RT-RPA- Pf Ago detection platform for one-tube simultaneous typing diagnosis of human respiratory syncytial virus

Abstract

Human respiratory syncytial virus (HRSV) is the most prevalent pathogen contributing to acute respiratory tract infections (ARTI) in infants and young children and can lead to significant financial and medical costs. Here, we developed a simultaneous, dual-gene and ultrasensitive detection system for typing HRSV within 60 minutes that needs only minimum laboratory support. Briefly, multiplex integrating reverse transcription-recombinase polymerase amplification (RT-RPA) was performed with viral RNA extracted from nasopharyngeal swabs as a template for the amplification of the specific regions of subtypes A (HRSV_A) and B (HRSV_B) of HRSV. Next, the Pyrococcus furiosus Argonaute (PfAgo) protein utilizes small 5'-phosphorylated DNA guides to cleave target sequences and produce fluorophore signals (FAM and ROX). Compared with the traditional gold standard (RT-qPCR) and direct immunofluorescence assay (DFA), this method has the additional advantages of easy operation, efficiency and sensitivity, with a limit of detection (LOD) of 1 copy/μL. In terms of clinical sample validation, the diagnostic accuracy of the method for determining the HRSV_A and HRSV_B infection was greater than 95%. This technique provides a reliable point-of-care (POC) testing for the diagnosis of HRSV-induced ARTI in children and for outbreak management, especially in resource-limited settings.

Keywords: human respiratory syncytial virus (HRSV); point of care (POC); pyrococcus furiosus argonaute; reverse transcription recombinase polymerase amplification (RT-RPA); typing diagnosis.

Figure 1

Schematic illustration of the RT-RPA-PfAgo platform.

Figure 2

The strategy of best primer pair and gDNA-probe pair screening. (A) The strategy for screening the best primer pairs for HRSV_A. In step 1, a downstream primer was selected to match all the upstream primers. Based on the intensity of the bands, the brightest band is optimal. In step 2, the best upstream primers were used to match all the downstream primers. Finally, the best primer was determined. (B) The strategy for screening the best primer pairs for HRSV_B. (C) The strategy for screening the best gDNA-probe pairs for HRSV_A. The combination of the fastest fluorescence signal growth and the highest final fluorescence intensity was selected to determine the best gDNA-probe pair. HRSV_A-C1 represents the combination of gDNA1, gDNA2, and probeA1. HRSV_A-C2 represents the combination of gDNA3, gDNA4 and probeA2. (D) The strategy for screening the best gDNA-probe pairs for HRSV_B. HRSV_B-C1 represents the combination of gDNA5, gDNA6 and probeB1. HRSV_B-C2 represents the combination of gDNA7, gDNA8 and probeB2.

Figure 3

The optimization of parameters in the RT-RPA-PfAgo platform. (A) Optimization of PfAgo. The optimal condition is the one with the highest fluorescence intensity in the error bar chart. A reaction tube exhibiting fluorescence under UV light is considered positive. (B) Optimization of guide DNA. (C) Optimization of Mn²⁺. (D) Optimization of the template. (E) The final reaction system. It is interpreted in conjunction with Figures (A–D) The numbers represent the number of tubes. For example, “1” represents 2μL PfAgo, 0.2μM gDNA, 0.5μL Mn²⁺, and 0.5μL template. The pink dots were the optimal condition. (F) The Urea-PAGE gel electropherogram of RT-RPA-PfAgo cleavage products.

Figure 4

Evaluation of the RT-RPA-PfAgo platform. (A) Evaluation of the sensitivity of the RT-RPA-PfAgo platform. The results showed that the LOD of the platform could reach 1 copy/μL. (B) Evaluation of the specificity of the RT-RPA-PfAgo platform. The fluorescence curves and images under UV light revealed no positive results for other common respiratory virus samples.

Figure 5

Comparison of the diagnostic capability of the RT-RPA-PfAgo platform, RT-PCR and DFA. (A) Three methods, including direct immunofluorescence assays, RT-PCR and RT-RPA-PfAgo platform, were selected to test 150 clinical samples. TFhe overlapping area represents the number of samples that have the same results across the corresponding detection methods. Compared to DFA, the RT-RPA-PfAgo platform is more consistent with RT-PCR. (B) Pictures of positive and negative samples from the DFA assays under a fluorescence microscope; no fluorescent signal is seen in the negative results, and cells infected by HRSV are seen in the positive results, with neighboring cells fusing with each other to form syncytial structures (C) The fluorescence intensity of twenty clinical samples which were randomly selected. (D) The comparison of fluorescence intensity between positive and negative samples for HRSV_A and HRSV_A infection shows statistically significant differences. (E) The fluorescence curve of the 20 random clinical samples. Elevated fluorescence levels in the FAM channel indicate HRSV_A infection, whereas elevated ROX fluorescence values indicate HRSV_B infection. "****" represents P<0.001.