Development of a real-time immuno-PCR assay for the quantification of environmental contaminants

Abstract

This thesis outlines the development of two universal real-time immuno-PCR (RT-iPCR) assays for application in agriculture. The first RT-iPCR was developed for the sensitive quantification of 17β-estradiol in water. Using a universal iPCR method and polyclonal antibodies, 17β-estradiol was accurately quantified at concentrations ranging from 1 pg mL-1 to 10 μg mL-1, with a limit of detection of 0.7 pg mL-1. The RT-iPCR assay provided an 800-fold increase in sensitivity as well as an expanded working range compared to the corresponding enzyme-linked immunosorbent assay. Antibodies were swapped with antibodies specific to P. brassicae for the quantification of clubroot resting spores using the same RT-iPCR assay. P. brassicae resting spores were quantified in the range of 50 to 10 000 spores, with a detection limit of 29 spores. Both RT-iPCR showed equal or improved sensitivity compared to other published analytical methods, with expanded linear working ranges and high-throughput capabilities.