Quantification of measles, mumps and rubella viruses using real-time quantitative TaqMan-based RT-PCR assay

Ammour, Yulia; Файзулоев, Е. Б.; Borisova, T. K.; Nikonova, Alexandra; Ga, Dmitriev; Lobodanov, S.; Zverev, Vitaly

doi:10.1016/j.jviromet.2012.09.011

Cited by 20 publications

(14 citation statements)

References 24 publications

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“…The spiking experiment extends the suitability of the MagNAPure technology as a validated automatic nucleic acid extraction method for viral RNA [13] to RV diagnostics, which was so far mainly based on reports employing sample processing by QIAamp Viral RNA Mini kit [3,12]. The detection limit of 200 virus particles per ml is in agreement with the detection limit of our TaqMan PCR of one to five copies per reaction, which equals to 100 to 400 copies per milliliter of original sample.…”

Section: Discussionsupporting

confidence: 69%

“…This is also the case during the exponential phase of RV replication. Therefore the already discussed cell destruction [3] appears to be a likely contributor for the discrepancy between these two parameters, but through this study such an association can be assigned as rather strain-specific. However, most of the clinical RV strains lack signs of cytopathogenicity on susceptible cell lines, thus genome copy number in clinical samples likely correlates with the amount of infectious virus particles.…”

Section: Discussionmentioning

confidence: 53%

“…In this study, the number of PFU or FFU per ml, as a calculation of infectious virus particles, was contrasted over time of culture to genome copies per ml for different RV strains with or without cytopathogenicity on Vero cells. We could extend the initial report on viral genome to CCID50 ratio [3] through the observation that genome to PFU/FFU ratio is considerably low, but only for a non-cytopathogenic RV strain. This is also the case during the exponential phase of RV replication.…”

Section: Discussionmentioning

confidence: 89%

“…This study presents the first TaqMan-based RT-qPCR using p90 as a template region and extends established protocols based on E1 [31,32], the capsid protein region [3] or p150 [2,12,19]. This highly conserved region within p90 has a GC content of 54 to 65%, which, in contrast to the average value of 70%, is more suitable for a TaqMan probe design.…”

Section: Discussionmentioning

confidence: 98%

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

et al. 2016

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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.

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Section: Discussionsupporting

confidence: 69%

Section: Discussionmentioning

confidence: 53%

Section: Discussionmentioning

confidence: 89%

Section: Discussionmentioning

confidence: 98%

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

et al. 2016

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“…Also, Ammour et al, 19 reported that qRT-PCR was successfully used for estimating the titers of measles, A B…”

Section: Discussionmentioning

confidence: 99%

Correlation between live attenuated measles viral load and growth inhibition percentage in non-small cell lung cancer cell line

Obaid

Obied

Al-Jibouri

et al. 2018

J. contemp. med. sci.

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, 10 −5 of virus stock (8.860 log 10 RNA copies/µl). The virus cytopathic effect (CPE) was studied at 24, 48, 72, and 96 hr. Oncolytic effects of the virus were assessed by cytotoxicity assay, apoptosis analysis, and proliferation marker Ki-67. The growth inhibition percentage was determined by Enzyme-Linked Immunosorbent Assay (ELISA) reader using crystal violet staining assay and compared with the viral load measured by quantitative real time-PCR. Results The results of the study showed that measles virus had an inhibitory effect on the growth of human lung cancer cell line compared with untreated control group. There was highly significant positive correlation between viral load and PGI at 24, 48, 72, 96 hr (P < 0.001). The growth inhibition rate of a cell was associated with a decrement in the expression of the Ki-67 protein and increase in the ratio of early and late apoptotic cells in comparison with untreated control group. Conclusion Live attenuated measles virus strain induced cytotoxic effect against human lung cancer cell line (A549) by induction of apoptosis as an important mechanism of anti-tumor activity, in addition, it indicates a correlation between the quantity of MV genomes and percentage of growth inhibition. This relation has proved that measles virus had anticancer effect.

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Mumps

Mankertz

2014

S2k-Leitlinie - Labordiagnostik Schwangerschaftsrelevanter Virusinfektionen

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Quantification of measles, mumps and rubella viruses using real-time quantitative TaqMan-based RT-PCR assay

Cited by 20 publications

References 24 publications

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

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

Correlation between live attenuated measles viral load and growth inhibition percentage in non-small cell lung cancer cell line

Mumps

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