Rapid visual detection of hepatitis E virus combining reverse transcription recombinase-aided amplification with lateral flow dipstick and real-time fluorescence

Fig 1. Illustration of reverse transcription recombinase-aided amplification with lateral flow dipstick (RT-RAA-LFD) and quantitative real-time reverse transcription recombinase-aided amplification (qRT-RAA) reaction. (a) Flowchart of the RT-RAA-LFD and qRT-RAA reactions. (b) Schematic representation of the RT-RAA-LFD reaction. The recombinase forms complexes with the primer or probe. The recombinase-primer complex then recognizes and binds to a homologous sequence on the DNA template. Simultaneously, the SSBs stabilize the complementary strand in its single-stranded state. Following this, the recombinase dissociates from the primer and makes it accessible for DNA polymerase to bind to the 3′ end of the primer. This binding initiates a strand displacement reaction and allows rapid amplification of a specific DNA fragment. The amplification product serves as a new template for hybridization with a nfo-probe under the action of recombinase. Endonuclease IV (nfo) cleaves the probe at the tetrahydrofuran (THF) residue and creates a new 3′ end that serves as the initiation site for DNA polymerase to synthesize a new DNA strand. This cleavage transforms the probe into a primer. The final…

Fig 2. Screening of the optimal primer pair for the RT-RAA assays. (a) Schematic diagram of primary primer screening. Seven forward primers (F1–F7) and seven reverse primers (R1–R7) are indicated by arrows, with their relative positions in the HEV ORF2 gene. (b) Primary screening results analyzed by agarose gel electrophoresis for the optimal reverse primer. RT-RAA reactions were conducted by utilizing F6 to screen all the reverse primers (R1–R7). (c) Primary screening results analyzed by agarose gel electrophoresis for the optimal forward primer. RT-RAA reactions were conducted by utilizing R3 to screen all the forward primers (F1–F7). (d) Schematic diagram of secondary primer screening. Twenty-two forward primers (F6-1–F7-10) and 17 reverse primers (R3-1–R3-16) are indicated by arrows, with their relative positions in the HEV ORF2 gene. (e) Secondary screening results analyzed by agarose gel electrophoresis for the optimal forward primer. RT-RAA reactions were conducted by utilizing R3 to screen all the forward primers (F6-1–F7-10). (f) Secondary screening results analyzed by agarose gel electrophoresis for the optimal reverse primer. RT-RAA reactions were conducted by utilizing F7-3…

Fig 3. Optimization of the RT-RAA-LFD and qRT-RAA assay reaction conditions for HEV detection. (a) Reaction temperature optimization of the RT-RAA-LFD assay. The reaction mixtures were incubated at 30°C, 33°C, 36°C, 39°C, and 42°C for 20 minutes, and the amplification products were visualized using LFD. (b) Reaction time optimization of the RT-RAA-LFD assay. The reaction mixtures were incubated at 39°C for 0, 5, 10, 15, and 20 minutes, and the amplification products were visualized using LFD. (c–f) Reaction temperature optimization of the qRT-RAA assay. The reaction mixtures were incubated at 36°C, 38°C, 40°C, and 42°C for 25 minutes, with data collected in the FAM channel every 30 seconds using the real-time PCR instrument.

Fig 4. Specificity analysis of the RT-RAA-LFD and qRT-RAA assays for HEV detection. (a) Specificity analysis of the RT-RAA-LFD assay. The amplification products were visualized using LFD. (b–c) Specificity analysis of the qRT-RAA assay. Data collection in the FAM channel occurred every 30 seconds using the real-time PCR instrument. The amplification products were observed under excitation light. Curves 1–6: nucleic acids of HEV, H1N1, PRRSV, ASFV, PCV, and negative control. NTC, no template control.

Fig 5. Sensitivity analysis of the RT-RAA-LFD and qRT-RAA and qRT-PCR assays for HEV detection. (a) Sensitivity analysis of the RT-RAA-LFD assay. (b) Probit regression analysis of the RT-RAA-LFD assay. The 95% LOD (247 copies/μL) is depicted by a red dot. (c and d) Sensitivity analysis of the qRT-RAA assay. Curves 1–8: 2.9 × 105–2.9 × 10−1 copies/μL of plasmid concentration and negative control, respectively. (e) Probit regression analysis of the qRT-RAA assay. The 95% LOD (25 copies/μL) is depicted by a red dot. (f) Sensitivity analysis of the qRT-PCR assay. Curves 1–8: 2.9 × 105–2.9 × 10−1 copies/μL of plasmid concentration and negative control, respectively. (g) Probit regression analysis of the qRT-PCR assay. The 95% LOD (25 copies/μL) is depicted by a red dot. NTC, no template control.