Comparison of electron microscopy, ELISA, real time RT-PCR and insulated isothermal RT-PCR for the detection of Rotavirus group A (RVA) in feces of different animal species

•Agreement between molecular tests for Rotavirus group A (RVA) detection was 80–92%.•The agreement between all assays was 81–100% in samples containing high viral loads.•The sensitivity of RVA RT-iiPCR was 3–4 copies of in vitro transcribed dsRNA.•The field-deployable RT-iiPCR system holds promise f...

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Bibliographic Details
Published in:Journal of virological methods 2016-09, Vol.235, p.99-104
Main Authors: Soltan, Mohamed A., Tsai, Yun-Long, Lee, Pei-Yu A., Tsai, Chuan-Fu, Chang, Hsiao-Fen G., Wang, Hwa-Tang T., Wilkes, Rebecca P.
Format: Article
Language:English
Subjects:
Animals
Cattle
Cattle Diseases - diagnosis
Cattle Diseases - virology
Diagnosis
Diarrhea - virology
Enzyme-Linked Immunosorbent Assay
Feces - virology
Horse Diseases - diagnosis
Horse Diseases - virology
Horses
Insulated isothermal PCR
Microscopy, Electron
Point of need
Point-of-Care Systems
Reagent Kits, Diagnostic
Real-Time Polymerase Chain Reaction
Real-time RT-PCR
Reverse Transcriptase Polymerase Chain Reaction
RNA, Double-Stranded
RNA, Viral - genetics
RNA, Viral - isolation & purification
Rotavirus
Rotavirus - genetics
Rotavirus - isolation & purification
Rotavirus Infections - diagnosis
Rotavirus Infections - veterinary
Rotavirus Infections - virology
Sensitivity and Specificity
Temperature
Viral Load
Viral Nonstructural Proteins - genetics
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Summary:•Agreement between molecular tests for Rotavirus group A (RVA) detection was 80–92%.•The agreement between all assays was 81–100% in samples containing high viral loads.•The sensitivity of RVA RT-iiPCR was 3–4 copies of in vitro transcribed dsRNA.•The field-deployable RT-iiPCR system holds promise for on-site detection of RVA. 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.
ISSN:0166-0934
1879-0984
DOI:10.1016/j.jviromet.2016.05.006