Development of Poly (A)-Tailed Universal Reverse Transcription PCR Method for Sequence-Independent Amplification of Rearranged Rotavirus

Group A Rotaviruses (GARV) are the main viral cause of acute gastroenteritis, leading to 870,000 deaths annually in the developing world and representing a major health problem. Therefore, diagnosis and treatment of this disease are crucial. Gene rearrangement within segmented viruses as well as rotavirus is seen throughout chronic rotavirus infection in immunodeficient young children and through serial passage of rotavirus in cell culture at a high multiplicity of infection. Detailed knowledge of rotavirus biology allows design of a vaccine against rotavirus by engineered antigens. The aim of this study was to develop Poly (A) -Tailed universal Reverse Transcription Polymerase Chain Reaction (RT-PCR) method and compare the efficacy of this procedure with specific multiplex PCR protocol for detecting normal and rearranged segments.
Virus was propagated on confluent monolayer of MA-104 cells and aliquots of each passage were kept frozen for further RNA genomic profiles analysis by polyacrylamide gel electrophoresis. Purified Rota virus RNA was polyadenylated and used for the amplification and detection of normal and rearranged segments of rotavirus using RT-PCR.
The generation of gene rearrangement through multiple serial passages of rotavirus was shown using MOI ≥ 1 . The rearranged RNA segments of NSP1 and NSP3 genes with different migration pattern in PAGE were detected by poly(A)RT-PCR.
In the current research, a novel Poly(A) -Tailed Universal Reverse Transcription PCR method was introduced for the high throughput amplification and analysis of the informative untranslated regions of the rotavirus genome.