Chen Yizhou, Bogema Daniel R, Barchia Idris M, Herron Grant A
Elizabeth Macarthur Agricultural Institute, NSW Department of Primary Industries, Menangle, New South Wales, Australia.
PLoS One. 2014 Mar 10;9(3):e91104. doi: 10.1371/journal.pone.0091104. eCollection 2014.
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.
METHODOLOGY/PRINCIPAL FINDINGS: 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/SIGNIFICANCE: 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.
农药抗性监测是农业生产系统实现可持续害虫综合治理（IPM）的关键部分。节肢动物种群抗性监测最初通过生物测定法进行，该方法检测对农药的表型反应。当确定抗性的致病突变后，可开发出提高速度和成本的分子诊断测定法。然而，由于基因分型方法不能准确应用于混合DNA，通量的提高受到限制。从混合DNA中定量等位基因频率将有助于更快、更便宜地监测农药抗性。
方法/主要发现：我们展示了一种通过TaqMan测定法从混合昆虫中定量抗性等位基因频率（RAF）的新方法，即利用原始荧光数据基于四参数S形曲线计算拐点处的转化荧光比率k'。我们的结果表明，k'具有可重复性，且与RAF高度相关（r>0.99）。我们还证明k'与RAF呈非线性关系，五个标准点足以构建预测模型。此外，我们确定了k'在不同运行之间的非线性关系，允许在单次分析中将多个运行的样本合并。
结论/意义：转化荧光比率（k'）方法可用于IPM中监测农药抗性，并从混合样本中准确量化等位基因频率。我们已确定五个标准（0.0、0.2、0.5、0.8和1.0）足以进行准确预测，并且在统计学上等同于实验中使用的13个标准点。
