Evaluation of a rapid and sensitive RT-qPCR assay for the detection of Ebola Virus

Biava, Mirella; Colavita, Francesca; Marzorati, A.; Russo, Denise; Pirola, Danilo; Cocci, Alessandro; Petrocelli, A.; Guanti, Michela Delli; Cataldi, G.; Kamara, T.A.; Kamara, Abdul; Konneh, Kelfala; Cannas, Angela; Coen, Sabrina; Quartu, Serena; Meschi, Silvia; Valli, Maria Beatrice; Mazzarelli, Antonio; Venditti, Carolina; Grassi, Germana; Rozera, Gabriella; Castilletti, Concetta; Mirazimi, Alì; Capobianchi, Maria Rosaria; Ippolito, Giuseppe; Miccio, Rossella; Di, Antonino

doi:10.1016/j.jviromet.2017.11.009

Cited by 24 publications

(14 citation statements)

References 12 publications

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“…An optical detector integrated for PCR fluorescence acquisition revealed a high analytical sensitivity with HCV RNA positive samples ranging from 6,000,000 IU/mL down to approximately 45 IU in serum (equivalent to 4500 IU/mL) with good correlation with standard laboratory methods for PCR [ 217 ] ( Figure 11 A). Based on an optical detection method and a silicon disposable lab-on-chip cartridge, Biava et al used a highly compact platform [ 218 , 219 ], for the real-time detection of viral nucleic acids extracted from plasma, urine, and oral swabs [ 220 ] ( Figure 11 B–D), resulting in a robust and easy-to-use tool.…”

Section: Exploring the Content Of Vesiclesmentioning

confidence: 99%

“… ( A ) A sample-to-answer qRT-PCR system for the detection of RNA from Hepatitis C virus (HCV) developed by Shin and co-workers using a droplet magnetofluidics tool in plastic cartridges [ 217 ]; ( B – D ) Q3 compact platform developed by Biava and co-workers for the real-time detection of viral nucleic acids extracted from plasma, urine, and oral swabs. Reproduced with permission from reference [ 220 ]. …”

Section: Figurementioning

confidence: 99%

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Lab-on-Chip for Exosomes and Microvesicles Detection and Characterization

Chiriaco

Bianco

Nigro

et al. 2018

Sensors

127

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Interest in extracellular vesicles and in particular microvesicles and exosomes, which are constitutively produced by cells, is on the rise for their huge potential as biomarkers in a high number of disorders and pathologies as they are considered as carriers of information among cells, as well as being responsible for the spreading of diseases. Current methods of analysis of microvesicles and exosomes do not fulfill the requirements for their in-depth investigation and the complete exploitation of their diagnostic and prognostic value. Lab-on-chip methods have the potential and capabilities to bridge this gap and the technology is mature enough to provide all the necessary steps for a completely automated analysis of extracellular vesicles in body fluids. In this paper we provide an overview of the biological role of extracellular vesicles, standard biochemical methods of analysis and their limits, and a survey of lab-on-chip methods that are able to meet the needs of a deeper exploitation of these biological entities to drive their use in common clinical practice.

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Section: Exploring the Content Of Vesiclesmentioning

confidence: 99%

Section: Figurementioning

confidence: 99%

Lab-on-Chip for Exosomes and Microvesicles Detection and Characterization

Chiriaco

Bianco

Nigro

et al. 2018

Sensors

127

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“…There are a growing number of molecular tests described in the literature. The tests that were selected in this study have been widely used not only in epidemic situations but also in external quality assessment studies [9][10][11][12][13][14][15][16][17][18][19][20]. Dual-target assays are increasingly used in commercial and in-house assays [6,[21][22][23].…”

Section: Discussionmentioning

confidence: 99%

Development and Evaluation of a Duo Zaire ebolavirus Real-Time RT-PCR Assay Targeting Two Regions within the Genome

et al. 2019

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Preparedness and response actions to mitigate Ebola virus disease (EVD) outbreaks rely on rapid diagnosis to be implemented locally to sort suspect patients attending health centers. Our aim was (i) to develop and evaluate an RT-qPCR assay combining primers and probes derived from two reference assays targeting different genomic regions; (ii) to study whether sensitivity and specificity of this dual-target assay were at least equal or better to the parental assays; (iii) to implement this dual-target assay onto the Cepheid GeneXpert open cartridge as a proof of principle for technological transfer aiming at bedsite testing locally. To do so, three home-made published RT-qPCR assays were selected to be compared with the RealStar® Filovirus Screen RT-PCR kit 1.0 (Altona Diagnostics, Hamburg, Germany), a technique that was largely deployed during the 2014–2015 West African EVD outbreak. Primers and probes sequences of the custom-made assays were analyzed in silico against a multiple sequence alignment, including >250 complete sequences corresponding to strains that have caused EVD epidemics in the past. Genomic RNA purified from the Mekambo strain of Zaire ebolavirus (EBOV) was used to study the sensitivity of the five methods. Based on these results, two in-house methods were selected and adapted to design the dual-target assay, which performances were compared to those of the parental assays using a synthetic RNA control. The dual-target assay showed better sensitivity and limit of detection (LoD95 at 0.4 copies/µL) than the parental methods (1.7 and 2.2 copies/µL). Ultimately, the dual-target assay was transferred onto the GeneXpert Flex-03 open cartridge, demonstrating a LoD95 at 0.75 copies/µL. Together these results indicate that EBOV dual-target assay has the potential to be used during EVD outbreak in the laboratory having performed molecular testing during the recent outbreaks.

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“…Virtually any qPCR protocol developed for larger, standard qPCR instruments can be smoothly ported to Q3. In fact, some applications of Q3 have already been demonstrated [ 11 , 12 , 13 ] and several assays are currently under development.…”

Section: Q3: System Descriptionmentioning

confidence: 99%

Q3: A Compact Device for Quick, High Precision qPCR

Cereda

Cocci

Cucchi

et al. 2018

Sensors

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An accurate and easy-to-use Q3 system for on-chip quantitative real-time Polymerase Chain Reaction (qPCR) is hereby demonstrated, and described in detail. The qPCR reactions take place inside a single-use Lab-on-a-Chip with multiple wells, each with 5 to 15 µL capacity. The same chip hosts a printed metal heater coupled with a calibrated sensor, for rapid and accurate temperature control inside the reaction mixture. The rest of the system is non-disposable and encased in a 7 × 14 × 8.5 (height) cm plastic shell weighing 300 g. Included in the non-disposable part is a fluorescence read-out system featuring up to four channels and a self-contained control and data storage system, interfacing with an external user-friendly software suite. Hereby, we illustrate the engineering details of the Q3 system and benchmark it with seamlessly ported testing protocols, showing that Q3 equals the performance of standard commercial systems. Overall, to the best of our knowledge, this is one of the most mature general-purpose systems for on-chip qPCR currently available.

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Evaluation of a rapid and sensitive RT-qPCR assay for the detection of Ebola Virus

Cited by 24 publications

References 12 publications

Lab-on-Chip for Exosomes and Microvesicles Detection and Characterization

Lab-on-Chip for Exosomes and Microvesicles Detection and Characterization

Development and Evaluation of a Duo Zaire ebolavirus Real-Time RT-PCR Assay Targeting Two Regions within the Genome

Q3: A Compact Device for Quick, High Precision qPCR

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