Real-time, MinION-based, amplicon sequencing for lineage typing of infectious bronchitis virus from upper respiratory samples

Salman L. Butt; Eric C. Erwood; Jian Zhang; Holly S. Sellers; Kelsey Young; Kevin K. Lahmers; James B. Stanton

The copyright holder for this preprint (which was not peer-reviewed) is the author/funder. . https://doi.org/10.1101/634600 doi: bioRxiv preprint Currently, pan-IBV RT-qPCR is used to rapidly detect IBV from clinical samples for screening purposes, 4 but this requires additional serotype-specific RT-qPCRs to genotype positive samples, including samples containing more than one genotype. Additionally, if unidentified IBVs are in the sample, then current detection and characterization may also require egg culture followed by various PCRs. 8 The AmpSeq method described in this report detected IBV in 13 of 14 IBV-positive and detected all the mixed isolates in four of seven samples containing two or more isolates. While mixed isolates were not 100% identified (0/2 in #1, 2/4 in #2, and ½ in #3) in three samples with the AmpSeq method, detection of those mixed isolates required several non-AmpSeq assays. Although, there is room for improving this new AmpSeq method, it represents a promising, single-step assay that can be used without egg culture. Thus, it is another tool for the detection of IBV, especially in cases when multiple isolates may be present or when genotyping is especially important.