Novel high-coverage primers for detection of canine morbillivirus by end-point and real-time RT-PCR assays

J Virol Methods. 2023 Nov 16:114853. doi: 10.1016/j.jviromet.2023.114853. Online ahead of print.ABSTRACTCanine distemper virus (CDV) is a major threat to domestic dogs and wildlife worldwide. Molecular assays are the most sensitive and specific tests to diagnose the disease, however, the high CDV genetic variability may compromise laboratory diagnosis. Herein, we designed a high-coverage primer set for end-point (RT-PCR) and real-time (RT-qPCR) for CDV detection. Initially, we collected 194 complete/near-complete CDV genomes (GenBank) and analyzed them for highly conserved regions for primer design. We then assessed the in silico coverage, analytical sensitivity, specificity and diagnostic performance of RT-PCR/RT-qPCR reactions based on our primers. Furthermore, the coverage of our primers, as well as their analytical sensitivity and diagnostic performance, were compared to a commonly used primer set for CDV detection (named PP-I). Our forward (F) and reverse (R) primers fully matched 100% (194/194) and 99% (192/194) of the analyzed sequences, whereas the PP-I F and R primers fully matched 15% (29/194) and 9% (18/194) sequences, respectively. The detection limit of our RT-PCR and RT-qPCR was equivalent to that of PP-I primers (0.01 TCID50/mL). Out of 70 clinical samples tested, 38 were positive by our RT-PCR/RT-qPCR assays, whereas reactions with primers PP-I failed to detect 9/28 (32%) positive samples selected for comparison purposes. In addition, our assays did not amplif...
Source: Journal of Virological Methods - Category: Virology Authors: Source Type: research