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Rapid and visual detection of monkey B virus based on recombinase polymerase amplification

September 22nd, 2023
Rapid and visual detection of monkey B virus based on recombinase polymerase amplification
Schematic of RPA-VF reaction and vertical flow visualization strip. In short, the recombinase, strand-displacing DNA polymerase and primers initiate the amplification. Then, the special endonuclease cleaves the THF site when the probe hybridizes to the target sequence. As a result, the C3 spacer is dissociated and the probe extension is activated. After amplification, the reaction tube is placed into the closed vertical flow visualization strip device for detecting the RT-RPA products. Credit: Zoonoses (2023). DOI: 10.15212/ZOONOSES-2023-0031

Monkey B virus (BV) infection in humans and other macaque species has a mortality rate of approximately 80%. Because BV infects humans through bites, scratches, and other injuries inflicted by macaques, the simple and rapid diagnosis of BV in field laboratories is of great importance to protect veterinarians, laboratory researchers, and support personnel from the threat of infection.

Two recombinase polymerase amplification (RPA) assays with a closed vertical flow (VF) visualization strip (RPA-VF-UL27 and RPA-VF-US6) were developed that target two conserved genes combined with a one-off, closed visualization strip device. The sensitivities and specificities of the two assays were compared after optimization of the reaction conditions. The performance of RPA-VF-US6 at room temperature was determined to evaluate its potential in point-of-care (POC) testing.

The technology is published in the journal Zoonoses.

RPA-VF-US6 specifically detected the positive plasmid control (rather than nucleic acids of herpesviruses) with a detection limit of 28 copies, while RPA-VF-UL27 had cross-reactivity with HSV-1, but even 3.4 copies of plasmid standards were readout by this assay. Moreover, RPA-VF-US6 had excellent performance at room temperature (the detection limit was 2,800 plasmid copies), indicating the potential of RPA-VF-US6 in POC testing.

Two RPA assays for BV visualization diagnosis were developed. RPA-VF-US6 is a simple, rapid, and specific detection method for BV. The entire reaction can be performed at a constant temperature within 30 min, suggesting the potential of RPA-VF-US6 for POC testing in field laboratories without sophisticated instruments.

More information:
Xinlan Chen et al, Rapid and Visual Detection of Monkey B Virus Based on Recombinase Polymerase Amplification, Zoonoses (2023). DOI: 10.15212/ZOONOSES-2023-0031

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