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Development of a Digital RT-PCR Method for Absolute Quantification of Bluetongue Virus in Field Samples.

ABSTRACT: Bluetongue (BT) is a major Office International des Epizooties (OIE)-listed disease of wild and domestic ruminants caused by several serotypes of Bluetongue virus (BTV), a virus with a segmented dsRNA genome belonging to the family Reoviridae, genus Orbivirus. BTV is transmitted through the bites of Culicoides midges. The aim of this study was to develop a new method for quantification of BTV Seg-10 by droplet digital RT-PCR (RTdd-PCR), using nucleic acids purified from complex matrices such as blood, tissues, and midges, that notoriously contain strong PCR inhibitors. First, RTdd-PCR was optimized by using RNAs purified from serially 10-fold dilutions of a BTV-1 isolate (105.43TCID50/ml up to 10-0.57 TCID50/ml) and from the same dilutions spiked into fresh ovine EDTA-blood and spleen homogenate. The method showed a good degree of linearity (R 2 ? 0.995). The limit of detection (LoD) and the limit of quantification (LoQ) established were 10-0.67TCID50/ml (0.72 copies/?l) and 100.03TCID50/ml (3.05 copies/?l) of BTV-1, respectively. Second, the newly developed test was compared, using the same set of biological samples, to the quantitative RT-PCR (RT-qPCR) detecting Seg-10 assay widely used for the molecular diagnosis of BTV from field samples. Results showed a difference mean of 0.30 log between the two assays with these samples (p < 0.05). Anyway, the analysis of correlation demonstrated that both assays provided similar measurements with a very close agreement between the systems.

SUBMITTER: Rocchigiani AM

PROVIDER: S-EPMC7186476 | biostudies-literature | 2020

REPOSITORIES: biostudies-literature

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Development of a Digital RT-PCR Method for Absolute Quantification of Bluetongue Virus in Field Samples.

Rocchigiani Angela M AM Tilocca Maria G MG Portanti Ottavio O Vodret Bruna B Bechere Roberto R Di Domenico Marco M Savini Giovanni G Lorusso Alessio A Puggioni Giantonella G

Frontiers in veterinary science 20200421

Bluetongue (BT) is a major Office International des Epizooties (OIE)-listed disease of wild and domestic ruminants caused by several serotypes of Bluetongue virus (BTV), a virus with a segmented dsRNA genome belonging to the family Reoviridae, genus Orbivirus. BTV is transmitted through the bites of Culicoides midges. The aim of this study was to develop a new method for quantification of BTV Seg-10 by droplet digital RT-PCR (RTdd-PCR), using nucleic acids purified from comp ...[more]

PMID: 32373633

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Project description:BackgroundDigital polymerase chain reaction (dPCR) is an increasingly popular technology for detecting and quantifying target nucleic acids. Its advertised strength is high precision absolute quantification without needing reference curves. The standard data analytic approach follows a seemingly straightforward theoretical framework but ignores sources of variation in the data generating process. These stem from both technical and biological factors, where we distinguish features that are 1) hard-wired in the equipment, 2) user-dependent and 3) provided by manufacturers but may be adapted by the user. The impact of the corresponding variance components on the accuracy and precision of target concentration estimators presented in the literature is studied through simulation.ResultsWe reveal how system-specific technical factors influence accuracy as well as precision of concentration estimates. We find that a well-chosen sample dilution level and modifiable settings such as the fluorescence cut-off for target copy detection have a substantial impact on reliability and can be adapted to the sample analysed in ways that matter. User-dependent technical variation, including pipette inaccuracy and specific sources of sample heterogeneity, leads to a steep increase in uncertainty of estimated concentrations. Users can discover this through replicate experiments and derived variance estimation. Finally, the detection performance can be improved by optimizing the fluorescence intensity cut point as suboptimal thresholds reduce the accuracy of concentration estimates considerably.ConclusionsLike any other technology, dPCR is subject to variation induced by natural perturbations, systematic settings as well as user-dependent protocols. Corresponding uncertainty may be controlled with an adapted experimental design. Our findings point to modifiable key sources of uncertainty that form an important starting point for the development of guidelines on dPCR design and data analysis with correct precision bounds. Besides clever choices of sample dilution levels, experiment-specific tuning of machine settings can greatly improve results. Well-chosen data-driven fluorescence intensity thresholds in particular result in major improvements in target presence detection. We call on manufacturers to provide sufficiently detailed output data that allows users to maximize the potential of the method in their setting and obtain high precision and accuracy for their experiments.

Project description:IntroductionToxigenic Vibrio cholerae serogroup O1 and O139 are the pathogens responsible for the global cholera epidemic. V. cholerae can settle in the water and spread via the fecal-oral route. Rapid and accurate monitoring of live V. cholerae in environmental water has become an important strategy to prevent and control cholera transmission. Conventional plate counting is widely used to detect viable bacteria but requires time and effort.MethodsThis study aims to develop a new assay that combines triplex droplet digital PCR (ddPCR) with propidium monoazide (PMA) treatment for quantitatively detecting live V. cholerae O1/O139 and cholera enterotoxin. Specific primers and probes were designed according to the conserved regions of gene rfb O1, rfb O139, and ctxA. The amplification procedures and PMA treatment conditions were optimized. The specificity, sensitivity, and ability of PMA-ddPCR to detect viable bacteria-derived DNA were evaluated in simulated seawater samples.Results and discussionThe results revealed that the optimal primer concentrations of rfb O1, rfb O139, and ctxA were 1 μM, while the concentrations of the three probes were 0.25, 0.25, and 0.4 μM, respectively. The best annealing temperature was 58°C to obtain the most accurate results. The optimal strategy for distinguishing dead and live bacteria from PMA treatment was incubation at the concentration of 20 μM for 15 min, followed by exposure to a 650-W halogen lamp for 20 min. In pure culture solutions, the limit of detection (LODs) of V. cholerae O1 and O139, and ctxA were 127.91, 120.23 CFU/mL, and 1.5 copies/reaction in PMA-triplex ddPCR, respectively, while the LODs of the three targets were 150.66, 147.57 CFU/mL, and 2 copies/reaction in seawater samples. The PMA-ddPCR sensitivity was about 10 times higher than that of PMA-qPCR. When detecting spiked seawater samples with live bacterial concentrations of 1.53 × 102 and 1.53 × 105 CFU/mL, the assay presented a higher sensitivity (100%, 16/16) than qPCR (50.00%, 8/16) and a perfect specificity (100%, 9/9). These results indicate that the developed PMA-triplex ddPCR is superior to the qPCR regarding sensitivity and specificity and can be used to rapidly detect viable toxigenic V. cholerae O1 and O139 in suspicious seawater samples.

Dataset Information

Development of a Digital RT-PCR Method for Absolute Quantification of Bluetongue Virus in Field Samples.

Publications

Development of a Digital RT-PCR Method for Absolute Quantification of Bluetongue Virus in Field Samples.

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