Sidebar: PCR Multiplexing
Scientists at Biosearch Technologies say that dramatic improvements in detecting pathogens in food and agricultural products and the environment can be obtained by multiplexing PCR assays. Spectrally distinct fluorophores and quenchers provide the ability to detect genetic signatures and distinguish closely related strains, all within the same reaction chamber, according to the researchers.
In a poster entitled “Intricacies of PCR Multiplexing as Revealed through a Pathogen Detection Assay,” the Biosearch team reported the development of a quadruplexed assay that distinguishes several strains of Bacillus anthracis from a similar organism, B. cereus. The scientists relied on careful selection of target sequences, fluorescent reporters, and a real-time PCR instrument for detection.
They used RealTimeDesign™ software to generate TaqMan® assays to the species-specific sequences. To multiplex these assays together the scientists characterized dye crosstalk, confirmed that each assay had a high amplification efficiency, and identified their detection limit, especially in the context of disproportionate targets.
“For situations where one sequence may be in vast excess over the others, we show the benefit of supplementing master mix components so that depleted reagents don’t limit detection sensitivity,” they wrote. “Multiplexing demands increased effort to characterize amplification performance but is ideally suited to interrogate multiple genetic signatures from small quantities of sample DNA."