Design and evaluation of primer pairs for efficient detection of avian rotavirus

Abstract

The use of molecular methods for rotavirus characterisation provides increased sensitivity for typing and allows the identification of putative reassortant strains. Reagents and methods for genotyping the virus need constant modification because of the reassortant nature of the virus. This study was aimed at designing and evaluating new oligonucleotide degenerate primer pairs that provide increased sensitivity and specificity for detecting avian rotavirus. Gene-specific primer pairs were designed by analysing different rotavirus strains isolated during the last decade by downloading them from the GenBank. The alignments were generated using clustal analysis from the BioEdit program. Degenerate nucleotides were included due to the reassortant nature of rotavirus. The consensus sequences were aligned using the BioEdit program and then treated with the Fast PCR software to derive the primers. The derived primer sequences were submitted for a BLAST search to ensure alignment was exclusive to the desired target genes. The designed primers had specific bands and were efficient in detecting rotavirus in faecal samples than previously published primers. Thus, a successful surveillance of rotaviruses requires that primer pairs be updated regularly in order to detect the emergence of novel or "unusual types", which have occurred by genetic drift causing nucleotide changes at the primer binding sites that result in typing failures. We recommend the use of the proposed primers in molecular surveillance studies for efficient detection of avian rotavirus.

Epitope-Based Peptide Vaccine against Glycoprotein G of Nipah Henipavirus Using Immunoinformatics Approaches

“…Conservation regions were determined using multiple sequence alignments with the help of Clustal-W in the BioEdit software version 7.2.5 [33]. Epitope conservancy prediction for individual epitopes was then calculated using the IEDB Analysis Resource.…”

Epitope - based peptide vaccine against glycoprotein G of Nipah henipavirus using immunoinformatics approaches

“…Conservation regions were determined using multiple sequence alignment with the help of Clustal-W in the Bio-edit software version [33] . Epitope conservancy prediction for individual epitopes was then calculated using the IEDB analysis resource.…”

Development and Application of Rapid Clinical Visualization Molecular Diagnostic Technology for Cryptococcus neoformans/C. gattii Based on Recombinase Polymerase Amplification Combined With a Lateral Flow Strip

“…We designed multiple pairs of forward and reverse primers on target gene and obtained specific primer pairs by RPA amplification ( Fischer et al., 2016 ). Approximately 15 bases are pulled-down after the upstream primers as probes, and the possible formation of cross dimers by the probe and downstream primers was predicted by Primer Premier 5.0 and base substitution was performed until all possible cross dimers were eliminated ( Oni et al., 2018 ). Subsequently, multiple upstream primers were designed upstream of the probe, and the best primer-probe combination was obtained by combining different upstream primers with downstream primers two by two ( Piepenburg et al., 2006 ; Wang et al., 2019 ).…”