Fig. 2: Nested recombinase polymerase amplification (RPA) increased the detection sensitivity for SARS-CoV-2 RNA.

a Schematic diagram of nested RPA primer design and workflow. RNA samples were amplified by the first RPA with outer primers (indicated by blue arrows) followed by the second RPA with inner primers and probe (indicated by red and purple arrows). b LOD for RT-qPCR (right) and nested RPA assay (left). Ct values and LFA results were measured with purified IVD 0–5 copies RNA samples of SARS-CoV-2 E gene, N gene, and ORF1ab gene (n = 3 for each assay and the error bar was presented as standard deviation ND indicated Ct value above 45. c The dynamics of primers, targeted products and non-specific products (labeled as NS products or by-products in picture) illustrated by polyacrylamide gel electrophoresis at 0, 10, 20 and 30 min of traditional RPA reaction. The quantitative analysis was measured by gray values (n = 2). The positive control (PC) experiment is done at 500 copies of target input while negative control (NC) is done at 0 copies of input. d The effect of primers and ATP supplements on the amplification result. F1R1 represents the outer forward/reverse primer pair and F2R2 represents the inner forward/reverse primer pair. e The efficacy of RNA release by Triton X-100/NP40 lysis buffer on pseudovirus was tested using RNase A aided RT-qPCR assay (left), and a comparison was made between Triton X-100/NP40 lysis buffer and commercialized Trizol reagent on cultured SRAS-CoV-2 (right). The designation “virus & RNase” indicates that the virus sample was directly treated with RNase, while “virus & lysis & RNase” indicates that the virus sample was treated with RNase in lysis buffer containing Triton and NP-40. The dilution ratios of SRAS-CoV-2 culture solution, denoted as “E-3, E-4, E-5,” were 1000, 10,000, and 100,000, respectively. Data are presented in term of the mean ± standard deviation with n = 3.