Development of a qPCR assay to detect and quantify ichthyotoxic flagellates along the Norwegian coast, and the first Norwegian record of Fibrocapsa japonica (Raphidophyceae)

Abstract Using a sensitive RT-QPCR assay, we analyzed the regulatory effects of sex and different dosage compensation mutations in Drosophila. To validate the assay, we showed that regulation for several genes indeed varied with the number of functional copies of that gene. We then confirmed that dosage compensation occurred for most genes we examined in male and female flies. Finally, we examined the effects on regulation of several genes in the MSL pathway, presumed to be involved in sex-dependent determination of regulation. Rather than seeing global alterations of either X chromosomal or autosomal genes, regulation of genes on either the X chromosome or the autosomes could be elevated, depressed, or unaltered between sexes in unpredictable ways for the various MSL mutations. Relative dosage for a given gene between the sexes could vary at different developmental times. Autosomal genes often showed deranged regulatory levels, indicating they were in pathways perturbed by X chromosomal changes. As exemplified by the BR-C locus and its dependent Sgs genes, multiple genes in a given pathway could exhibit coordinate regulatory modulation. The variegated pattern shown for expression of both X chromosomal and autosomal loci underscores the complexity of gene expression so that the phenotype of MSL mutations does not reflect only simple perturbations of genes on the X chromosome.

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Evaluation of a fully automated high-throughput SARS-CoV-2 multiplex qPCR assay with built-in screening functionality for del-HV69/70- and N501Y variants such as B.1.1.7

Journal of Clinical Virology ◽

10.1016/j.jcv.2021.104894 ◽

2021 ◽

pp. 104894

Author(s):

Dominik Nörz ◽

Moritz Grunwald ◽

Flaminia Olearo ◽

Nicole Fischer ◽

Martin Aepfelbacher ◽

...

Keyword(s):

High Throughput ◽

Qpcr Assay ◽

Multiplex Qpcr

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Optimized protocol for a quantitative SARS-CoV-2 duplex RT-qPCR assay with internal human sample sufficiency control

Journal of Virological Methods ◽

10.1016/j.jviromet.2021.114174 ◽

2021 ◽

pp. 114174

Author(s):

Aileen G. Rowan ◽

Philippa May ◽

Anjna Badhan ◽

Carolina Herrera ◽

Patricia Watber ◽

...

Keyword(s):

Qpcr Assay ◽

Optimized Protocol ◽

Human Sample

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Optimisation of a PMAxx™-RT-qPCR Assay and the Preceding Extraction Method to Selectively Detect Infectious Murine Norovirus Particles in Mussels

Food and Environmental Virology ◽

10.1007/s12560-020-09454-w ◽

2021 ◽

Author(s):

Ravo M. Razafimahefa ◽

Louisa F. Ludwig-Begall ◽

Françoise S. Le Guyader ◽

Frédéric Farnir ◽

Axel Mauroy ◽

...

Keyword(s):

Extraction Method ◽

Qpcr Assay ◽

Murine Norovirus

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Performance of BioFire array or QuickVue influenza A + B test versus a validation qPCR assay for detection of influenza A during a volunteer A/California/2009/H1N1 challenge study

Virology Journal ◽

10.1186/s12985-021-01516-0 ◽

2021 ◽

Vol 18 (1) ◽

Author(s):

David R. McIlwain ◽

Han Chen ◽

Maria Apkarian ◽

Melton Affrime ◽

Bonnie Bock ◽

...

Keyword(s):

Influenza A ◽

Rapid Test ◽

Influenza Viruses ◽

Qpcr Assay ◽

Superior Performance ◽

Qrt Pcr ◽

Challenge Study ◽

Test Kit ◽

2009 H1n1 ◽

B Test

Abstract Background Influenza places a significant burden on global health and economics. Individual case management and public health efforts to mitigate the spread of influenza are both strongly impacted by our ability to accurately and efficiently detect influenza viruses in clinical samples. Therefore, it is important to understand the performance characteristics of available assays to detect influenza in a variety of settings. We provide the first report of relative performance between two products marketed to streamline detection of influenza virus in the context of a highly controlled volunteer influenza challenge study. Methods Nasopharyngeal swab samples were collected during a controlled A/California/2009/H1N1 influenza challenge study and analyzed for detection of virus shedding using a validated qRT-PCR (qPCR) assay, a sample-to-answer qRT-PCR device (BioMerieux BioFire FilmArray RP), and an immunoassay based rapid test kit (Quidel QuickVue Influenza A + B Test). Results Relative to qPCR, the sensitivity and specificity of the BioFire assay was 72.1% [63.7–79.5%, 95% confidence interval (CI)] and 93.5% (89.3–96.4%, 95% CI) respectively. For the QuickVue rapid test the sensitivity was 8.5% (4.8–13.7%, 95% CI) and specificity was 99.2% (95.6–100%, 95% CI). Conclusion Relative to qPCR, the BioFire assay had superior performance compared to rapid test in the context of a controlled influenza challenge study.

