Fumian TM, Leite JPG, et al. (2016) Performance of a one-step quantitative duplex RT-PCR for detection of rotavirus A and noroviruses GII during two periods of high viral circulation. J Virol Methods, 228:123–129. doi:10.1016/j.jviromet.2015.11.008.
Multiplex PCR is ideal for limited samples
In this paper, Fumian et al. describe a sensitive duplex RT-qPCR method that uses fluorescently labeled qPCR probes for pathogen detection. Multiplex RT-qPCR offers several technical advantages when working with limited samples, while continuing to provide the same level of sensitivity as singleplex reactions. As little as nanogram amounts of sample can be used to assay multiple targets in a single reaction well. Here, the researchers used a one-step RT-qPCR protocol to assay just 5 ng of total RNA for each duplex RT-qPCR. Multiplex reactions also reduce the number of pipetting steps, limiting technical errors, experimental variation, and reaction cross contamination.
Quantitative standards for multiplex RT-qPCR
In their assay design, the researchers used a single, synthetic dsDNA fragment containing both NoV and RVA viral target regions—ordered as a gBlocks® Gene Fragment (IDT)—as their quantitative standard. Having control amplicons for both target genes in an exact 1:1 ratio allows accurate optimization of both assays in the reaction. The single control template ensures amplification efficiencies are equal, a requirement for multiplex RT-qPCR to provide quantitative results. gBlocks Gene Fragments make it easy to generate multiplex standards, because they are entirely synthetic—control amplicon sequences are simply designed on the same dsDNA molecule.
Increased sensitivity for viral particle detection
In a comparison with commonly used enzyme immunoassays (EIA), the researchers showed that their multiplex RT-qPCR method is up to 11% more sensitive in detecting viral particles in the diluted fecal samples used in this study, with a limit of detection of 8 genome copies. The assay is also easier to perform than current EIA methods or multiple singleplex reactions. While Fumian et al. focused on only 2 targets, some qPCR instruments are capable of simultaneously distinguishing greater numbers of fluorophores, enabling assessment of a larger number of targets in similar multiplex amplification reactions.