South Wales, Australia.
authors.forename: Yizhou
authors.initials: Y
authors.lastname: Chen
authors.affiliation: Elizabeth Macarthur Agricultural Institute, NSW Department of Primary Industries, Menangle, New South Wales, Australia.
authors.forename: Daniel R
authors.initials: DR
authors.lastname: Bogema
authors.affiliation: Elizabeth Macarthur Agricultural Institute, NSW Department of Primary Industries, Menangle, New South Wales, Australia.
authors.forename: Idris M
authors.initials: IM
authors.lastname: Barchia
authors.affiliation: Elizabeth Macarthur Agricultural Institute, NSW Department of Primary Industries, Menangle, New South Wales, Australia.
authors.forename: Grant A
authors.initials: GA
authors.lastname: Herron
mesh_terms: D000818:Animals; D001042:Aphids / Q000187:drug effects / Q000235:genetics*; D001315:Australia; D002219:Carbamates / Q000633:toxicity*; D005453:Fluorescence; D005787:Gene Frequency / Q000235:genetics*; D060005:Genotyping Techniques / Q000379:methods*; D003368:Gossypium / Q000469:parasitology*; D007305:Insecticide Resistance / Q000187:drug effects / Q000235:genetics*; D010957:Plasmids / Q000378:metabolism; D016133:Polymerase Chain Reaction; D012150:Polymorphism, Restriction Fragment Length; D020641:Polymorphism, Single Nucleotide / Q000235:genetics*; D011743:Pyrimidines / Q000633:toxicity*; D012015:Reference Standards; D012621:Seasons; D019914:Taq Polymerase / Q000378:metabolism
chemical_list: D002219:Carbamates; D011743:Pyrimidines; C011994:pirimicarb; D019914:Taq Polymerase