Table 1.
Target sequences and optimized primer/probe sequences (PCR/RPA). Nucleotide substitutions relative to MACV are bold.
Table 2.
Cross-reactivity assessment of MACV RT-PCR and RT-RPA assays against a panel of non-MACV viruses.
Fig 1.
Sequence alignment of target variants.
Nucleotide substitutions relative to MACV are highlighted. Binding sites for real-time RT-PCR and RT-RPA primers/probes are indicated.
Fig 2.
Real-time RT-PCR amplification plots using the mixed primer set on ARPs containing a) MACV, b) MACV_2, c) MACV_10, and d) MACV_14 target sequences.
Serial 10-fold dilutions are shown (copies/ml): 5ⅹ101 (brown), 5ⅹ102 (red), 5ⅹ103 (orange), 5ⅹ104 (yellow), 5ⅹ105 (green), 5ⅹ106 (blue), 5ⅹ107 (purple), and no template control (NTC, black). Panel e) shows standard curve with mean Ct values (± SD, n = 3) versus log10(ARP concentration).
Fig 3.
a) Real-time RPA kinetics comparing probes 1prb and 2prb. b) heatmap of fluorescence intensity (F-F0, background-subtracted) for primer pair screening (forward: 1F, 2F, 3F; reverse: 4R, 5R, 6R) with probe 2prb. c) real-time RPA kinetics using primer/probe set 1F/4R/2prb on serially diluted DNA template. Data represent mean ± SD (n = 2 replicate reactions).
Fig 4.
Real-time RT-RPA assay with ARPs.
a) Relative fluorescence (F-F0) at endpoint (20 min) for RT-RPA assays using SuperScript IV RT supplemented with varying concentrations of RNase H (0 - 5 U/µl). b) real-time RT-RPA amplification kinetics using optimized conditions (M-MuLV RT or SuperScript IV RT + 0.5 U/µl RNase H) and ARPs (1.5 × 103–2.5 × 104 copies/ml). Data represent mean ± SD (n = 2).
Fig 5.
Heatmap of endpoint fluorescence intensity (F-F0) for redesigned primer pairs screened with probe 2prb across target variants.
Primer variants: forward (1F.1g, 1F.gg, 1F.2g, 1F.ga, 1F.tg) and reverse (4R.2t, 4R.gtc). Numbers within cells (x/y) indicate substitutions in forward (x) and reverse (y) primers relative to original sequences. Panels: a) MACV, b) MACV_14, c) MACV_2 and MACV_10.
Fig 6.
Real-time RT-RPA amplification kinetics with ARP serial dilutions containing: a) MACV, b) MACV_2, c) MACV_10, d) MACV_14.
The optimized dual primer/probe set mixture was used (1F/4R/2prb + 1F.gtag/4R.gctc/2prb.ga, each at half concentration). LOD is indicated (5 × 103 copies/ml). Panel e) linear dependence of fluorescence signal on log10(ARP concentration). Data represent mean ± SD (n = 2).
Fig 7.
Evaluation of RT-RPA assay on an Axxin T16-ISO using serum-spiked samples.
a) Amplification kinetics for MACV ARPs spiked into human serum and negative controls. Corresponding control samples with ARPs diluted in nuclease-free water are shown for comparison. b) Mean endpoint fluorescence for the samples shown in panel (a). c) Determination of the assay’s LOD for ARPs spiked into serum, demonstrating a LOD of 5 × 103 copies/ml. Data represent mean ±SD (n = 5 biological replicates for panel (b); n = 2 reaction replicate for panel (c)).
Table 3.
Comparative analysis of the real-time RT-PCR and RT-RPA assays.