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Correction to: Performance of BioFire array or QuickVue influenza A + B test versus a validation qPCR assay for detection of influenza A during a volunteer A/California/2009/H1N1 challenge study

Virology Journal ◽

10.1186/s12985-021-01531-1 ◽

2021 ◽

Vol 18 (1) ◽

Author(s):

David R. McIlwain ◽

Han Chen ◽

Maria Apkarian ◽

Melton Affrime ◽

Bonnie Bock ◽

...

Keyword(s):

Influenza A ◽

Qpcr Assay ◽

Challenge Study ◽

2009 H1n1 ◽

B Test

An amendment to this paper has been published and can be accessed via the original article.

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Development of a SYBR Green-based RT-qPCR assay for the detection of Indian citrus ringspot virus

3 Biotech ◽

10.1007/s13205-021-02903-8 ◽

2021 ◽

Vol 11 (7) ◽

Author(s):

Amol D. Kokane ◽

Kapil Lawrence ◽

Sunil B. Kokane ◽

Mrugendra G. Gubyad ◽

Pragati Misra ◽

...

Keyword(s):

Sybr Green ◽

Qpcr Assay ◽

Ringspot Virus

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Analytical validation of a real-time hydrolysis probe PCR assay for quantifying Plasmodium falciparum parasites in experimentally infected human adults

Malaria Journal ◽

10.1186/s12936-021-03717-y ◽

2021 ◽

Vol 20 (1) ◽

Author(s):

Claire Y. T. Wang ◽

Emma L. Ballard ◽

Zuleima Pava ◽

Louise Marquart ◽

Jane Gaydon ◽

...

Keyword(s):

Clinical Trials ◽

Plasmodium Falciparum ◽

18S Rrna ◽

Drug Efficacy ◽

18S Rrna Gene ◽

Molecular Techniques ◽

Qpcr Assay ◽

Rrna Gene ◽

Analytical Sensitivity ◽

Blood Samples

Abstract Background Volunteer infection studies have become a standard model for evaluating drug efficacy against Plasmodium infections. Molecular techniques such as qPCR are used in these studies due to their ability to provide robust and accurate estimates of parasitaemia at increased sensitivity compared to microscopy. The validity and reliability of assays need to be ensured when used to evaluate the efficacy of candidate drugs in clinical trials. Methods A previously described 18S rRNA gene qPCR assay for quantifying Plasmodium falciparum in blood samples was evaluated. Assay performance characteristics including analytical sensitivity, reportable range, precision, accuracy and specificity were assessed using experimental data and data compiled from phase 1 volunteer infection studies conducted between 2013 and 2019. Guidelines for validation of laboratory-developed molecular assays were followed. Results The reportable range was 1.50 to 6.50 log10 parasites/mL with a limit of detection of 2.045 log10 parasites/mL of whole blood based on a parasite diluted standard series over this range. The assay was highly reproducible with minimal intra-assay (SD = 0.456 quantification cycle (Cq) units [0.137 log10 parasites/mL] over 21 replicates) and inter-assay (SD = 0.604 Cq units [0.182 log10 parasites/mL] over 786 qPCR runs) variability. Through an external quality assurance program, the QIMR assay was shown to generate accurate results (quantitative bias + 0.019 log10 parasites/mL against nominal values). Specificity was 100% after assessing 164 parasite-free human blood samples. Conclusions The 18S rRNA gene qPCR assay is specific and highly reproducible and can provide reliable and accurate parasite quantification. The assay is considered fit for use in evaluating drug efficacy in malaria clinical trials.

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Dynamics of Specific cfDNA Fragments in the Plasma of Full Marathon Participants

Genes ◽

10.3390/genes12050676 ◽

2021 ◽

Vol 12 (5) ◽

pp. 676

Author(s):

Takehito Sugasawa ◽

Shin-ichiro Fujita ◽

Tomoaki Kuji ◽

Noriyo Ishibashi ◽

Kenshirou Tamai ◽

...

Keyword(s):

Exercise Physiology ◽

High Sensitivity ◽

Genomic Region ◽

Qpcr Assay ◽

Strongly Correlated ◽

Healthy Humans ◽

Blood Cell And Plasma ◽

Next Generation Sequencing Ngs ◽

Taqman Qpcr Assay ◽

Taqman Qpcr

Plasma cell-free DNA (cfDNA) is frequently analyzed using liquid biopsy to investigate cancer markers. We hypothesized that this concept might be applicable in exercise physiology. Here, we aimed to identify specific cfDNA (spcfDNA) sequences in the plasma of healthy humans using next-generation sequencing (NGS) and clearly define the dynamics regarding spcfDNA-fragment levels upon extreme exercises, such as running a full marathon. NGS analysis was performed using cfDNA of pooled plasma collected from healthy participants. We confirmed that the TaqMan-qPCR assay had high sensitivity and found that the spcfDNA sequence abundance was 16,600-fold higher than that in a normal genomic region. We then used the TaqMan-qPCR assay to investigate the dynamics of spcfDNA-fragment levels upon running a full marathon. The spcfDNA fragment levels were significantly increased post-marathon. Furthermore, spcfDNA fragment levels were strongly correlated with white blood cell and plasma myoglobin concentrations. These results suggest the spcfDNA fragments identified in this study were highly sensitive as markers of extreme physical stress. The findings of this study may provide new insights into exercise physiology and genome biology in humans.

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