Abstract
There is no gold standard for detection of Rotavirus Group A (RVA), one of the main causes of diarrhea in neonatal animals. Sensitive and specific real-time RT-PCR (rtRT-PCR) assays are available for RVA but require submission of the clinical samples to diagnostic laboratories. Patient-side immunoassays for RVA protein detection have shown variable results, particularly with samples from unintended species. A sensitive and specific test for detection of RVA on the farm would facilitate rapid management decisions. The insulated isothermal RT-PCR (RT-iiPCR) assay works in a portable machine to allow sensitive and specific on-site testing. The aim of this investigation was to evaluate a commercially available RT-iiPCR assay for RVA detection in feces from different animal species. This assay was compared to an in-house rtRT-PCR assay and a commercially available rtRT-PCR kit, as well as an ELISA and EM for RVA detection. All three PCR assays targeted the well-conserved NSP5 gene. Clinical fecal samples from 108 diarrheic animals (mainly cattle and horses) were tested. The percentage of positive samples by ELISA, EM, in-house rtRT-PCR, commercial rtRT-PCR, and RT-iiPCR was 29.4%, 31%, 36.7%, 51.4%, 56.9%, respectively. The agreement between different assays was high (81.3–100%) in samples containing high viral loads. The sensitivity of the RT-iiPCR assay appeared to be higher than the commercially available rtRT-PCR assay, with a limit of detection (95% confidence index) of 3–4 copies of in vitro transcribed dsRNA. In conclusion, the user-friendly, field-deployable RT-iiPCR system holds substantial promise for on-site detection of RVA.
Highlights
Rotavirus is classified as a member of family Reoviridae, genus Rotavirus
Host species barriers and host range restriction exist in rotavirus, reassortment can result in interspecies transmission, which contributes to the diversity and evolution of rotavirus (Martella et al, 2010; Zhou et al, 2015)
Nucleic acids were extracted with an automated nucleic acid extraction system according to the manufacturer’s instructions from all 108 samples for molecular testing
Summary
Rotavirus is classified as a member of family Reoviridae, genus Rotavirus. It is non-enveloped, 60–80 nm in diameter, and the genome length is approximately 18.5 Kb. The genome is composed of 11 segments of double-stranded RNA and encodes six structural proteins (VP1–4, 6 and 7) and six non-structural proteins (NSP16) (Desselberger, 2014; Zhou et al, 2015). Rotaviruses are characterized by relatively high antigenic and genetic diversity, as a result of accumulation of point mutations (genetic drift), and/or reassortment of genomic segments (genetic shift) (Matthijnssens et al, 2012). Host species barriers and host range restriction exist in rotavirus, reassortment can result in interspecies transmission, which contributes to the diversity and evolution of rotavirus (Martella et al, 2010; Zhou et al, 2015)
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