Frontiers in Bioengineering and Biotechnology (Nov 2021)

RAP: A Novel Approach to the Rapid and Highly Sensitive Detection of Respiratory Viruses

  • Guohao Fan,
  • Ruiqing Zhang,
  • Xiaozhou He,
  • Fengyu Tian,
  • Mingzhu Nie,
  • Xinxin Shen,
  • Xuejun Ma,
  • Xuejun Ma

DOI
https://doi.org/10.3389/fbioe.2021.766411
Journal volume & issue
Vol. 9

Abstract

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Recombinase aided amplification (RAA) is an emerging isothermal amplification method used for detecting various pathogens. However, RAA requires a complex and long probe to ensure high sensitivity during fluorescence assay. TaqMan probe used for quantitative PCR (qPCR) is simple and universal. Herein, we developed a new approach for detecting nucleic acids of pathogens, known as RAP (Recombinase aided PCR). The method combines RAA and qPCR to ensure a rapid and highly sensitive detection using a conventional qPCR device. RAP is a two-stage amplification process performed in a single tube within 1 hour. The method involves an RAA reaction for 10 min at 39°C (first stage) followed by 15 cycles of qPCR (second stage). Using human adenovirus 3 (HADV3) and human adenovirus 7 (HADV7) plasmids, the sensitivities of RAP assays for detecting HADV3 and HADV7 were 6 and 17 copies per reaction, respectively. The limit of RAP detection was at least 16-fold lower than the corresponding qPCR, and no-cross reaction with other respiratory viruses was observed. The results of RAP analysis revealed 100% consistency with qPCR assay. This study shows that RAP assay is a rapid, specific, and highly sensitive detection method with a potential for clinical and laboratory application.

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