Strategic Approach To Produce Low-Cost, Efficient, and Stable Competitive Internal Controls for Detection of RNA Viruses by Use of Reverse Transcription-PCR

Quantitative real-time RT-PCR assay for research studies on enterovirus infections in the central nervous system

“…The IC RNA included in the assay is amplified competitively with the same set of primers dedicated to the specific amplification of the EV template. Another major interest of the IC RNA is its incorporation directly in the biological specimens to be tested before RNA extraction, which allows close monitoring of the entire sample process (Naze et al, 2009;Villanova et al, 2007;Xiao et al, 2009). Because of its competitive property, particular attention has been paid to the molecular design of IC to avoid cross hybridization with the EV template genome and to ensure optimal amplification of both IC and EV templates.…”

PCR inhibitors - occurrence, properties and removal

“…; Villanova et al . ). One limiting factor of this application may be the concurrent use of primers, which may lead to a general decrease in PCR sensitivity (Hoorfar et al .…”

“…; Villanova et al . ). A possible disadvantage of non‐competitive controls is the use of separate primers, which may behave different than those used for the target sequence (Villanova et al .…”

“…A possible disadvantage of non‐competitive controls is the use of separate primers, which may behave different than those used for the target sequence (Villanova et al . ). Generally, by using internal controls in multiplex PCR assays, the co‐amplification of the control may inhibit the amplification of the target sequence, which might be solved by limiting the concentration of the control primers and control RNA (Hofmann ; Gall et al .…”

Routine use of duplex real-time PCR assays including a commercial internal control for molecular diagnosis of opportunistic DNA virus infections

“…The only point is that each laboratory has to determine its own limits and establish its own procedure for the validation of PCR results obtained with its laboratory-developed PCR techniques. Of note, the RNA SEAC is also available, which may facilitate the improvement of laboratory-made RT-PCRs (Villanova et al, 2007). Another advantage is the cost-effectiveness of this SEAC.…”