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

Angela M Rocchigiani,Maria G Tilocca,Roberto Bechere,Giantonella Puggioni,Bruna Vodret,Ottavio Portanti,Alessio Lorusso,Giovanni Savini,Marco Di Domenico

doi:10.3389/fvets.2020.00170

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 (R2 ≥ 0.995). The limit of detection (LoD) and the limit of quantification (LoQ) established were 10−0.67TCID50/ml (0.72 copies/μl) and 100.03TCID50/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.

Highlights

Digital polymerase chain reaction is a recent technology enables an accurate absolute quantification of target nucleic acids
We aimed to develop a new method for quantification of Bluetongue virus (BTV) Seg-10 by droplet digital RT-PCR (RT-dd-PCR), using nucleic acids purified from complex matrices such as blood, tissues, and midges, that notoriously contain strong PCR inhibitors
The primers and probe concentrations were optimized by using RNA purified from dilution of BTV-1 from 102.43 to 10−0.57

Summary

Introduction

Digital polymerase chain reaction (dPCR) is a recent technology enables an accurate absolute quantification of target nucleic acids. The principle of dPCR was first described in the 1990s [1, 2]. The dPCR approach combines limiting dilutions, PCR, and Poisson distribution to quantitate the total number of amplifiable targets within a sample [1]. Digital technology is based on end-point PCR that provides the direct measure of nucleic acids without relying on a standard curve [3]. In a dPCR assay, the sample is randomly partitioned into individual reactions, such that some contain no nucleic acid template and others contain one or more template copies [4]. The very high number of partitions of the sample allows a significant precision on results [5]

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