Development and evaluation of a rapid detection assay for severe fever with thrombocytopenia syndrome virus based on reverse-transcription recombinase polymerase amplification

Abstract

Development and evaluation of a rapid detection assay for severe fever with thrombocytopenia syndrome virus based on reverse-transcription recombinase polymerase amplification, Molecular and Cellular Probes (2020), doi: https://doi.Table of Abbreviations: dT-FAM Thymidine nucleotide carrying FAM fluorophore dT-BHQ1 Thymidine nucleotide carrying BHQ-1 quencher HHV Human herpesvirus LAMP loop-mediated isothermal amplification LOD Limit of detection PIV Parainfluenza virus RPA Recombinase polymerase amplification RT-LAMP Reverse-transcription loop-mediated isothermal amplification RT-PCR Reverse-transcription polymerase chain reaction RT-qPCR Real-time reverse-transcription polymerase chain reaction RT-RAA Reverse-transcription recombinase-aided amplification RT-RPA Real-time reverse-transcription recombinase polymerase amplification SFTS Severe fever with thrombocytopenia syndrome SFTSV Severe fever with thrombocytopenia syndrome virus SSB Single-stranded DNA binding Abstract 1 Rapid detection of severe fever with thrombocytopenia syndrome virus (SFTSV) is 2 crucial for its control and surveillance. In this study, a rapid isothermal real-time 3 reverse-transcription recombinase polymerase amplification (RT-RPA) assay was 4 developed for the detection of SFTSV. The detection limit at 95% probability was 241 5 copies per reaction. A test of 120 serum samples of suspected severe fever with 6 thrombocytopenia syndrome (SFTS) patients revealed that the sensitivity and 7 specificity of the RT-RPA assay was approximately 96.00% (95%CI: 80.46%-99.79%) 8 and 98.95% (95% CI: 94.28%-99.95%), respectively; the kappa value was 0.9495 (P 9 ＜0.001). The Bland-Altman analysis showed that 87.50% of the different data points 10 were located within the 95% limits of agreement, indicating a good correlation 11 between the results from RT-RPA assays and those of RT-qPCR assays. In conclusion, 12 the rapid and efficient RT-RPA assay can be a promising candidate for point-of-care 13 detection method of SFTSV. 14 15 Keywords: Severe fever with thrombocytopenia syndrome virus; Recombinase 16 polymerase amplification; Detection assay Severe fever with thrombocytopenia syndrome virus (SFTSV), belonging to the 19 family Phenuiviridae, the genus Banyangvirus, the species Huaiyangshan 20 banyangvirus, was first identified as the causative pathogen of the outbreak of severe 21 fever with thrombocytopenia syndrome (SFTS) in the central and northeast regions of 22 China in 2009 [1]. During the following decade, this emerging endemic tick-borne 23 disease has been reported in many countries worldwide, including South Korea, Japan, 24 United Arab Emirates, and the United States [2-5]. Delayed admission due to 25 nonspecific clinical manifestations, along with rapid progression to multiple organ 26 dysfunction in severe cases led to a high case fatality rate of 6.40~30% in SFTS 27 patients [6, 7]. 28 Currently, detection methods of SFTSV comprise nucleic acid testing, serological 29 detection, and virus isolation [1, 8, 9]. Due to the long turnaround...

Dual-gene detection in a single-tube system based on CRISPR-Cas12a/Cas13a for severe fever thrombocytopenia syndrome virus

“…At present, the most focused molecular detection methods, such as polymerase chain reaction (PCR) and quantitative real-time polymerase chain reaction (qPCR), are not suitable for on-site detection of SFTSV, due to the requirements of sophisticated instruments and skilled personnel ( Sun et al, 2012 ). A series of isothermal amplification techniques have also been established to detect SFTSV, including loop-mediated isothermal amplification (LAMP) and recombinase polymerase amplification (RPA; Zhou et al, 2020 ; Sano et al, 2021 ). But these amplification technologies still show limitations in sensitivity and specificity.…”

Accuracy of reverse‐transcription polymerase chain reaction and loop‐mediated isothermal amplification in diagnosing severe fever with thrombocytopenia syndrome: A systematic review and meta‐analysis

“…After reading the full text of the articles, 103 were discarded, including 48 that did not use clinical patient samples or lacked data, 35 that were not on nucleic acid‐related diagnostic techniques, 14 that lacked reference standards, and 6 that were not on or related to SFTS. A final total of 16 articles were included, seven on RT‐PCR 19 , 20 , 24 , 25 , 26 , 27 , 28 and seven on RT‐LAMP, 16 , 21 , 29 , 30 , 31 , 32 , 33 and two that were on both RT‐PCR and RT‐LAMP. 15 , 34 A flowchart of the research process is shown in Figure 1 .…”

Advancements in the Worldwide Detection of Severe Fever with Thrombocytopenia Syndrome Virus Infection from 2009 to 2023

“…Detection of SFTSV genome could be achieved by different nucleic acid detection techniques such as RT-PCR (13,(40)(41)(42)(43), real-time RT-PCR (23,28,41,(44)(45)(46)(47)(48), LAMP (24,(49)(50), as well as RPA (25,(51)(52).…”

“…After combining with the fluorescent detection reagent (FDR) method, results could be determined by observing a color change within 30 min (64). Jang et al developed a multiplex RT-LAMP to identify larger segments and GroES genes for SFTSV and Orientia tsutsugamushi (OT) (24). The sensitivity of the multiplex SFTSV/OT/Internal control (IC) RT-LAMP assay proved comparable to that of the commercial PowerChek™ SFTSV Real-time PCR (91.3% vs. 95.6%).…”

“…Pathogenic detection includes virus isolation via cell culture and electron microscopy techniques ( 3 , 20 - 21 ). Nucleic acid amplification techniques such as reverse transcription-polymerase chain reaction (RT-PCR) ( 22 - 23 ), loop-mediated isothermal amplification (LAMP) ( 24 ), and recombinase polymerase amplification (RPA) ( 25 ). Rapid diagnostic tests, such as lateral flow assays, provide prompt results and prove beneficial in resource-limited environments ( 3 ).…”