Abstract
The combination of recombinase polymerase amplification (RPA) and lateral flow test (LFT) is a strong diagnostic tool for rapid pathogen detection in resource-limited conditions. Here, we compared two methods generating labeled RPA amplicons following their detection by LFT: (1) the basic one with primers modified with different tags at the terminals and (2) the nuclease-dependent one with the primers and labeled oligonucleotide probe for nuclease digestion that was recommended for the high specificity of the assay. Using both methods, we developed an RPA-LFT assay for the detection of worldwide distributed phytopathogen—alfalfa mosaic virus (AMV). A forward primer modified with fluorescein and a reverse primer with biotin and fluorescein-labeled oligonucleotide probe were designed and verified by RPA. Both labeling approaches and their related assays were characterized using the in vitro-transcribed mRNA of AMV and reverse transcription reaction. The results demonstrated that the RPA-LFT assay based on primers-labeling detected 103 copies of RNA in reaction during 30 min and had a half-maximal binding concentration 22 times lower than probe-dependent RPA-LFT. The developed RPA-LFT was successfully applied for the detection of AMV-infected plants. The results can be the main reason for choosing simple labeling with primers for RPA-LFT for the detection of other pathogens.
Highlights
RNA3 and subgenomic-proceeded RNA4 are most presented in the host cell, so they are preferred targets for detection
reverse transcription (RT)-Recombinase polymerase amplification (RPA)-lateral flow test (LFT) with F4-R4 primers were performed based on two different methods of labeled amplicons
We found that the presence of the DNA/RNA of the potato plant in the RT-RPA-LFT assay eliminated nonspecific background signal and improved the specificity and sensitivity of the approach based on labeled primers
Summary
Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations. This approach is actively applied for the RPA-LFT detection of pathogens and species identification [8,9,10,11]. There is about no rethe direct comparison of based on the generation of labeled amplicons through search about the direct comparison of RPA-LFT based on the generation of labeled ampliforward and reverse primers and through the and oligonucleotide and reverse probe primer.and. Figure of of labeled amplicons andand their detection, based on Figure 1
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