Detection of low-load Epstein-Barr virus in blood samples by enriched recombinase aided amplification assay

Abstract

Epstein-Barr virus (EBV), a common human γ-herpesvirus, infects more than 90% of adults worldwide. The purpose of this study was to establish a novel EBV detection method by combining the recombinase aided amplification (RAA) assay with an initial enrichment step that utilizes magnetic beads coated with a recombinant human mannan-binding lectin (rhMBL, M1 protein). An M1 protein–protein A magnetic bead complex (M1 beads) was prepared and used to achieve separation and enrichment of EBV from blood. After nucleic acid extraction, DNA was amplified by RAA. Using 388 whole blood samples and 1 serum sample, we explored the specificity, sensitivity and applicability of the newly developed detection method and compared it with commercial quantitative real-time polymerase chain reaction (qPCR) following M1 bead enrichment, traditional qPCR and traditional RAA. After enrichment, the positivity rate of EBV was increased from 15.94% to 17.74% by RAA (P < 0.05) and from 7.20% to 15.17% by qPCR (P < 0.05). The viral loads after enrichment were increased by 1.13 to 23.19-fold (P < 0.05). Our data demonstrates that an RAA assay incorporating M1 bead enrichment is a promising tool for detecting low EBV viral loads in blood samples that will facilitate an early response to EBV infection. Graphical abstract

Development of a Rapid Epstein–Barr Virus Detection System Based on Recombinase Polymerase Amplification and a Lateral Flow Assay

“…The isothermal amplification of nucleic acids using RAA has been applied in a manner similar to RPA, including in a study by Yuan Gao et al [ 18 ] to detect the sensitivity and specificity of RAA for detecting EBV in extracted nucleic acids. Additionally, Jing-yi Li et al used the RAA method in combination with magnetic beads enriched with recombinant human mannan-binding lectin to detect low-carbon-load EBV in blood [ 33 ]. However, the RPA method was developed first and is more mature and stable than the RAA method, which does have some advantages.…”

Development of a Rapid Epstein-Barr Virus Detection System Based on Recombinase Polymerase Amplification and a Lateral Flow Assay

“…Isothermal amplification of nucleic acids using RAA has been applied in a manner similar to RPA, including in a study by Yuan Gao et al [33] to detect the sensitivity and specificity of RAA for detecting EBV in extracted nucleic acids. Additionally, Jing-yi Li et al used the RAA method in combination with magnetic beads enriched with recombinant human mannan-binding lectin to detect low-carbon-load EBVs in blood [34]. However, the RPA method was developed first and is more mature and stable than the RAA method, which does have some advantages.…”