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Abstract
Rapid and correct diagnosis of rotavirus infection is very important to offer immediate treatment. In the present study, ribonucleic acid-polyacrylamide gel electrophoresis (RNA-PAGE) and agarose gel electrophoresis (AGE) methods were standardised and compared for the detection of rotaviruses. Double-stranded RNA (dsRNA) of rotavirus was extracted from five positive faecal samples by QIAamp Viral RNA Mini Kit and subjected to RNA-PAGE where the concentration of separating gel was varying, i.e. 7.0%, 8.5% and 12.5%. The bands of the RNA segments were clearly separated on 7.0% and 8.5% separating gel. Extracted dsRNA was also subjected to AGE where the concentration of gel was varying at 0.8%, 1.2%, 1.6% and 2.0% containing ethidium bromide. All the 11 bands of 11 segments of dsRNA were clearly visualised in all the concentrations of gel while 1.2% gel showed clear and separated bands of ladder as well as samples. Different concentrations of rotavirus, i.e. 100–1000 ng, were also loaded on standardised RNA-PAGE and AGE, separately. However, both the methods showed same detection level up to 100 ng particles of dsRNA. Further, the time required for extraction of dsRNA from five samples, their PAGE running and staining by silver stain took around 6–7 h which was comparatively more than the AGE separation, took only 2–3 h for complete procedure. Extracted dsRNA of rotavirus from 100 stool samples of children and 42 faecal samples of piglets were subjected to standardised RNA-PAGE and AGE separately for detection of rotavirus. Interestingly, 38 (38%) samples from children and 3 (7.14%) from piglet were found to be positive for rotavirus by AGE. However, RNA-PAGE could detect only 34 (34%) samples from children and 3 (7.14%) from piglets. Out of 38 positive samples from children, 32 showed long electrophoretic patterns and remaining 6 showed short electrophoretic pattern while that for 3 piglet samples, all showed long electrophoretic pattern. Thus, from our results, it can be concluded that the AGE is highly sensitive (91.17%), marginally less specific (90.90%), reproducible, superior, efficient, less laborious, cost-effective and time-saving than the RNA-PAGE for rapid diagnosis of rotavirus from faecal samples of humans as well as animals.
Acknowledgements
The authors are thankful to the Director, ICAR Research Complex for NEH Region, Umiam, Meghalaya, India for providing the necessary facilities for the study.
Funding
The work was supported by grants from the Indian Council of Agricultural Research, New Delhi, to ZBD.