Development, validation and evaluation of a homogenous one-step reverse transcriptase-initiated PCR assay with competitive internal control for the detection of hepatitis C virus RNA

Abstract

Nucleic acid amplification testing for hepatitis C virus (HCV) RNA has become an essential tool for the prevention and clinical management of hepatitis C. We describe the development, validation and evaluation of a homogenous reverse transcriptase-initiated HCV-PCR assay with competitive internal control that is applicable to both the quantitative detection of HCV genomes in single patient samples and the screening of blood donations by mini-pool testing. For the implementation of a positive run control, a HCV RNA-positive plasma sample was calibrated against an international HCV RNA standard preparation. For quantification purposes, an in vitro-transcribed RNA calibrator sequence was used. The detection limit of the assay (95% positive cut-off) was determined by probit analysis and was calculated as 114 IU/mL. Comparable sensitivity to different HCV template sequences was verified for HCV genotypes 1-5. Quantitative test results correlated well with viral loads that had been previously determined by the Bayer VERSANT HCV RNA 3.0 bDNA assay (n=53, R=0.943, p<0.001). During more than 5 years of blood donation testing, the specificity of the assay was found to be 99.51%. All assay components showed constant performance during this time period. In conclusion, we introduce a well-proven method that allows fast and reliable quantification of HCV genomes.

Competitive RT-PCR Strategy for Quantitative Evaluation of the Expression of Tilapia (Oreochromis niloticus) Growth Hormone Receptor Type I

“…In general, lower target quantities give higher CVs. Our variability is in the range of the variability of the PCR assay [17,18]. In general, CVs corresponding to reaction conditions, in which the quantity of target and competitor was equivalent or nearby, were under 10%, and CVs corresponding to reaction conditions, in which the ratio C/T was 10 or 1/10, were between 10% and 35%.…”

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

“…In this paper, we described the construction of a Q␤ recombinant derivative to be used as a CIC in RT-PCR detection assays. rQ␤ was developed to overcome the weaknesses derived from using naked RNA as an IC (13,17,21,30,33) and the different amplification efficiencies of stable noncompetitive ICs with respect to the target (6, 7) and as an alternative strategy to armored RNA technology (25).…”

RNA diagnostics: real‐time RT‐PCR strategies and promising novel target RNAs

“…Absolute quantification requires a well‐defined calibrator to determine the number of initial nucleic acid targets in the sample reactions. A log‐linear interpolation between initial copy numbers of the calibration curve and the corresponding CP/Ct values observed is used for calculation of the initial copy numbers present in the unknowns 7,8…”

“…The multiplexed detection of endogenous glyceraldehyde‐3‐phosphatedehydrogenase (GAPDH) mRNA, co‐purified during RNA isolation, is a suitable method to monitor the performance of RNA isolation, RT, and subsequent amplification 13. In order to completely eliminate interindividual variations within the internal control reactions, however, the implementation of an artificial control sequence added to samples during RNA‐purification is superior to this strategy7,14 (Figure 2(b)).…”