Detection and differentiation of low virulence and virulent avian orthoavulavirus javaense using a molecular beacon with RT-LAMP

Authors: Mears, Megan; Olivier, Timothy; Williams Coplin, Tina; Espinoza, Edna; Bakre, Abhijeet

Submitted to: Scientific Reports
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 7/29/2024
Publication Date: 8/5/2024
Citation: Mears, M.C., Olivier, T.L., Williams Coplin, T.D., Espinoza, E., Bakre, A.A. 2024. Detection and differentiation of low virulence and virulent avian orthoavulavirus javaense using a molecular beacon with RT-LAMP. Scientific Reports. 2024(14):18047. https://doi.org/10.1038/s41598-024-68816-7.
DOI: https://doi.org/10.1038/s41598-024-68816-7

Interpretive Summary: Newcastle disease is a commercially important poultry disease; current containment efforts are based on detection of virulent avian orthoavulavirus 1 (AOAV-1) strains and these efforts can be time and resource intensive. Assays that can rapidly detect and differentiate AOAV-1 in samples can help control spread of the virus especially in resource poor countries where large scale culling is not economically feasible. In this manuscript, we developed a molecular beacon based colorimetry assay that can detect and differentiate AOAV-1 in samples in a single tube approaching the current quantitative PCR based assay in sensitivity and specificity.

Technical Abstract: Newcastle disease (ND), an economically important disease in poultry, is caused by virulent strains of the genetically diverse Orthoavulavirus javaense (OAVJ). Laboratories rely on quantitative real-time reverse transcription PCR (qRT-PCR) to detect OAVJ and differentiate between OAVJ pathotypes. This study demonstrates that a fusion cleavage site based molecular beacon with reverse transcription loop mediated isothermal amplification (MB-RT-LAMP) assay can detect and differentiate OAVJ pathotypes in a single assay. Data show that the assay can rapidly identify diverse OAVJ genotypes with sensitivity only one log-fold lower than the current fusion qRT-PCR assay (104 copies), exhibits a high degree of specificity for OAVJ, and the molecular beacon can differentiate mesogenic/velogenic sequences from lentogenic sequences. Further, data show that a two-minute rapid lysis protocol preceding MB-RTLAMP can detect and differentiate OAVJ RNA from both spiked samples and oropharyngeal swabs without the need for RNA isolation. As the MB-RT-LAMP assay can rapidly detect and discriminate between lentogenic and mesogenic/velogenic sequences of OAVJ within one assay, without the need for RNA isolation, and is adaptable to existing veterinary diagnostic laboratory workflow without additional equipment, this assay could be a rapid primary screening tool before qRT-PCR based validation in resource limited settings.