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

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.

Prevalence of Cyclospora cayetanensis and other enteropathogen among children under the age of 15 years in Biratnagar, Nepal

“…Serological tests and antibiotic susceptibility tests for the isolates were done according to the procedure described by World Health Organization [16]. The ELISA was used for detection of rotavirus [17]. Verbal informed consent was taken from study population.…”

Application of Transmission Electron Microscopy Techniques in the Veterinary Diagnosis of Viral Gastroenteritis in Livestock Animals

“…Typically, coronavirus particles can be demonstrated in fecal samples by direct electron microscopy ( Figure 5), immune electron microscopy or immunolabeling with colloidal gold particles ( Figure 6) [27,[98][99][100][101][102].…”

Rapid Detection of Type 2 Porcine Reproductive and Respiratory Syndrome Virus by a Duplex Reverse Transcription Insulated Isothermal PCR on a Field-Deployable System

“…Clinical performance of various iiPCR for various bacterial and viral pathogens in companion animals, livestock animals, and aquaculture animals, food safety, and health care has been demonstrated to be comparable to that of the reference nested PCR, real-time PCR, and/or virus isolation method [28,[30][31][32][33][34][35][36][37][38][39][40][41]. In several cases, clinical sensitivity slightly higher than that of the reference realtime PCR methods was observed for targets with notable sequence variations; clinical specificity can be maintained by careful design of the primer and probe [36,[42][43][44].…”