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

Autor: Rocchigiani AM; Department of Sanità Animale, Istituto Zooprofilattico Sperimentale Della Sardegna, Sassari, Italy., Tilocca MG; Department of Sanità Animale, Istituto Zooprofilattico Sperimentale Della Sardegna, Sassari, Italy., Portanti O; OIE Reference Laboratory for Bluetongue, Istituto Zooprofilattico Sperimentale Abruzzo e Molise, Teramo, Italy., Vodret B; Department of Sanità Animale, Istituto Zooprofilattico Sperimentale Della Sardegna, Sassari, Italy., Bechere R; Department of Sanità Animale, Istituto Zooprofilattico Sperimentale Della Sardegna, Sassari, Italy., Di Domenico M; OIE Reference Laboratory for Bluetongue, Istituto Zooprofilattico Sperimentale Abruzzo e Molise, Teramo, Italy., Savini G; OIE Reference Laboratory for Bluetongue, Istituto Zooprofilattico Sperimentale Abruzzo e Molise, Teramo, Italy., Lorusso A; OIE Reference Laboratory for Bluetongue, Istituto Zooprofilattico Sperimentale Abruzzo e Molise, Teramo, Italy., Puggioni G; Department of Sanità Animale, Istituto Zooprofilattico Sperimentale Della Sardegna, Sassari, Italy.
Jazyk: angličtina
Zdroj: Frontiers in veterinary science [Front Vet Sci] 2020 Apr 21; Vol. 7, pp. 170. Date of Electronic Publication: 2020 Apr 21 (Print Publication: 2020).
DOI: 10.3389/fvets.2020.00170
Abstrakt: 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 (10 5.43 TCID 50 /ml up to 10 -0.57 TCID 50 /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 -0.67 TCID 50 /ml (0.72 copies/μl) and 10 0.03 TCID 50 /ml (3.05 copies/μl) 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.
(Copyright © 2020 Rocchigiani, Tilocca, Portanti, Vodret, Bechere, Di Domenico, Savini, Lorusso and Puggioni.)
Databáze: MEDLINE
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