An inter-platform repeatability study investigating real-time amplification of plasmid DNA

Donald, Carol E.; Qureshi, Fizza; Burns, Malcolm; Holden, Marcia J.; Blasic, Joseph R.; Woolford, Alison J.-A.

doi:10.1186/1472-6750-5-15

Cited by 13 publications

(4 citation statements)

References 27 publications

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“…Therefore, using C T values for comparison of viral loads between studies might be misleading. 24,25 According to preliminary studies, short-term viremia and low viral loads complicate Zika virus detection in blood. 13,26 Since Zika virus RNA has been detected in urine from Zika virus-infected patients for two weeks or longer, 26 we investigated whether blood or urine were the most suitable clinical specimens for diagnoses of acute Zika virus infection.…”

Section: Resultsmentioning

confidence: 99%

Assay optimization for molecular detection of Zika virus

Corman

Rasche

Baronti

et al. 2016

Bull World Health Organ

141

160

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ObjectiveTo examine the diagnostic performance of real-time reverse transcription (RT)-polymerase chain reaction (PCR) assays for Zika virus detection.MethodsWe compared seven published real-time RT–PCR assays and two new assays that we have developed. To determine the analytical sensitivity of each assay, we constructed a synthetic universal control ribonucleic acid (uncRNA) containing all of the assays’ target regions on one RNA strand and spiked human blood or urine with known quantities of African or Asian Zika virus strains. Viral loads in 33 samples from Zika virus-infected patients were determined by using one of the new assays.FindingsOligonucleotides of the published real-time RT–PCR assays, showed up to 10 potential mismatches with the Asian lineage causing the current outbreak, compared with 0 to 4 mismatches for the new assays. The 95% lower detection limit of the seven most sensitive assays ranged from 2.1 to 12.1 uncRNA copies/reaction. Two assays had lower sensitivities of 17.0 and 1373.3 uncRNA copies/reaction and showed a similar sensitivity when using spiked samples. The mean viral loads in samples from Zika virus-infected patients were 5 × 104 RNA copies/mL of blood and 2 × 104 RNA copies/mL of urine.ConclusionWe provide reagents and updated protocols for Zika virus detection suitable for the current outbreak strains. Some published assays might be unsuitable for Zika virus detection, due to the limited sensitivity and potential incompatibility with some strains. Viral concentrations in the clinical samples were close to the technical detection limit, suggesting that the use of insensitive assays will cause false-negative results.

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Section: Resultsmentioning

confidence: 99%

Assay optimization for molecular detection of Zika virus

Corman

Rasche

Baronti

et al. 2016

Bull World Health Organ

141

160

View full text Add to dashboard Cite

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“…The difference between in situ analysis and the archived samples was not due to a lower extraction efficiency of the MFB module compared to the benchtop extraction. Instead, the lower in situ values may be due to software or hardware differences in calculating cycle thresholds between the MFB and the laboratory based qPCR analysis, which impact qPCR standards and copy number estimates 17,18 . Alternatively, the difference could also be linked to the behavior of mackerel, which displayed strong semidiurnal migrations between the bottom and surface of the mesocosm, triggered by an artificial sunrise at 09:00 each day.…”

Section: Resultsmentioning

confidence: 99%

Remote, autonomous real-time monitoring of environmental DNA from commercial fish

