Development of a universal internal positive control

Abstract

Described here is a novel, universal exogenous internal positive control (IPC), which is fully synthetic for unparalleled quality control. The IPC was rationally designed, is small and efficiently amplified, has been successfully utilized alone or in triplex qPCR reactions, and is not crossreactive to human DNA or to any of the numerous non-human DNA samples tested.It was sensitive to inhibition by at least four commonly encountered amplification inhibitors. The IPC was used in a hydrolysis probe qPCR assay; however, it may be adaptable for other applications such as intercalating dye qPCR, PCR, isothermal amplification and reverse transcription-PCR.

A Pipeline for Faecal Host DNA Analysis by Absolute Quantification of LINE-1 and Mitochondrial Genomic Elements Using ddPCR

“…4), this is small relative to the observed day-to-day variations. However in future studies, variation in DNA recovery efficiency could be measured and corrected for using synthetic DNA of artificial sequence spiked into the samples prior to DNA extraction 37 . Taken together, our data suggest that the majority of observed day-to-day variation is likely biological in origin, perhaps related to variation in diet, GI tract environment, GI inflammation or other factors.…”

Developmental data for several human mitochondrial DNA (mtDNA) long amplification targets

“…The novel IPC template similarly consisted of two complementary, PAGE-purified synthetic oligonucleotides (Ultramers; Integrated DNA Technologies) which were reconstituted in TE, quantified by absorbance at 260 nm using the extinction coefficients 628,700 and 621,800 L/(mole•cm), respectively, prepared as a duplex in equimolar proportions, and diluted to 1,250 copies/µL [2 , 3] . HL60 DNA (ATCC), diluted to 20 pg/µL in TE, served as the calibrator in triplex reactions [2] .…”

“…The data was used to develop a triplex qPCR method, which includes amplification of one of the long targets, to quantify and assess degradation of human mtDNA, the results of which were previously published [2] . That triplex method also incorporated an internal positive control to test for the presence of amplification inhibitors in the sample [3] . The data presented herein may be used to develop alternative amplification methods for user-specific biomedical applications.…”

A short interspersed nuclear element-based quantitative PCR assay for simultaneous human and dog DNA detection and quantification

“…The canine target is the SINEC Cf element, which has approximately 200,000 copies per cell and is detected in the assay with a 99-bp amplicon and a VIC-labeled probe with an MGBNFQ quencher [3]. The IPC assay is a universal exogenous assay developed by Mark F Kavlick consisting of forward and reverse primers, a NED-labeled probe with an MGBNFQ quencher and a 65-bp synthetic template that does not share sequence homology with human and various non-human genomes [7]. The target amplicons are small, similarly sized DNA fragments that are in the common size range for forensic samples and that can be prone to degradation because of environmental conditions.…”

“…TaqMan probes were ordered HPLC-purified in 1x TE buffer from Applied Biosystems. Aliquots of the primers, NED-labeled probe and template for the IPC were provided by Mark F Kavlick [7]. The PCR cocktail consisted of 5 μl of TaqMan fast advanced master mix (Applied Biosystems), 0.4 μl of 10 μM human forward primer, 0.8 μl of 10 μM human reverse primer, 0.4 μl of 10 μM dog forward primer, 0.1 μl of 10 μM dog reverse For the human and dog assays, the threshold was manually set to 0.4, and the baseline was manually set from three cycles to the highest cycle prior to which no signal appeared to rise above the baseline.…”