Multiplex Assay of Viruses Integrating Recombinase Polymerase Amplification, Barcode-Anti-Barcode Pairs, Blocking Anti-Primers, and Lateral Flow Assay

A multiplex assay based on recombinase polymerase amplification (RPA) and lateral flow test (LFT) is a desirable tool for many areas. This multiplex assay could be efficiently realized using single-stranded (ss) DNAs located in separate zones on the test strip and bound complementary ssDNA tags of double-stranded (ds) DNA amplicons. Here, we investigate how to enrich multiplex assay capabilities using ssDNAs. Bifunctional oligonucleotide probes integrating (1) a forward primer for RPA, (2) a C9 spacer to stop polymerase, and (3) a ssDNA tag for binding at test strip are developed. The amplicons have a unique individual ssDNA tag at one end and a universal label of fluorescein introducing through a reverse primer at the other end. A conjugate of gold nanoparticles (GNP) with antibodies to fluorescein is used to detect all amplicons. The remainder of primers after RPA interacting with GNP conjugate was found to be a limiting factor for sensitive and specific multiplex assay. The addition of anti-RPA-primers before the use of test strips was proposed to simply and effectively eliminate remaining primers. This approach was successfully applied for the detection of three priority plant RNA viruses: potato virus Y (PVY), -S (PVS) and potato leafroll virus (PLRV). The total time of the assay is 30 min. The multiplex RPA-LFT detected at least 4 ng of PVY per g of plant leaves, 0.04 ng/g for PVS, and 0.04 ng/g for PLRV. The testing of healthy and infected potato samples showed concordance between the developed assay and reverse transcription-polymerase chain reaction. Thus, the capabilities of the proposed universal modules (ssDNA anchors, bifunctional probes, and blocking anti-primers) for multiplex detection of RNA analytes with high specificity and sensitivity were demonstrated.