Reverse transcription recombinase polymerase amplification assay for rapid detection of canine associated rabies virus in Africa

Wahed

et al. 2021

Sci Rep

Rabies is a generally fatal encephalitis caused by a negative-sense single-stranded RNA lyssavirus transmitted to humans mainly from dog bite. Despite the recommendation by WHO and OIE to use the direct immunofluorescence test as standard method, molecular diagnostic assays like reverse transcription quantitative polymerase chain reaction (RT-qPCR) are increasing as a confirmatory method. However, both technologies are inaccessible in resource-limited settings. Moreover, the available point-of-need molecular assay is of poor detection limit for African strains. Herein, we developed a reverse transcription recombinase polymerase amplification (RT-RPA) assay as potential point-of-need diagnostic tool for rapid detection of various strains of rabies virus including locally isolated African strains. The sensitivity and specificity of the method was evaluated using a molecular RNA standard and different Rabies-related viruses belonging to the Rhabdoviridea family, respectively. The RABV-RPA performances were evaluated on isolates representative of the existing diversity and viral dilutions spiked in non-neural clinical specimen. The results were compared with RT-qPCR as a gold standard. The RABV-RPA detected down to 4 RNA molecules per reaction in 95% of the cases in less than 10 min. The RABV-RPA assay is highly specific as various RABV isolates were identified, but no amplification was observed for other member of the Rhabdoviridea family. The sample background did not affect the performance of the RABV-RPA as down to 11 RNA molecules were identified, which is similar to the RT-qPCR results. Our developed assay is suitable for use in low-resource settings as a promising alternative tool for ante-mortem rabies diagnosis in humans for facilitating timely control decisions.

Section: Discussionmentioning

confidence: 87%

A recombinase polymerase amplification assay for rapid detection of rabies virus

Wahed

et al. 2021

Sci Rep

Molecular and Cellular Probes

“…In detection of clinical samples, the RT-RPA assay has a certain probability to produce false-negative results [32]. To improve the clinical sensitivity of RT-RPA, investigation into the influence of the amount and distribution of mismatches on RPA and the employment of degenerate primers [33] is in need. Besides, with better fluorescence detection equipment, this technique can be integrated into a phone-size portable device and be easily used for field detection in SFTS-prevalent areas.…”

Section: Discussionmentioning

confidence: 99%

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

Zhou

Wang

Zhu

et al. 2020

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...

“…Similarly, RPA, an isothermal alternative of polymerase chain reaction (PCR), is a sensitive molecular diagnostic technology, which is rapid, portable, and can be multiplexed for several pathogens (Rostron et al, 2019 ). RPA has been used as reliable and rapid diagnostics for several viruses such as the human immunodeficiency virus (Crannell et al, 2014 ), Zika virus (Vasileva Wand et al, 2019 ), avian influenza A (Waheda et al, 2015 ), rabies virus (Coertse et al, 2019 ), Potato virus Y and Wheat dwarf virus (Glais and Jacquot, 2015 ), Tomato yellow leaf curl virus (Londoño et al, 2016 ), Apple stem grooving virus (Kim et al, 2018 ), and Banana bunchy top virus (Kapoor et al, 2017 ). Although these techniques proved better POCT techniques, detailed analysis of these molecular diagnostic mechanisms shows their low sensitivity and low throughput (Huang et al, 2020 ).…”

Section: Introductionmentioning

confidence: 99%

Next-Generation Molecular Diagnostics Development by CRISPR/Cas Tool: Rapid Detection and Surveillance of Viral Disease Outbreaks

Srivastava

Upadhyay

Srivastava

2020

Front. Mol. Biosci.

Virus disease spreads effortlessly mechanically or through minute insect vectors that are extremely challenging to avoid. Emergence and reemergence of new viruses such as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), H1N1 influenza virus, avian influenza virus, dengue virus, Citrus tristeza virus, and Tomato yellow leaf curl virus have paralyzed the economy of many countries. The cure for major viral diseases is not feasible; however, early detection and surveillance of the disease can obstruct their spread. Therefore, advances in the field of virus diagnosis and the development of new point-of-care testing kits become necessary globally. Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein (Cas) is an emerging technology for gene editing and diagnostics development. Several rapid nucleic acid diagnostic kits have been developed and validated using Cas9, Cas12, and Cas13 proteins. This review summarizes the CRISPR/Cas-based next-generation molecular diagnostic techniques and portability of devices for field-based utilization.