Development of a Quantitative Real-Time Polymerase Chain Reaction (RT-PCR) Assay for Plant Species

In order to facilitate optimal plant DNA quantitation and identification, an assay has been developed that uses generic plant PCR primers that amplify a region in the chloroplast genome of plant samples. The assay uses the SYBR green detection dye to detect the PCR product with a universal PCR primer set to the large subunit of ribulose bisphosphate carboxylase, rbcL, but can be used with any of the universal barcode primers for land plants (rbcL, matK, trnH, psbA). Standard dilutions of control wheat DNA of varying concentrations were tested to create a standard curve. Several plant DNA extractions of different species of unknown concentrations were also tested and the concentrations were quantified from the standard curve. This paper discusses the experimental procedures used to develop and optimize a real-time PCR assay for plants in order to detect plant species that is modeled after the human DNA detection system called Quantifiler for forensic applications. In principle, this can be used to quantitate DNA from any chloroplast containing plant species that is present as trace material at a crime scene, grass stains, or for food and drug analysis