Evaluation of two Real-Time, TaqMan Reverse Transcription-PCR assays for detection of rabies virus in circulating variants from Argentina: influence of sequence variation

CARABALLO, D. A.; LOMBARDO, M. A.; BECKER, P.; SABIO, M. S.; LEMA, C.; MARTÍNEZ, L. M.; LI, Y.; CISTERNA, D. M.; BELTRÁN, F. J.

Ciudad de Mexico

Congreso; XXXI Rabies in the Americas; 2020

In rabies diagnosis, it is essential to count on a rapid test to give a quick response. Although the direct fluorescent antibody test (FAT) is the gold standard technique, the combined sensitivity and robustness of the TaqMan RT-PCR assays (qRT-PCR) have made these methods a valuable alternative for rabies virus (RABV) detection. We faced a study to test the applicability of two widely used qRT-PCR assays targeting the nucleoprotein gene and Lead sequences of RABV genomes, in all variants circulating in Argentina. A total of 44 FAT and conventional RT-PCR-positive samples corresponding to bats, dogs, cattle, and horses, were used in the study. All variants were successfully detected by the pan-lyssavirus LN34 qRT-PCR assay. The Lyssavirus Genotype 1 (LysGT1) assay positively detected variants V1, V2, V3, V4, Eptesicus, and Histiotus, while V3A, V6, and Myotis samples remained undetectable. We further sequenced the region targeted by LysGT1 and demonstrated that the presence of 3 or more mismatches with respect to the primers and probe sequences precludes viral detection. We conclude that the LysGT1 assay is prone to yield variant-dependent false-negative test results and, in consequence, the LN34 assay should be adopted in diagnostic laboratories for the detection of RABV in Argentina.