Magnetic Field-Enhanced Agglutination Readout Combined With Isothermal Reverse Transcription Recombinase Polymerase Amplification for Rapid and Sensitive Molecular Detection of Dengue Virus.

Fanny Leon,Jean-Pierre Molès,Chantal Fournier-Wirth,Elena Pinchon,Aurélien Daynès,Jean-François Cantaloube,François Morvan,Charly Mayran

doi:10.3389/fchem.2021.817246

Abstract

Among the numerous molecular diagnostic methods, isothermal reverse transcription recombinase polymerase amplification (RT-RPA) is a simple method that has high sensitivity and avoids the use of expensive instruments. However, detection of amplified genomes often requires a fluorescence readout on costly readers or migration on a lateral flow strip with a subjective visual reading. Aiming to establish a new approach to rapidly and sensitively detect viruses, we combined RT-RPA with a magnetic field-enhanced agglutination (MFEA) assay and assessed the ability of this method to detect the dengue virus (DENV). Magnetization cycles accelerated the capture of amplified DENV genomes between functionalized magnetic nanoparticles by a fast chaining process to less than 5 min; the agglutination was quantified by simple turbidimetry. A total of 37 DENV RNA+ and 30 DENV RNA− samples were evaluated with this combined method. The sensitivity and specificity were 89.19% (95% CI, 72.75–100.00%) and 100% (95% CI, 81.74–100.00%), respectively. This approach provides a solution for developing innovative diagnostic assays for the molecular detection of emerging infections.

Highlights

The sensitivity and specificity of molecular methods used in laboratories and based on reverse transcription quantitative polymerase chain reaction (RT-qPCR) analysis for detection of viral RNA are excellent, but these methods have a long turnaround time and require trained personnel and costly equipment available only in major medical centers and not compatible with point-of-careAbbreviations: reverse transcription RPA (RT-RPA), reverse transcription recombinase polymerase amplification; MNP, magnetic nanoparticles; MFEA, magnetic field-enhanced agglutination; RT-qPCR, reverse transcription quantitative polymerase chain reaction; Ct, cycle threshold.Innovative Readout for DNA Detection testing
A reverse transcription RPA (RT-RPA) assay developed and applied to dengue virus (DENV) detection showed a good concordance with RT-qPCR assays
Negative controls were 30 plasma samples from individuals who had donated blood and who had no history of arbovirus contact (DENV RNA− samples); these were provided by the French national blood service (Etablissement Français du Sang, EFS, Montpellier, France)

Summary

Introduction

The sensitivity and specificity of molecular methods used in laboratories and based on reverse transcription quantitative polymerase chain reaction (RT-qPCR) analysis for detection of viral RNA are excellent, but these methods have a long turnaround time and require trained personnel and costly equipment available only in major medical centers and not compatible with point-of-careInnovative Readout for DNA Detection testing. The recombinase polymerase amplification (RPA) assay is an alternative method of isothermal amplification that can detect nucleic acids (Piepenburg et al, 2006) With this technique, it is not necessary to melt the DNA in order that the primers are directed to the complementary target sequences. A reverse transcription RPA (RT-RPA) assay developed and applied to DENV detection showed a good concordance with RT-qPCR assays This RT-RPA assay demonstrated various advantages versus RT-LAMP assays; these included an easy design of primers (only two for RT-RPA), a faster assay run time at a lower temperature, a higher sensitivity, and a relative ease of performance (Teoh et al, 2015; Xi et al, 2019; Xiong et al, 2020)

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Results

Conclusion