Development and performance evaluation of a streamlined method for nucleic acid purification, denaturation, and multiplex detection ofBluetongue virusandEpizootic hemorrhagic disease virus

Abstract

Abstract. Bluetongue virus (BTV) and Epizootic hemorrhagic disease virus (EHDV) possess similar structural and molecular features, are transmitted by biting midges (genus Culicoides), and cause similar diseases in some susceptible ruminants. Generally, BTV causes subclinical disease in cattle, characterized by a prolonged viremia. EHDV-associated disease in cattle is less prominent; however, it has emerged as a major economic threat to the white-tailed deer (Odocoileus virginianus) industry in many areas of the United States. The recent emergence of multiple BTV and EHDV serotypes previously undetected in the United States demonstrates the need for robust detection of all known serotypes and differential diagnosis. For this purpose, a streamlined workflow consisting of an automated nucleic acid purification and denaturation method and a multiplex one-step reverse transcription quantitative polymerase chain reaction for the simultaneous detection of BTV serotypes 1-24 and EHDV serotypes 1-7 was developed using previously published BTV and EHDV assays. The denaturation of double-stranded (ds) BTV and EHDV RNA was incorporated into the automated nucleic acid purification process thus eliminating the commonly used separate step of dsRNA denaturation. The performance of this workflow was compared with the World Organization of Animal Health BTV reference laboratory (National Veterinary Services Laboratory, Ames, Iowa) workflow for BTV and EHDV detection, and high agreement was observed. Implementation of the workflow in routine diagnostic testing enables the detection of, and differentiation between, BTV and EHDV, and coinfections in bovine blood and cervine tissues, offering significant benefits in terms of differential disease diagnosis, herd health monitoring, and regulated testing.Key words: Bluetongue virus; Epizootic hemorrhagic disease virus; polymerase chain reaction. Schroeder et al. 710and/or EHDV infection is most often seen in white-tailed deer and includes fever, excessive salivation and nasal discharge, and hemorrhaging from oral and nasal tissue.46 Serotypes of BTV found in the United States do not typically cause clinical disease in cattle. 25 However, exotic BTV serotypes are known to cause clinical signs, and occasionally clinical signs are also seen with U.S. serotypes. In late summer through early fall of 2012, a significant number of epizootic hemorrhagic disease cases in cattle were confirmed in the northern U.S. states of Nebraska, South Dakota, Wyoming, and Iowa, and it is believed that the disease was spread from deer to cattle by insect vectors (Wilson D: 2013, Epizootic hemorrhagic disease update. Calif Vet Jan/Feb: 44-45). In general, cattle most often act as reservoirs for BTV and EHDV due to a prolonged cell-associated viremia, which contributes significantly to the epidemiology of the disease. 46Bluetongue virus and EHDV infections can have a negative economic impact on the cattle and deer industry. 19,30,41,46 The emergence of exotic strains of BTV and EHDV, as well as the appearance ...

Detection of African swine fever, classical swine fever, and foot-and-mouth disease viruses in swine oral fluids by multiplex reverse transcription real-time polymerase chain reaction

“…The mRT-qPCR consisted of previously well-characterized and published assays 1,4,21,31 that, at the time of this publication, were in use as single-plex RT-qPCR or qPCR at the USDA-NVSL-FADDL and within the U.S. National Animal Health Laboratory Network (NAHLN). These assays were combined into the multiplex format (Table 2), and the linear dynamic range and relative analytical sensitivity of the mRT-qPCR assay was determined using serial dilutions of pooled purified nucleic acid (purNA) from culture stock viruses.…”

“…Primer and TaqMan probe sequences for detection of ASFV, CSFV, FMDV, and XIPC were taken from previous publications, 1,4,20,21 and optimal oligonucleotide concentrations were determined through empirical testing. Fluorescence and/or quencher molecules for oligonucleotide probes for ASFV and FMDV were modified from their original USDA designs in order to make the mRTqPCR compatible for simultaneous discrimination of 4 different targets.…”

“…Nucleic acid was purified using a commercial RNA isolation kit c and workflow process as previously described. 21 Five hundred microliters of clarified lysate was transferred to a 96-well, deep-well sample plate containing 20 µL of magnetic bead mix (10 µL of lysis/binding enhancer and 10 µL of RNA binding beads), and 300 µL of isopropanol was added. The following plates were loaded onto the automated particle processor d,e for nucleic acid purification: sample plate, wash solution 1 plate (300 µL/ well), wash solution 2 plate (300 µL/well), and elution buffer plate (90 µL/well).…”

“…a Nucleic acid purification was performed using 150 µL of swab suspension using the reagents and methods described above, except that the 150 µL was added directly into the sample plate containing the 20-µL magnetic bead mix and processed directly as previously described. 21 In parallel, all OF samples were nucleic acid purified using a secondary manual low-throughput nucleic acid purification method.…”

“…21 The positive amplification control (PAC) was prepared by combining ASFV target containing plasmid DNA with in vitro transcribed CSFV, FMDV, and XIPC target RNA at 1,000 copies/µL each. PAC was used for each mRT-qPCR run to ensure functionality of the mRT-qPCR conditions.…”