Hansen

Jacobsen

Middelboe

et al. 2020

Sci Rep

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Environmental DNA (eDNA) is increasingly used for monitoring marine organisms; however, offshore sampling and time lag from sampling to results remain problematic. In order to overcome these challenges a robotic sampler, a 2nd generation Environmental Sample Processor (ESP), was tested for autonomous analysis of eDNA from four commercial fish species in a 4.5 million liter mesocosm. The ESP enabled in situ analysis, consisting of water collection, filtration, DNA extraction and qPCR analysis, which allowed for real-time remote reporting and archival sample collection, consisting of water collection, filtration and chemical preservation followed by post-deployment laboratory analysis. The results demonstrate that the 2G ESP was able to consistently detect and quantify target molecules from the most abundant species (Atlantic mackerel) both in real-time and from the archived samples. In contrast, detection of low abundant species was challenged by both biological and technical aspects coupled to the ecology of eDNA and the 2G ESP instrumentation. Comparison of the in situ analysis and archival samples demonstrated variance, which potentially was linked to diel migration patterns of the Atlantic mackerel. The study demonstrates strong potential for remote autonomous in situ monitoring which open new possibilities for the field of eDNA and marine monitoring. Analysis of aquatic environmental DNA (eDNA) has gained momentum in recent years due to the method´s potential for rapid and cost-effective biomonitoring circumventing collection and visual identification of living specimens 1-3. This has led to much research over the past decade in relation to detection and quantification of aquatic macro-organisms in both freshwater 2,4,5 and more recently in marine environments 1,3,6. Despite the apparent benefits, eDNA surveys still require manual sampling of water at designated sampling sites, which constrains eDNA based marine monitoring in remote and offshore areas due to the associated boat-costs 7,8. Such limitations also prevent the acquisition of standardized eDNA time series on a scale from days to months, potentially leading to failure to detect important temporal and spatial changes in species occurrence and abundance, which is of particular relevance for rare and migratory species 9. Moreover, there is presently a considerable time lag in eDNA from field sampling to the result is available which delays and hampers management efforts. Despite the apparent need for time series and real-time data to accurately monitor and manage larger organisms in the oceans, completely autonomous in situ sampling, analysis and remote reporting of eDNA has not been conducted so far 10. One field of marine monitoring where the potential of eDNA has been highlighted as a valuable supplement or even replacement for existing monitoring methods is in relation to marine fisheries 3,11. Here, extensive monitoring is conducted worldwide in order to describe spatial and temporal dynamics of fish stocks, which is key for sustainable resource m...

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“…Also, low‐copy PCRs are stochastically limited, and LODs of <3 copies per PCR are reportedly not possible [Bustin et al, ]. Sensitivity and assay comparison are influenced by different real‐time PCR platforms and PCR primer pairs, detection chemistries, and plasmid DNA copy number used during quantitation [Donald et al, ].…”

Section: Discussionmentioning

confidence: 99%

Varicella zoster virus quantitation in blood from symptomatic and asymptomatic individuals

Toi

Lay

Lucas

et al. 2013

Journal of Medical Virology

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Primary infection with varicella zoster virus (VZV) occurs in immunocompromised and immunocompetent individuals. Clinical and asymptomatic reactivation with shedding of infectious virus and viremia may occur. The prevalence of VZV viremia is unknown. The aim of this study was to detect VZV viremia and quantify VZV DNA using quantitative polymerase chain reaction (qPCR) in blood from different populations. A qPCR-based method using EvaGreen® was used to quantify VZV DNA in 491 samples, including whole blood, plasma and buffy-coat, from patients hospitalized with varicella-associated disease (Group 1, n=10) and three groups with no VZV disease: individuals with a first clinical diagnosis of central nervous system demyelination (Group 2, n=213) with their age and sex-matched controls (Group 3, n=218); and HIV-infected individuals (Group 4, n=50). VZV-specific IgG antibody titres were measured in Group 3. The proportion positive for viremia and mean detectable VZV DNA load (copies/ml) were: Group 1: 100% (10/10) and 4.6 × 10(6) ± 1.4 × 10(7) ; Group 2: 4% (9/213) and 1.5 × 10(3) ± 1.8 × 10(4) ; Group 3: 8% (17/218) and 1.1 × 10(3) ± 7.8 × 10(3) ; Group 4: 12% (6/50) and 7.7 × 10(1) ± 2.8 × 10(2) . VZV DNA load and IgG titres were not significantly correlated (Group 3 only). VZV load in Group 1 was significantly elevated compared to Groups 2-4 (P<0.001); the latter were not significantly different from each other (P=0.05). VZV genotypes from clades 1-5 were identified in Group 1. VZV DNA was detected but at low frequency and viral load in both immunocompetent and immunocompromised individuals asymptomatic for VZV infection, compared to individuals with active VZV infection.

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An inter-platform repeatability study investigating real-time amplification of plasmid DNA

Abstract: Background: The wide variety of real-time amplification platforms currently available has determined that standardisation of DNA measurements is a fundamental aspect involved in the comparability of results.

Cited by 13 publications

References 27 publications

Assay optimization for molecular detection of Zika virus

Assay optimization for molecular detection of Zika virus

Remote, autonomous real-time monitoring of environmental DNA from commercial fish

Varicella zoster virus quantitation in blood from symptomatic and asymptomatic individuals

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