A sensitive one-step TaqMan amplification approach for detection of rubella virus clade I and II genotypes in clinical samples

Abstract

Although teratogenic rubella virus (RV) causes a vaccine-preventable disease, it is still endemic in several countries worldwide. Thus, there is a constant risk of RV importation into non-endemic areas. RV monitoring, especially during measles and Zika virus outbreaks, requires reliable diagnostic tools. For this study, a TaqMan-based one-step reverse transcription-quantitative PCR (RT-qPCR) assay, with the p90 gene as a novel and so far unexplored target for detection of clade I and II genotypes, was developed and evaluated. Automated nucleic acid extraction was carried out. Performance characteristics of the TaqMan RT-qPCR assay were determined for a RV plasmid standard and RNA extracted from virus-infected cell culture supernatants representing clade I and II genotypes. Diagnostic specificity and sensitivity were validated against other RNA and DNA viruses, relevant for RV diagnostic approaches and for RV-positive clinical samples, respectively. The assay is specific and highly sensitive with a limit of detection as low as five to one copies per reaction or 200 infectious virus particles per ml. The coefficients of variation (CV) were specified as intra-(within one run) and inter-(between different runs) assay variation, and calculated based on the standard deviations for the obtained Ct values of the respective samples. Intraand inter-assay CV values were low, with a maximum of 3.4% and 2.4%, respectively. The assay was shown to be suitable and specific for the analysis of clinical samples. With p90 as a novel target, the highly sensitive and specific TaqMan assay outlined in this study is suitable for RV diagnosis worldwide.

Alterations in Cell Mechanics by Actin Cytoskeletal Changes Correlate with Strain-Specific Rubella Virus Phenotypes for Cell Migration and Induction of Apoptosis

“…Due to the cell detachment during RV infection, only a minor portion of dead cells can be detected in the infected cell monolayer ( Figure 1 A(iii)). Moreover, CPE development could contribute to an increase in the ratio of total number of particles to infectious particles, which was noted for the high-cytopathogenic strains Wb-12 and Therien, but not for the low-cytopathogenic strain 03-03703 [ 23 ].…”

“…For mRNA expression studies, MOI 5 was applied and samples were collected at 72 hpi to maximize any possible RV-associated effects. Virus titration was performed by plaque (RV strains Wb-12, RA27/3, Therien, and Rub 1B) or focus-forming assay (all remaining RV strains) as published [ 23 ].…”

Rubella Virus Strain-Associated Differences in the Induction of Oxidative Stress Are Independent of Their Interferon Activation

“…The CPE-positive Therien and Wb-12 RV strains were titred on Vero cells by standard plaque assay (titre expressed as plaque forming unit, PFU per mL). Titre of low cytopathogenic 03-03703 and 07-00426 was determined by a focus—forming assay (titre given as focus forming unit, FFU per mL) as described previously [ 23 ] with the exception that TrueBlue™ peroxidase was used as the substrate (Medac GmbH, Wedel, Germany).…”

Rubella Viruses Shift Cellular Bioenergetics to a More Oxidative and Glycolytic Phenotype with a Strain-Specific Requirement for Glutamine

“…Quantification of viral RNA genome copies. TaqMan-based one-step RT-qPCR was performed as described previously (45). Briefly, total RNA was extracted by MagnaPure technology.…”

“…Thereafter, the virus inoculum was replaced with fresh medium, and samples were incubated until further analysis. Standard plaque and focus-forming assays were used as described previously for the titer determination of RV strains with and without notable CPE induction, respectively (45).…”