Novel triplex nucleic acid lateral flow immuno-assay (NALFIA) for rapid detection of Nipah virus, Middle East respiratory syndrome coronavirus and Reston ebolavirus

We report the development of the first triplex Nucleic Acid Lateral Flow Assay (NALFIA) for detection of genomes of Nipah virus (NiV), Middle East respiratory syndrome coronavirus (MERS-CoV) and Reston ebolavirus (REBOV), intended for screening of bats as well as other hosts and reservoirs of these three viruses. Our triplex NALFIA is a two-step assay format wherein the target nucleic acid in sample is first amplified using tagged primers, and the tagged ds DNA amplicons are captured by immobilized antibodies on NALFIA device resulting to signal development from binding of streptavidin-colloidal gold conjugate to biotin tag on the captured amplicons. Triplex amplification of N gene of NiV, UpE gene of MERS-CoV, and Vp40 gene of REBOV was optimized using primers adapted from validated real-time RT-PCR assays of previous studies and the compatible combinations of hapten-labels and antibodies for triplex NALFIA device were identified. Digoxigenin, rhodamine red and alexa fluor 488 were identified as suitable 5’ labels on forward primers. The lowest copy number detected by the triplex NALFIA with 2 μl of triplex RT-PCR product were up to 8.21e4 for NiV N target, 7.09e1 for MERS-CoV UpE target, and 1.83e4 for REBOV Vp40 target. Using simulated samples and Taqman real-time RT-PCR as standard, the sensitivity and positive predictive values were found to be 100% for MERS-CoV UpE and REBOV Vp40 targets and 91% for NiV N target while the specificity and negative predictive values were 100% for MERS-CoV UpE targets and REBOV Vp40 , and 93.3% for NiV N target.